Abstract
Abstract ID: 100
P. J. Higgins
Center for Cell Biology and Cancer Research, Albany Medical College
Angiogenesis
Plasminogen activator inhibitor type-1 (PAI-1), the major regulator of plasmin generation, is required for tumor invasion and the angiogenic response. Plasmin is utilized by both tumor cells and activated endothelial cells to locomote through complex extracellular matrix/stromal tissue barriers. PAI-1 synthesis must be tightly controlled in these processes as deficient PAI-1 expression (as in the PAI-1-/- mouse) or excessive PAI-1 production is incompatible with maintenance of a matrix “scaffold” structure permissive for tumor invasion or formation of functional neocapillaries. To assess PAI-1 function in cell migration, 800 bp of the human PAI-1 promoter were cloned upstream of a GFP insert. PAI-1 transcription in transfectants was confirmed by Northern blotting and, at the single-cell level, by GFP reporter activity after the switch from a sessile to motile phenotype. GFP expression was activated (as was the endogenous gene) within the real time of induced motility and only in the migrating cohort. To evaluate this relationship more directly, a second vector was developed in which 800 bp of the PAI-1 promoter was cloned upstream of an insert encoding a PAI-1-GFP chimeric protein. De novo–synthesized PAI-1/GFP was tracked in situ by fluorescence microscopy. Such PAI-1 expressing cells were highly motile in 2-D and 3-D migration assays. Moreover, invasive traits could be “rescued” in PAI-1-/- cells upon transfection with PAI-1–PAI-1/GFP expression vectors. The level of chimeric protein expressed correlated with cellular migratory activity and attenuation of PAI-1 synthesis by constitutive or inducible PAI-1 antisense vectors ablated the migratory response. These findings illustrate the usefulness of this approach to image transcriptional and migratory events in single cells using promoters from genes implicated in cellular invasive traits. (Supported by grants from the NIH (GM57242) and the Department of the Army.)
Abstract ID: 101
E. Brown D. Fukumura L. Munn R. K. Jain
Radiation Oncology, Massachusetts General Hospital
Angiogenesis
The process of tumor angiogenesis involves the growth and invasion of endothelial and perivascular cells from the host organism into the tumor. Using transgenic mice and multiphoton laser scanning microscopy (MPLSM) in living tumors, our laboratory has recently demonstrated that perivascular cells of host origin can express vascular endothelial growth factor (VEGF) and can form three-dimensional sleeve-like structures around tumor vasculature, as well as filamentous structures with no apparent blood flow. One interesting scientific question remains: What is the dynamic ordering of the construction of these vascular structures? The MPLSM allows us to monitor this dynamic evolution of tumor vasculature because of its noninvasive high-resolution imaging ability. In this poster, we explore the evolution of tumor vasculature, specifically following VEGF–GFP expressing host perivascular cells, endothelial cells, and the resultant blood flow.
Abstract ID: 102
R. Tong R. K. Jain
Radiation Oncology, Massachusetts General Hospital
Angiogenesis
Many scientists and researchers around the world suggest and show that antiangiogenic therapy can have a synergistic effect in combining with chemotherapy or radiation therapy. However, this is counterintuitive because chemotherapy and radiation therapy rely on blood vessels to delivery anticancer drugs and oxygen, respectively, to cancer cells. At the same time, the “holy grail” of the antiangiogenic therapy is to eliminate all the tumor blood supply within the tumor. Thus, it appears that antiangiogenic therapy should have a negative effect on either chemotherapy or radiation therapy. In 2001, Rakesh Jain put forth a hypothesis suggesting that during an antiangiogenic treatment, the tumor blood vessels first become “normalized” before the tumor reaches a point where there are inadequate blood vessels to support tumor growth. When immature and ineffective blood vessels are pruned away by the therapy, the remaining vasculature becomes more “normal” and efficient in delivering therapeutics globally. By using a multiphoton microscopy together with chronic animal window models, one can observe tumor blood vessels formation (or pruning) in vivo. Furthermore, red blood cell velocity and blood vessel permeability measurements are also feasible using the multiphoton microscopy. Interstitial fluid pressure and microvascular pressure can be measured using the micropipet method. All these data will give us insightful information about angiogenesis and antiangiogenic therapy. Hopefully, by incorporating the data with mathematical models, one can formulate the optimal schedule and dosage for the combinational therapy.
Abstract ID: 103
J. P. Weichert A. R. Moser M. A. Longino F. T. Lee, Jr.
Radiology, University of Wisconsin
Angiogenesis
Abstract ID: 105
P. M. Winter S. D. Caruthers A. H. Schmieder T. D. Harris L. Chinen T. Williams M. P. Watkins J. S. Allen S. A. Wickline G. M. Lanza
Cardiology, Washington University
Angiogenesis
The avB3-integrin has been implicated in tumor growth and metastasis and is highly expressed on many tumor cells and their vasculature. The objective of this research is to noninvasively image avB3-integrin expression by angiogenic vessels with MRI at 1.5 T using targeted paramagnetic nanoparticles.
Abstract ID: 106
Y. Ma X. Y. Wang J. H. Yang, et al.
Department of Pathology
Angiogenesis
Abstract ID: 107
Alexei Bogdanov, Jr. Alexander Petrovsky Dana Hu-Lowe David Shalinsky Ralph Weissleder
Radiology, Center for Molecular Imaging Research
Angiogenesis
Several novel antiangiogenic agents induce a targeted antiproliferative effect in endothelial cells by inhibiting VEGF-receptor 2-tyrosine kinase (KDR) activity. Although morphometry of tumors (i.e., tumor volume measurement) can serve as a technique for validation of antiangiogenic effects, we hypothesized that the MR determination of hemodynamic properties can potentially provide a substantially earlier measurement of angiogenesis surrogate markers. Using an experimental VEGFR-KDR inhibitor, we demonstrated that measurements of intravascular volume fraction by the steady-state T1-weighted MRI allows the assessment of very early effects (after 1 day of treatment) before the tumor size changes become obvious. The tested VEGF-RTKI inhibitor induced a highly pronounced antiangiogenic effect as determined by (1) a rapid decrease of rVVF determined by MRI after three doses of the inhibitor, and (2) a significant retardation of the tumor growth at later time points. These studies indicate that MR imaging can be used as a functional method to monitor the treatment efficacy within a short period of time after treatment initiation.
Abstract ID: 108
D. Fukumura Y. Izumi E. di Tomaso R. K. Jain
Radiation Oncology, Harvard Medical School and Massachusetts General Hospital
Angiogenesis
Nitric oxide (NO) is known to regulate physiological functions such as angiogenesis, blood flow, and vascular permeability, and thus is considered to facilitate tumor growth. We have previously shown that endothelial NO synthase (eNOS) plays a predominant role in vascular endothelial growth factor-induced angiogenesis and that tissue NO levels correlate with angiogenesis and metastatic potential of murine melanomas grown in primary and metastasis sites. Here we determined the causal relationship between NO and angiogenesis and the contribution of host stromal NOS. We found homogeneous expression of eNOS in vascular endothelial cells in both normal brain and B16F10 tumors. eNOS was also detected in some tumor cells, while iNOS was expressed heterogeneously in tumor cells but was not detectable in normal brain. The expression of iNOS was also found in some host cells, presumably infiltrated inflammatory cells in the tumors.
Abstract ID: 109
D. Dolmans A. Kadambi J. S. Hill D. Fukumura, et al.
Radiation Oncology, Harvard Medical School and Massachusetts General Hospital
Angiogenesis
The antivascular effects of photodynamic therapy (PDT) and their mechanisms are not clearly understood. Here we examined the effects of PDT with a novel photosensitizer MV6401 on microvasculature in normal tissue and a mouse mammary tumor (MCaIV) grown in a mouse dorsal skinfold chamber. Fifteen minutes after intravenous injection, MV6401 was shown by fluorescence microscopy and immunohistochemistry to colocalize with the endothelial cell marker CD31, suggesting that at this time point, it was only present in the vascular compartment. PDT mediated by MV6401 at this time point induced vascular permeability increases, a tumor growth delay, and a dose-dependent biphasic blood flow stasis. This biphasic response was classified into two components, an acute response observed immediately post-PDT, and a long-term response observed at times greater than 3 hr post-PDT. The acute temporal vascular effects were characteristic of vasoconstriction but not thrombus formation. However, the long-term vascular shutdown was mediated by thrombus formation as evidenced by histological evaluation and inhibition with heparin, but with no increase in tumor interstitial pressure. The effects on normal vessels were minimal at the two lower doses, but were similar to the effects in tumor at the highest (0.072 mg/kg) dose. Collectively, these data suggest that PDT with MV6401 induces relatively selective long-term tumor blood flow stasis via thrombus formation and leads to tumor growth delay. This study provides mechanistic insights in antitumor vascular effects of PDT and suggests novel strategies for tumor treatments with PDT.
Abstract ID: 110
D. Dolmans A. Kadambi J. Hill J. Walker, et al.
Radiation Oncology, Harvard Medical School and Massachusetts General Hospital
Angiogenesis
We examined the antivascular and antitumor effects of photodynamic therapy (PDT) on MCaIV mammary carcinoma implanted orthotopically in the mammary fat pad of female SCID mice. When tumors reached 3 mm in diameter, the animals were treated with PDT using four different drug doses of MV6401: 0, 0.03, 0.06, or 0.12 mg/kg BW. The tumors were irradiated 15 min post-MV6401 administration with 5 J/cm2 of 664 nm light. At this time, the photosensitizer was localized within the blood vessel compartment as shown with fluorescence microscopy. Tumor size and tumor blood flow were measured by intravital microscopy before and various time points after the PDT. In each group (n = 5), 25 vessels per animal were observed at every time point. Tumors grown in the mammary fat pad showed a drug dose-dependent growth delay following PDT. Complete tumor growth arrest up to 21 days was observed only in the highest drug dose group. Regardless of the drug dose, tumor blood flow stasis was observed in all regions examined during and immediately after PDT. However, persistence of blood flow shutdown was drug dose-dependent. Three weeks after PDT, 68%, 24%, and 0% of the regions were perfused in the 0.03, 0.06, 0.12 mg/kg BW treated tumors, respectively. Drug alone and light alone did not exhibit altered blood flow compared to the nontreated animals. In summary, drug dose-dependent antivascular and antitumor effects by PDT were observed in clinically relevant orthotopic mammary tumor. Vascular targeting with PDT is a potentially useful treatment strategy for breast cancer.
Abstract ID: 111
C. Perlitz K. Licha P. Hauff S. Wisniewski M. Schirner
Ultrasound and Optical Imaging, Schering
Angiogenesis
As living tissue is relatively transparent to light in the near-infrared (NIR) region of 700–900 nm, tissue probing with NIR light provides the opportunity to detect tumors up to several centimeters below the tissue surface. Targeting of a specific molecule in a certain disease status via an antibody linked to an NIR absorbing dye provides the chance to develop new imaging agents. These agents may exert a high sensitivity to detect lesions in certain clinical settings such as under intraoperative conditions, in endoscopical approaches or in optical mammography. We are currently investigating two different approaches in several mouse tumor models. The first approach is based on the permeability of tumor blood vessels leading to the accumulation of fluorescent dyes in the tumor interstitium. In the second approach, the same hydrophilic indotricarbocyanine is linked to a single-chain antibody targeting a marker molecule indicative of tumor angiogenesis. In vivo imaging is performed on tumor-bearing mice. It is achieved with a tunable, pulsed solid-state laser system used for excitation and an intensified charge-coupled device (CCD) camera for detection. A long-wave pass filter is used to cut off reflected excitation light. Though applying different modes of action, both approaches show significant accumulation of diagnostic dyes in the tumor compared to a control region over time. Besides the comparison of these two approaches, we focus on currently evolving issues such as the need for high standardization in these experiments, problems that arise with the coupling of dyes to biomolecules (e.g., fluorescence quenching), and the evaluation of possible marker molecules for the specific approach.
Abstract ID: 112
T. P. L. Roberts K. Turetschek A. Preda V. Novikov, et al.
Medical Imaging, University of Toronto
Angiogenesis
Abstract ID: 113
Y. R. Kim A. Petrovsky D. Shalinsky H. L. Lowe R. Weissleder A. Bogdanov
Radiology, CMIR/HMS
Angiogenesis
Antiangiogenic therapy is emerging as an effective strategy to target and potentially eliminate neoplastic tumor vessels. The goal of this work was to establish whether magnetic resonance imaging (MRI) of tumor blood volume (Vb) can be used as an early predictor of angiogenic response to tumor treatment. In this study, we demonstrated a reliable and simple methodology for longitudinally measuring tissue Vb and intra/extravascular water exchange rate in response to antiangiogenic therapy. Using an absolute Vb measurement technique utilizing a strictly intravascular T1 contrast agent, MRI steady-state measurements of tumor and muscle tissues were comparatively analyzed for the assessment of changes in neoplastic vasculature. In particular, the feasibility and efficacy for monitoring vascular parameters were investigated using a noninvasive steady-state MRI technique. Mice with tumor implants derived from human colon carcinoma MV522 cell line were treated with AG013925 VEGF-R2 KDR inhibitor by oral administration (25 mg/kg) and were imaged after three treatments and 1-week course of 2 × BID. The measured Vb was 2.66 ± 0.99% for the striated muscle and 2.52 ± 1.47% for the tumor 1 week after the implantation. Tumor Vb markedly decreased after 1 day of treatment and remained low for the treatment period (approximately at 1%). The intra/extravascular water exchange rate of growing tumor vasculature was higher than that of striated muscle while the treatment significantly reduced the water exchange rate.
Abstract ID: 114
K. Camphausen T. Scott M. Sproull C. N. Coleman, C. Menard
Radiation Oncology Branch, ROSP/CCR/NCI/NIH
Angiogenesis
Abstract ID: 115
E. di Tomaso D. Capen Janet Hart R. K. Jain R. Jones L. L. Munn
Radiation Oncology, Massachusetts General Hospital
Angiogenesis
Abnormalities in the endothelium of tumors have been extensively studied, but the relationships between immunohistochemical and morphological defects have not been previously described. Although approximately 4% of the vessel surface area in human colon carcinoma xenografts in mice lacks CD31/CD105 immunoreactivity, the cellular architecture in these regions remains undefined. Our goal was to locate these defective regions by confocal microscopy and determine the morphology of the wall of the same vessel by electron microscopy. LS174T human adenocarcinomas were grown in SCID mice for 10 days to 2 weeks. Cy5-CD31/CD105 was injected intravenously 1 hr prior to perfusion–fixation. By EM, most regions of vessels that lacked CD31/CD105 immunoreactivity had thin processes of endothelial cells, some of these lacking overlapping junctions with neighboring endothelial cells. Focal sites lacking endothelium were also apparent in some regions and are being investigated further. More than 80% of the vessels lacking CD31/105 staining also lacked immunoreactivity for basement membrane proteins, suggesting that this disruption may result in, or may even be a prerequisite for, defects in the endothelial lining. The combination of the two techniques developed in this study allows simultaneous analysis of vessels by light, confocal, and electron microscopy, with little variation in structure over the 2-μm sections. We conclude that in this tumor model, functional vessels can have abnormal CD31/CD105 staining associated with abnormalities in the endothelium such as attenuated lining cell processes or possibly absence of endothelial cells. Further studies will show whether similar results are obtained in the case of orthotopic implantations.
Abstract ID: 116
P. Yalamanchili D. C. Onthank P. J. Silva N. Y. Sanabria, et al.
Discovery Biology, Bristol Myers Squibb Medical Imaging
Angiogenesis
The integrin avβ3 mediates diverse responses in vascular cells and tumor cells and is involved in tumor metastasis and neovascularization of tumors. In this study, we evaluated a nonpeptide small molecule antagonist (DPC A80351) of avβ3 for imaging tumors. The MMTV-neu transgenic mouse and a cell line derived from a mammary tumor of this mouse model were used for in vitro binding studies and in vivo imaging. DPC-A80351 binds to MMTV-neu cells with a Kd of 31 pM and a Bmax of 6392 sites/cell. In vitro internalization studies showed that bound DPC A80351 is internalized in a time-dependent receptor-specific process in MMTV-neu cells. The tissue distribution and scintigraphic imaging of DPC-A80351 in the c-neu Oncomouse model showed excellent tumor uptake over 2 hr (9.4 ± 0.6% ID/g) with low nontarget (muscle) tissue uptake (0.55 ± 0.11% ID/g). Tumor-to-muscle ratios derived from the tissue distribution were 17:1, 35:1, and 59:1 at 2, 24, and 96 hr, respectively. Tumor-to-blood ratios were 17:1, 58:1, 194:1 at 2, 24, and 96 hr, respectively. DPC-A80351 was predominantly eliminated by renal excretion. At 24 hr postinjection, approximately 83% of the injected dose was present in the urine and 1% was present in the feces. In conclusion, DPC-A80351 has high affinity for avβ3 receptor and has high tumor uptake with low uptake in nontarget organs and has great potential for imaging tumors that express avβ3 receptors.
Abstract ID: 117
E. E. Uzgiris L. Brogan K. M. Fish B. Moasser J. F. Smith B. Grimmond
Genomics and Molecular Imaging Laboratory, GE Global Research
Angiogenesis
Reptation, the process by which linear polymers can snake their way around obstacles and through small channels, allows extended linear polymer agents to enter the tumor interstitium through the tumor endothelium with much higher efficiency than is found with large globular agents or folded linear polymers. This entry and the concomitant high MR signals can be used as a measure of angiogenic blood vessel development. The high signal regions are correlated with blood vessel development in those regions as demonstrated by histological sectioning. In this case, the polymer is a DTPA conjugated polylysine chain which is in an extended conformation, a “worm chain,” when the conjugation is about 90% or higher. However, when the conjugation falls below this level, the polymer assumes a folded state with a tighter hydrodynamic radius as is observed with gel permeation chromatography. We examine the polymer length dependence on tumor signal levels, and in particular, we examine the limit of very short polymers. In this limit, the blood circulation time becomes quite short, but this effect is compensated to a degree by the decrease in the polymer friction coefficient with the decrease in length. We also examine the important issue of polymer charge. Previous investigations were of highly negatively charged polymers only. We examine the efficacy of uncharged polymers, slightly negatively charged polymers, and slightly positively charged polymers.
Abstract ID: 118
P. McQuade L. C. Knight D. McCarthy M. J. Welch
Radiology, Washington University in St. Louis
Angiogenesis
During angiogenesis, expression of β3 integrins on endothelial cells increases on new blood vessels. Bitistatin, a member of the disintegrin family, is a good marker for β3 integrins (LC Knight et al. J. Nucl. Med. 2000, 41, 1056). Bitistatin is constrained by seven disulfide bridges into a folded structure with multiple loops. The apex of one of these loops contains the Arg–Gly–Asp (RGD) moiety that is responsible for binding to β3 integrins. Previous work with bitistatin involved labeling with 125I. The biodistribution in EMT-6 bearing BALB/c mice displayed optimal tumor uptake at 2 hr postinjection (11.7 ± 4.6% ID/g; LC Knight et al. J. Labelled Compd. Radiopharm. 2001, 44, S84). This work was extended to label bitistatin with 64Cu, which would allow microPET imaging of the tumor. 64Cu was attached to bitistatin via 1,4,7,10-tetraazacyclododecane-N,Nʹ,Nʺ,N‴-tetraacetic acid (DOTA) that had been bound to a lysine residue. In EMT-6 bearing mice, the biodistribution differed from that of the 125I material, in that the maximum tumor uptake was lower (1.6 ± 0.2% ID/g) and took 6 hr to reach its peak. In an attempt to reduce steric interactions caused by lysine modification, the DOTA analog bro-moacetamidobenzyl-1,4,7,10-tetraazocyclododecane-N,N′,N″,N‴-tetraacetic acid (BAD) was examined. This resulted in higher tumor uptake (maximum 5.2 ± 0.6% ID/g at 6 hr), however, this was accompanied by unacceptably high renal retention. These data suggest that biodistribution of modified bitistatin is affected by both charge and steric factors on the modified side chains. Ongoing studies involve labeling bitistatin with 124I and 76Br.
Abstract ID: 119
B. Moffat D. Hall T. Chenevert B. Ross
Radiology, University of Michigan
Angiogenesis
The development of novel therapies for manipulating angiogenesis has necessitated the development of robust techniques for imaging surrogate markers of angiogenesis in both preclinical animal trials and in clinical settings. One of the potential surrogate markers of altered or manipulated angiogenesis is blood flow. Magnetic resonance perfusion imaging of blood flow has the potential to become an important noninvasive assay of angiogenesis since MRI is already an accepted conventional diagnostic tool with excellent anatomical contrast without the use of nonionizing radiation. Perfusion MRI of CBF using an endogenous contrast has been developed significantly for use with human patients, however, existing methodologies are largely unsuitable for routine assessment of blood flow in rodent models. We present here a new MRI technique for producing quantitative endogenous perfusion contrast in MR images. This method is robust from the point of view that it requires no special amplifiers, r.f. coils, decoupling, or complicated image processing that are not standard on most animal MR scanners, thus making it ideal for high throughput screening of therapeutic manipulation of angiogenesis. The perfusion contrast images are produced by preweighting a fast spin-echo sequence with a train of slice selective hyperbolic secant inversion pulses that are frequency shifted relative to the imaging slice. We will present the requirements for implementing the protocol within a routine imaging study, including optimization strategies, data acquisition, image processing, and data presentation (both flow maps and histogram analysis). The protocol was tested by applying it to study the effects of carbogen breathing on both CBF and brain tumor blood flow (TBF) in Fischer 344 rats with implanted 9L gliomas.
Abstract ID: 120
E. A. Schellenberger A. Bogdanov, Jr. A. Petrovsky, R. Weissleder L. Josephson
CMIR, MGH
Apoptosis
Annexin V, a protein used for the detection of apoptosis based on its ability to bind extracellular phosphatidylserine, was reacted with the near-infrared fluorescent (NIRF) dye Cy5.5TM. Annexin V was sensitive to reaction with Cy5.5, with activity decreasing sharply as progressively higher numbers of Cy5.5 were attached per mole of protein. However, a conjugate with 1.1 mol of Cy5.5 per mole of annexin V (Cy5.51.1–annexin V) was highly active, as shown by a 41-fold higher signal for apoptotic than nonapoptotic cells in flow cytometry. We therefore employed the Cy5.51.1–annexin V conjugate to determine the kinetics of caspase activation and phosphatidylserine exposure in populations of camptothecin-treated Jurkat T cells. Cy5.51.1–annexin V can be used with the large number of commercially available FITC probes of apoptosis in flow cytometry studies, because the two fluorochromes have extremely different optical properties. In addition, the NIRF of Cy5.51.1–annexin V permits visualization of probe accumulation through overlaying tissues. This property may allow Cy5.51.1–annexin V to be used for the optical imaging of apoptosis in vivo.
Abstract ID: 121
Alnawaz Rehemtulla
Radiation Oncology, University of Michigan
Apoptosis
Strict coordination of proliferation and programmed cell death (apoptosis) is essential for normal physiology. An imbalance in these two opposing processes results in various diseases including AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemia/reperfusion injury, cancer, autoimmune disease among others. Objective and quantitative noninvasive imaging of apoptosis would be a significant advance for rapid and dynamic screening as well as validation of experimental therapeutic agents. Here, we report the development of a novel hybrid luciferase reporter molecule, which when expressed in mammalian cells, has attenuated levels of reporter activity. This reporter molecule was stably expressed in human D54 glioma cells and implanted subcutaneously in nude mice. Upon treatment with TNF-alpha related apoptosis inducing agent (TRAIL), caspase-3 activation was observed and the tumors underwent apoptosis. Concurrent with the activation of caspase-3, cleavage of the reporter molecule to the 40-kDa luciferase polypeptide was observed. Restoration of luciferase activity was also observed which was detected in living animals using bioluminescence imaging (BLI). A 5–6-fold increase in the photon counts was detected from the tumor site after TRAIL treatment compared to saline-treated tumors. The ability to image apoptosis noninvasively and dynamically over time provides a new opportunity for high-throughput screening of pro- and antiapoptotic compounds and for target validation in vivo in both cell lines and transgenic animals.
Abstract ID: 122
H. Zhao C. H. Contag
Pediatrics, Stanford University
Apoptosis
Characterizing molecular events that mark specific steps in the cellular response to stress is crucial for the development of new imaging targets for the next generation of in vivo assays. Here, we examined the heme oxygenase-1 (Hmox-1) gene encoding a stress responsive protein induceable by hyperoxia (95% oxygen) and CdCl2 (10 mM). Rat glioma C6 cells exposed to either stressor were observed at 0, 6, 12, 24, and 48 hr. Cells showed morphological changes but only a very small percentage had disrupted mitochondria membrane potentials or DNA fragmentation at 24 hr posttreatment. Flow cytometric cell cycle analyses using propidium iodide revealed that most cells appeared to be arrested at G2/M phase at 24 hr. To follow Hmox-1 transcription levels in real time, we stably transfected C6 cells with a luciferase-YFP fusion reporter gene driven by the Hmox-1 15 kb upstream regulatory region. Reporter levels were elevated as early as 30 min postinduction with CdCl2, while peak induction (15–20 fold) occurred at 6–8 hr. This was consistent with the Hmox-1 mRNA levels (19-fold increase) as detected by real-time polymerase chain reaction (RT-PCR). However, luciferase activity decreased dramatically to basal levels by 24 hr while luciferase mRNA levels fell by only 26%, which may reflect a shut down of cellular translation coincident with cells entering G2/M phase, and/or preapoptotic reduced cellular ATP synthesis. These results support the importance of HO-1 as a marker of the time surrounding the turning point at which cells become committed to apoptotic cell death.
Abstract ID: 123
D. Piwnica-Worms
Radiology, Washington University School of Medicine
Cancer
Mechanisms of drug resistance can act directly on the disposition, targeting, and metabolism of the candidate drug or indirectly on downstream regulators and effectors of therapeutic drug action. In the context of anticancer drug resistance, we will discuss strategies for molecular imaging of two key mechanisms of multidrug resistance in cancer chemotherapeutics. The first is an example of a direct acting mechanism: the role of the multidrug resistance (MDR1) P-glycoprotein membrane transporter and the noninvasive imaging of P-glycoprotein-mediated drug transport activity with SPECT and PET agents. The second is an example of a downstream effector of drug resistance: p53, a transcription factor that when mutated enables cancer cells to traverse the usual checkpoints that cells use to assure the integrity of their DNA. The interaction of p53 with noncovalent protein binding partners can be noninvasively imaged in living animals using a novel mutant herpes simplex virus 1 thymidine kinase (HSV1-TK) reporter and microPET.
Abstract ID: 124
V. Raman D. Artemov Y. Mironchik A. Pathak Z. Bhujwalla
Radiology, Johns Hopkins University
Cancer
Some of the lethal phenotypic traits of cancer, such as invasion and metastasis, may arise from the unique physiological environment of tumors which is characterized by areas of poor flow, hypoxia, high lactate, and low pH. Here, for the first time, we have used a combined multiparametric approach of combined vascular, spectroscopic, and optical imaging to obtain vascular, metabolic, and hypoxic maps from colocalized regions within a tumor. We generated PC-3 tumors derived from cells stably transfected with the hypoxia response element (HRE) of human VEGF-A ligated to the enhanced green fluorescence protein (EGFP) gene. Increased GFP expression in these cells in response to hypoxia, as well as 10 μM cobalt chloride, was established in culture. Vascular and metabolic maps were obtained noninvasively from PC-3 tumors using MRI for vascular imaging and MRSI for metabolic imaging. Optical imaging of GFP expression in excised tumor sections was used to visualize hypoxia in relation to the noninvasively obtained MR vascular and metabolic maps. Distinct vascular, metabolic, and hypoxia distributions were detected in the tumors. Areas of low vascular volume measured by MRI were associated with high fluorescence in the optical images. These regions of low vascular volume and high fluorescence also exhibited high permeability in the MRI maps. A combined MRI/MRS and optical imaging approach of acquiring metabolic, vascular, and hypoxia images from colocalized regions, for tumors exhibiting significant differences in metastatic patterns, will provide further understanding of the dynamics between these parameters, and their role in cancer invasion and metastasis.
Abstract ID: 125
E. Sausville
Developmental Therapeutics Program, National Cancer Institutes
Cancer
Two distinct types of targets are relevant to defining novel cancer treatments: pathway-related targets are of functional importance to the biologic success of a tumor; cell differentiation or tumor physiology-related targets, which while not germane to the causation of the cancer may be an expression of tissue of origin or host reaction to the tumor. A desired ideal would be to define patients with a high likelihood of response to new treatments directed at both types of targets. A key issue is how to define the activation state of relevant pathways in situ in a living host, or the presence of intracellular differentiation/physiology-related descriptors of different tumor types. The current concept of pathway-related targets is that they will derive from common abnormalities in cell proliferative control (activation of oncogenes and loss of tumor suppressor genes); defective cell death mechanisms; delayed or absent cellular senescence; capacity to remodel tumor stroma, including the promotion of blood vessel growth, and tumor cell migration and metastasis. Examples of present opportunities will be considered, including tyrosine kinase-related pathways, MAP kinase activation state; mammalian target of rapamycin (mTOR) activity, and heat shock protein 90 (hsp90) modulation. Opportunities to contribute to the development of treatments related to cell differentiation or physiology endpoints would include the desirability of imaging transcription-coupled DNA repair potential and aryl hydrocarbon-receptor activation status as an expression of susceptibility to novel DNA-interactive agents and drug metabolizing pathways, respectively.
Abstract ID: 126
Jan E. Schnitzer
Vascular Biology, Sidney Kimmel Cancer Center
Cancer
Molecular medicine has discovered many targets leading to therapies that work in vitro but not in vivo where barriers restrict access. The poor accessibility of most tissue- or disease-specific molecules identified through genomic or other analysis has also hindered organ and tumor imaging of gene and protein expression in vivo. A strategy will be described that combines novel high-resolution proteomic mass spectrometric techniques and tissue subfractionation to map the accessible vascular proteome in vivo in order to identify readily accessible organ- and tumor-specific targets on blood vessels and to create novel targeting antibodies. By intravenous injection, these antibodies target the endothelial cell surface for tissue-directed delivery with up to 89% of the injected dose of antibody accumulating in a single tissue in just 30 min. Perhaps more importantly, these antibodies target tissue-specific proteins in caveolae for transport across the microvascular endothelium into the tissue parenchyma where they are internalized by the underlying tissue cells. Caveolae-mediated transcytosis can rapidly overcome the normally restrictive endothelial and even epithelial cell barriers and thus provide critical access into select tissues. Our analysis so far reveals considerable molecular heterogeneity for endothelia and caveolae in different tissues, thereby providing useful tissue-specific targets not only for drug and gene delivery in vivo but also for improved imaging of normal and diseased tissues in vivo. Our data support vascular targeting as a strategy that can achieve theoretical expectations for directed delivery in vivo. Now detailed molecular mapping analysis of endothelia is necessary to define the full complement of tissue-induced accessible targets.
Abstract ID: 127
J. P. Weichert A. R. Moser M. A. Longino R. E. Counsell
Radiology, University of Wisconsin
Cancer
Abstract ID: 128
S. M. Weber C. Qi J. P. Weichert
Radiology, University of Wisconsin
Cancer
Abstract ID: 129
J. P. Weichert S. M. Weber C. Qi J. Longo F. T. Lee, Jr.
Radiology, University of Wisconsin
Cancer
Abstract ID: 130
T. Padera A. Kadambi E. di Tomaso C. Mouta-Carreira, et al.
Radiation Oncology, Massachusetts General Hospital
Cancer
Lymphatic metastasis contributes to mortality from solid tumors. Whether metastasizing cancer cells reach lymph nodes via intratumor lymphatic vessels is unknown. Here we examine functional lymphatics associated with mouse tumors expressing normal or elevated levels of vascular endothelial growth factor-C (VEGF-C), a molecule that stimulates lymphangiogenesis. Although VEGF-C overexpression increased lymphatic surface area in the tumor margin and lymphatic metastasis, these tumors contained no functional lymphatics, as assessed by four independent functional assays and multiphoton laser scanning microscopy. These findings suggest that the functional lymphatics in the tumor margin alone are sufficient for lymphatic metastasis, and should be targeted therapeutically.
Abstract ID: 131
M. Yang P. Jiang N. Yamamoto R. M. Hoffman
AntiCancer
Cancer
Strong fluorescent labeling with green fluorescent protein (GFP) along with inexpensive video detectors, positioned external to the mouse, allows the monitoring of details of tumor growth, angiogenesis, and metastatic spread in mouse models. Opening a reversible skin-flap in the light path increases detection sensitivity to the single-cell level on internal organs. Single tumor cells, expressing GFP, seeded on the brain can be imaged through a scalp skin-flap. Lung tumor micro-foci representing a few cells are viewed through a skin-flap over the chest wall while contralateral micro-metastases were imaged through the corresponding skin-flap. Pancreatic tumors and their angiogenic micro-vessels were imaged via a peritoneal wall skin-flap. A skin-flap over the liver allowed imaging of physiologically relevant micro-metastases originating in an orthotopically implanted tumor. Single tumor cells on the liver arising from intraportal injection were also detectable (Yang, M., et al. Proc. Natl. Acad. Sci. USA 99, 3824–3829, 2002). Using the antiapoptotic gene bcl-2, dominant-negative caspase 9 (C9DN) and p16INK4a, we showed by whole-body GFP imaging that disruption of the apoptosis and senescence program confers tumor aggressiveness and drug resistance of GFP-labeled lymphomas, which arose in Eu-myc transgenic mice (Schmitt, C.A., et al. Cancer Cell 1, 289–298, 2002; Schmitt, C.A., et al. Cell 109, 335–346, 2002). Using the HT-1080 human fibrosarcoma labeled with GFP or RFP, mixed-color populations were injected intravenously into SCID mice. This resulted in individual green or red clones on the lung. These results indicate the possibility of color-coding cells of different genotype in vivo for studies such as those described above.
Abstract ID: 132
G. Caceres X. Zhu R. Zamkina A. Aller, et al.
Rumbaugh-Goodwin Institute for Cancer Research
Cancer
Bioluminescent imaging has been used to study the growth and metastasis of Photinus pyralis luciferase transfected MCF-7 breast and DU-145 prostate tumor cells in nude mice. Growth and metastasis of the human cell lines transfected with the luciferase gene were qualitatively and quantitatively analyzed in nude mice using the Night Owl Molecular Light Imager. Studies with 106–107 cells per mouse showed differences in tumor distribution and lower limits of detection in vivo depending on the route of inoculation. Cells injected intravenously were not detectable until metastasis was observed in the lymph nodes. Cells injected intraperitoneally were localized in the peritoneal cavity with spleen infiltration. Cells injected subcutaneously could be detected by imaging prior to observing a measurable tumor mass. Similar imaging studies have also been performed using green fluorescent protein (GFP) transfected human tumor cell lines.
Abstract ID: 133
M. Morrison S. Pullan H. Wolfe M. Mendizabal, et al.
R&D, Amersham Health
Cancer
GC-C receptor is expressed in adult humans only by intestinal epithelial cells, not by extraintestinal tissues. Significantly, expression of GC-C has been detected in all primary and metastatic colorectal tumors, but not in any extraintestinal tissues or tumors examined. Previous work carried out by the authors identified that 99m99mTc-mercaptoacetamidoadipoylglycylglycine (N3S-adipoyl) STa (5–18) and other derivatives (e.g., 99mTc-NC100673) bind with high affinity to GC-C. In order to extrapolate the data shown in animal models, and as receptor uptake and retention in the tumor is determined to a large extent by receptor concentration, it was necessary to compare the receptor expression in the animal models used with human tumor expression. GC-C receptor density of human liver metastases, xenograft tumors, and cell lines was measured. Results indicate that the mean receptor density of human tumors lies between that seen for T84 and LS180 tumors. In order to determine whether compound uptake varied with receptor density, biodistribution studies were undertaken in a range of xenograft tumors with different GC-C receptor expressions. The data demonstrated comparatively higher retention of 99mTc-NC100673 in CaCO2 and T84 xenograft tumors compared to the other tumor types. In conclusion, it has been shown that in both moderately differentiated tumors (e.g., T84, CaCO2) and less well differentiated tumors (e.g., LS180, HT29), specific uptake of active GC-C targeting agents is seen above that of the negative control. Therefore, differences in tumor differentiation and receptor.
Abstract ID: 134
K. Glunde S. Guggino Y. Ichikawa Z. M. Bhujwalla
Radiology Department, Johns Hopkins University School of Medicine
Cancer
Cancer cells invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The tumor microenvironment, frequently characterized by hypoxia and acidic extracellular pH, may play a role in cancer cell invasion and metastasis. Therefore, we investigated the impact of acidic extracellular pH levels on lysosomal trafficking in human breast cancer cells. Lysosomal trafficking was studied in vitro using immunofluorescence staining of lysosomal-associated membrane proteins (LAMPs), which are widely used as lysosomal markers. We also evaluated and validated a new method to optically image lysosomes, by labeling highly glycosylated lysosomal proteins with a dansylated glucosamine (6-O-dansyl-GlcNH2). This new method of tracking lysosomal trafficking enabled us to study extracellular acidification in living cells. The dansylated glucosamine was incorporated into breast cancer cells and was finally localized in small intracellular vesicles that colocalized with immunofluorescence staining of LAMPs in the lysosomes. An acidic extracellular pH of 6.4, maintained for 24 hr, altered the distribution of lysosomes. In control cells (pH 7.4), lysosomes were mainly found in the perinuclear region. In acidified cells (pH 6.4), the lysosomal distribution was more scattered than in control cells, and shifted more towards the cell periphery. Similar results were obtained in living cells using lysosomal labeling with 6-O-dansyl-GlcNH2 after 4 hr of exposure to acidic media (pH 6.4). These findings suggest altered lysosomal trafficking in the presence of an acidic extracellular environment often seen in metastatic tumors. This altered lysosomal trafficking can result in increased secretion of degradative enzymes.
Abstract ID: 135
A. H. Schmieder P. M. Winter S. D. Caruthers T. D. Harris L. Chinen T. Williams M. P. Watkins J. S. Allen S. A. Wickline G. M. Lanza
Biomedical Engineering, Washington University Medical School
Cancer
The avB3-integrin has been implicated in melanoma growth and metastasis and is highly expressed on both the tumor cells and neovasculature. The objective of this research is to noninvasively image avB3-integrin expression by melanoma neovasculature with MRI at 1.5 T using avB3-targeted nanoparticles.
Abstract ID: 136
T. D. McKee A. Pluen Y. Boucher S. Ramanujan, et al.
Radiation Oncology, Massachusetts General Hospital
Cancer
Abstract ID: 137
J. Bading
Radiology, University of Southern California
Cancer
When modeling radiotracer kinetics in tumors, investigators have generally used the classical FDG compartmental model, in which blood-borne activity is equated to arterial activity concentration × a volume factor, and the radiotracer is assumed to equilibrate rapidly between the tumor interstitial and intracellular spaces. The author has developed and validated a model for [F-18]5-fluorouracil (5-FU) in a rat colon tumor implanted peripherally in Fischer 344 rats. 5-FU tumor time–activity curves conformed very closely to a catenary, three-compartment model. Compartment 1 was shown to correspond to tumor extracellular space (intravascular plasma + interstitium) by comparing its distribution volume with that measured for the extracellular tracer Tc-99m DTPA. By comparison with direct assay of radiolabeled metabolites in excised tumors, Compartments 2 and 3 were shown to correspond, respectively, to an exchanging intracellular pool of free 5-FU + mono- and diphosphate nucleotides and to trapped triphosphate nucleotides + RNA. Model fits were improved slightly by adding a term to account for F-18 in red blood cells. The physiologic correlates (i.e., intravascular and interstitial spaces as well as transport processes) of the FDG model differ from those of the 5-FU model, in which the tumor cell membrane rather than the capillary wall constitutes a significant barrier to radiotracer penetration. The 5-FU model may serve as a better paradigm than the FDG model for describing the kinetics of small, hydrophilic molecules in solid, somatic tumors.
Abstract ID: 138
E. A. Chiocca, MD, PhD
Neurosurgery, Massachusetts General Hospital
Cancer
Imaging gene expression in vitro and in vivo can potentially provide diagnostic information on disease processes such as malignant brain tumors. This will have a profound impact on therapeutic endeavors, such as drug discovery, immunotherapies, and gene therapies. We will review how molecular imaging modalities are being used in animal models of brain tumors and how these can be translated into correlates of therapy for clinical trials of this and other neurological diseases.
Abstract ID: 139
B. Y. Croft
Biomedical Imaging Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute
Cancer
In the National Cancer Institute, the Biomedical Imaging Program (BIP) (http://cancer.gov/bip/) of the Division of Cancer Treatment and Diagnosis is responsible for the extramural grant portfolio and programs related to oncologic imaging. The Small Animal Imaging Resource Programs (SAIRPs) (http://www3.cancer.gov/bip/sairp.htm) serve to connect small animal imagers with cancer-related researchers. This program complements the NCI's Mouse Models of Human Cancer Consortium (http://emice.nci.nih.gov/) effort in which 20 research groups are creating mouse models of various cancers and depositing breeding stock in the NCI Repository in Frederick, MD, for the use of the bio-sciences community. The mice will be of special interest to preclinical trial investigators. The ACR Imaging Network (ACRIN) (http://www.acrin.org/) continues the trials in the clinical arena, accepting proposals for the support of clinical trials in cancer imaging for diagnosis and treatment. The NCI Office of Technology and the BIP, along with the National Electrical Manufacturers' Association (NEMA), sponsor the National Forum on Biomedical Imaging in Oncology, the third meeting of which is being held January 31–February 1, 2003, in Arlington, VA (http://www3.cancer.gov/dctd/forum/); the meeting brings together researchers, clinical investigators, manufacturers, and representatives of the NCI, FDA, and CMS to discuss the issues surrounding technology development and its introduction and use for patients. A significant action resulting from the Forum was the creation of the Interagency Council on Biomedical Imaging in Oncology, the purpose of which is to provide multiagency advice to academic and commercial technology developers on projects related to cancer (http://www3.cancer.gov/scienceresources/announcements/imaging.html).
Abstract ID: 140
M. G. Harisinghani Jelle O. Barentsz P. F. Hahn Willem Deserno R. Weissleder
Radiologist, Abdominal Imaging and Intervention
Cancer
Monocrystalline iron oxides (MION) and their derivatives represent unique platform reagents for high-resolution MR micro/macrovascular imaging, imaging of phagocytic function in different tissues, to modify nanoparticle with targeted affinity ligands and to use them as chemically sensitive nanosensors. The goal of the current study was to utilize a clinical preparation (Combidex) for imaging cellular uptake in lymph nodes of prostate cancer patients in an effort to increase the accuracy by which micrometastatic disease could be detected. We had previously observed in mouse model studies, that minimal nodal tumor deposits could cause either (1) function lymphatic abnormalities decreasing delivery of MION to involved nodes (micrometastases) or (2) cellular uptake in nodal macrophages be displaced by macroscopic tumor deposits. Sixty patients with primary prostate cancer were enrolled in the study and received an MR examination before and 24–36 hr following intravenous administration of the agent (2.6 mg Fe/kg) followed by radical prostatectomy, surgical lymph node exploration, and histological analysis. Of the evaluated 168 lymph node groups, 90 were benign and 78 malignant by histopathologic analysis. ROC analysis comparing precontrast (Az = 0.77) to combined pre- and postcontrast (Az = 0.98) images showed a statistically significant improvement in detection of nodal metastases (p < .0001). Sensitivity of the new technique was 92% and specificity was 93%. These studies confirm prior mouse model experiments and show unprecedented performance of an imaging test for the noninvasive detection of tumor metastases in lymph nodes.
Abstract ID: 141
B. Baggett R. Roy S. Momen R. J. Gillies
Biochemistry and Cancer Center, University of Arizona
Cancer
Firefly (Phytonis pyralis) luciferase is a useful reporter gene in vivo, allowing noninvasive imaging of tumor growth, tumor metastasis, efficacy of gene transfer, efficacy of drug treatment, and induction of expression. The luciferase protein is very heat labile with an in vitro half-life of approximately 3 min at 37°C. We have reasoned that this thermolability is an appropriate trait for monitoring induction or reduction of gene expression, but it may be less appropriate for monitoring of cell number or gene therapy, where maximum sensitivity is desirable. Six different heat-stable mutants have been created using combinations of point mutations. We have characterized these mutants in vitro, showing a range of half-lives, from 2- to 25-fold increases compared to wild type. SW-480 colon cancer cells were stably transfected with each mutant and wild-type luciferases. Cyclohexamide was used to monitor in vivo half-lives and showed increased thermostability in all mutants compared to wild-type. All proteins, including wild type, had increased in vivo half-lives. This was likely due to the Hsp90 chaperone activity, since luciferase levels were reduced by treatment with geldanamycin, an Hsp90 inhibitor. Mice were inoculated with SW-480 or MDA-mb-231 cells transfected with mutant and/or wild-type luciferase. The mice were imaged weekly and increased light production and sensitivity was observed in the tumors bearing thermostable luciferase. We conclude that the thermostable proteins increase sensitivity for imaging because more active protein is able to accumulate, therefore images can be acquired from a smaller number of mutant transfected cells than wild-type transfected cells.
Abstract ID: 142
J. H. Shin J.-K. Chung Y. J. Lee J. H. Kang K. I. Kim H. J. Oh J. M. Jeong D. S. Lee M. C. Lee
Department of Nuclear Medicine, College of Medicine, Seoul National University
Cancer
Abstract ID: 143
A. H. Jacobs J. Voges R. Reszka A. Gossmann C. Dittmar A. Winkeler K. Wienhard W.-D. Heiss
MPI for Neurological Research, University of Cologne
Cancer
Abstract ID: 144
A. H. Jacobs C. Dittmar A. Winkeler Ch. Kummer S. Vollmar R. Graf K. Wienhard W. D. Heiss
MPI for Neurological Research, University of Cologne
Cancer
Abstract ID: 145
A. H. Jacobs A. Winkeler C. Dittmar M. Hartung Ch. Kummer S. Vollmar R. Graf K. Wienhard W. D. Heiss
MPI for Neurological Research, University of Cologne
Cancer
Abstract ID: 146
G. Niu S. Jhiang M. Graham F. R. Domann
Radiation Oncology, University of Iowa
Cancer
The active transport of iodide into the thyroid is mediated by the sodium iodide symporter (NIS), an intrinsic membrane protein. To detect the transfer and expression of therapeutic genes via a noninvasive imaging technique using hNIS as a radiologically detectable reporter gene, we transferred hNIS genes into human mammary cancer cell line (MB-435 cells) by adenovirus carrier (Ad-hNIS). Using RT-PCR studies, we confirmed that the hNIS mRNA was expressed after human MB-435 cells were infected with Ad-hNIS while no hNIS mRNA was found in uninfected cells. In vitro assays showed that I-125 uptake in cells infected with Ad-hNIS increased significantly compared with uninfected cells. The uptake reached a maximum in 10 min and was about 20-fold that of uninfected cells or vector control cells. Autoradiographic analysis showed that Ad-hNIS infected cells could effectively sequester sufficient amounts of I-125 to expose X-ray film and to generate a readily detectable image. Through these primary investigations, we proved adequately that cancer cells infected with Ad-hNIS could significantly increase their iodide uptake ability. Thus, in vivo imaging using Ad-NIS will be feasible. The further steps of our study will include construction of new adenovirus carriers that contain both hNIS and another therapeutic gene expression cassette. In this way, we should be able to monitor the distribution and duration of therapeutic transgene expression in a noninvasive manner via SPECT or PET. (This work was supported by NIH grant CA91709.)
Abstract ID: 147
J. D. Hazle R. J. Stafford C. J. Rosser R. E. Price M. Tonaka
Oncological Diagnostics and Therapy, German Cancer Research Center
Cancer
Abstract ID: 148
E. H. Abdel-Dayem S. Pautler J. R. Vasselli W. M. Linehan P. L. Choyke
Diagnostic Radiology Department and the National Cancer Institute, National Institutes of Health
Cancer
Abstract ID: 149
G. Y. Stein1 K. Shohat2 M. Firon2 R. Ron3, M. Shaharabany3 J. H. Resau4 I. Tsarfaty2,4
1Cytoview, Israel 2Tel Aviv University, Israel 3fBIT, Israel
4Molecular Oncology, Van Andel Institute, Grand Raids, MI
Cancer
HGF/SF, and its receptor Met, plays an important role in normal cellular processes as well as in tumor development and progression. We have generated GFP-Met and DsRed-HGF/SF and used CLSM analysis to characterize the kinetics and spatial distribution of Met and HGF/SF. Both proteins exhibited fluorescence while retaining their biological activity. GFP-Met trafficking in 293 T was evident, 2 min following exposure to DsRed-HGF/SF and disappeared from the cell membrane after approximately 1 hr. Surprisingly, the interaction between Met and HGF/SF appears to preferentially localize in regions of cell–cell interaction at the cell membrane. The expression and phosphorylation of Ras-GAP and its association with Met was examined following a 10-min incubation with HGF/SF. We have developed novel algorithms, which quantify the interaction between the proteins and which revealed an association between Met and GAP-Ras upon HGF/SF treatment. Additionally, we have developed an antibody against the phosphorylated form of the Met receptor. We measured the relative levels of Met expression, phosphorylation (pMet), and colocalization of the Met/pMet in 80 breast tumors as compared with adjacent normal tissue. Using a quantitative index, we show that alteration of Met expression and phosphorylation predicts poor prognosis for overall survival and metastasis-free survival. Here we demonstrate that direct molecular imaging of a tyrosine kinase growth factor receptor in living cells can provide a new insight into the molecular mechanisms of protein's activity. Moreover, we show that study of the in situ expression patterns can serve as a prognostic marker for breast cancer.
Abstract ID: 150
I. Serganova V. Ponomarev L. Ageyeva M. Doubrovin T. Beresten E. Vider S. Soghomonian J. Balatoni R. Finn R. Blasberg J. Massague J. Gelovani Tjuvajev
Radiology and Neurology, Memorial Sloan Kettering Cancer Center
Cancer
The transforming growth factor b (TGFb) plays a significant role in oncogenesis. It stimulates proliferation of mesenchymal cells while inhibiting the growth of most normal epithelial cells. The TGF-b pathway involves a family of membrane receptor protein kinases and a family of receptor substrates, the Smad proteins, which move into the nucleus where they act as transcription factors. The objective of this study was to develop a reporter system for PET imaging of TGFb signaling pathway activity in oncogenesis and tumor maintenance. We developed a reporter vector that contains a dual-gene reporter herpes simplex virus 1 thymidine kinase/green fluorescent protein (TKGFP) fusion. This reporter gene was placed under control of an artificial enhancer derived from germline Iga promoter/enhancer (provided by Dr. J. Massague, MSKCC, NY). The enhancer contains multiple copies of the binding sites for the transcription factors of TGFb pathway, AML1 and Smad proteins. The reporter was activated in transduced MDA-MB-231 cells in vitro by TGFb-1 (2 ng/ml). Expression of TKGFP was visualized by fluorescence microscopy at 24 hr after exposure. The level of tk/GFP expression was assessed by FACS and a radiotracer assay with [14C]FIAU. A strong correlation between the transcriptional activation of TKGFP reporter gene and TGFb-1 activated genes (PTHRP and VEGF) was observed. [124I]FIAU PET images of TGFb signal transduction activity in transduced subcutaneous tumors in mice were obtained after induction with TGFb (2 ng/g iv BID, 2 days). These data demonstrate that Cis-TGFb/TKGFP reporter system can be used for noninvasive imaging of TGFb signal transduction pathway with [124I]FIAU and PET. Non-invasive monitoring of TGFb signal transduction pathway activity would provide a valuable information on the role of TGFb signaling in oncogenesis, tumor maintenance, and metastases. It could be used for the assessment of novel antitumor drugs, which act as specific inhibitors of TGFb signaling.
Abstract ID: 151
I. Serganova V. Ponomarev T. Beresten M. Doubrovin E. Vider L. Ageyeva S. Soghomonian J. Balatoni R. Finn R. Blasberg J. Gelovani Tjuvajev
Radiology and Neurology, Memorial Sloan Kettering Cancer Center
Cancer
Tumor hypoxia has been associated with the malignant progression and resistance to radiotherapy and chemotherapy. Studies on the molecular responses to hypoxia has identified the major mediator of cellular hypoxic responses, a transcriptional activator named hypoxia-inducible factor 1 (HIF-1). HIF-1 consists of two subunits, HIF-1a and HIF-1b. HIF-1a is an O2-regulated subunit that maintains (determines) the function of HIF-1. Under hypoxic conditions, HIF-1 binds to the hypoxia-responsive element (HRE) in the regulatory region of its target genes and initiates transcription. We aimed to develop an approach for monitoring hypoxia-induced up-regulation of different endogenous genes in situ and in vivo by the noninvasive imaging using a hypoxia-sensitive reporter system. We developed a retroviral vector bearing a dual-gene reporter herpes virus type 1 thymidine kinase and green fluorescent protein fusion (TKGFP) as a “sensor” controlled by the HRE of the erythropoietin gene. A dual-gene reporter red fluorescent protein and Escherichia coli xanthine phosphoribosyl transferase (RedXPRT) was expressed constitutively as a “beacon.” Hypoxia was induced by exposing cells to various concentrations of CoCl2 (1–500 μM) or 2% O2 during 24 hr. Expression of TK/GFP reporter gene is equally activated either by oxygen deficiency (2% O2) or by addition of CoCl2 (50–500 μM). Single cell-derived clones were obtained by consecutive selection for red and green fluorescence using fluorescence-activated cell sorting (FACS). A strong correlation was observed under hypoxic conditions between HIF-1a-induced TKGFP and VEGF at mRNA and protein levels. Using [124I]FIAU and micro-PET, the activity of the reporter system was visualized in rats bearing large subcutaneous tumor xenografts hypoxic core. In conclusion, we developed a novel hypoxia-sensitive reporter for noninvasive PET and optical imaging, which can be used for the assessment of novel antiangiogenic drugs and “direct” hypoxia-specific imaging tracers.
Abstract ID: 152
H. Karacay G. L. Griffiths W. J. McBride A. Sheerin R. M. Sharkey H. Hansen D. M. Goldenberg
Cancer
Pretargeting using a bispecific antibody (bsMAb) and a peptide labeled with Ga-67 was examined as a method for tumor imaging, with the goal of potentially applying this procedure with the positron emitter, Ga-68. The bispecific antibody was a chemical conjugate of Fab0 fragments of hMN-14, an anti-CEA monoclonal antibody, and m679, an anti-histamine–succinyl–glycine (HSG). The peptide, IMP 241, is composed of two HSGs for recognition by the bsMAb and DOTA for binding Ga-67. In nude mice, the peptide was cleared very rapidly from the blood (e.g., % ID/g at 1 hr = 0.11 ± 0.05) with the urinary excretion as the primary route of elimination. No appreciable normal tissue or tumor uptake was observed. For pretargeting, nude mice bearing human colonic xenografts were given 125I-bsMAb (6 mCi, 15 mg) followed 1 day later with 67Ga-IMP 241 (0.6 mCi, 1.5 × 10−11 mol). Tumor uptake was observed as early as 1 hr (13.17 ± 5.77% ID/g) postinjection and was maintained for at least 6 hr (16.05 ± 8.75% ID/g), which was nearly 50 times higher than that seen with the 67Ga-peptide alone. Tumor/nontumor ratios for the pretargeted 67Ga-peptide were 8.5, 2.5, and 13 for the blood, kidney, and liver, respectively, compared to ratios in these same organs of 5, 0.075, and 1.4 for the 67Ga-IMP-241 alone. These data show excellent targeting and tumor/nontumor ratios at very early times that suggest this procedure would be highly suitable for immuno-PET imaging. (Supported in part by grant from DOE DE-FG02-95ER62028.)
Abstract ID: 153
Maria Simonova Olena Shtanko Nikolai Sergeyev Ralph Weissleder Alexei Bogdanov, Jr.
Radiology, MGH-CMIR
Cancer
Quantification of vector delivery and gene expression is important in comparing and optimizing gene therapy strategies. Here we demonstrate that scintigraphic imaging with oxotechnetate (99mOT) can be used to image expression of novel cell-surface expressed fusion proteins and to utilize this imaging strategy to probe for phenotypic changes in genetically manipulated cells. The fusion proteins were designed as “artificial receptors,” typically not expressed by eukaryotic cells, for binding clinically approved imaging agents. These markers were engineered as Type I and/or II membrane proteins and consisted of (1) a 99mOT binding domain (metallothionein [MT]) and (2) a membrane anchoring domain. The MT Type I membrane protein had an N-terminal signal peptide (human antitrypsin) fused to MT and a C-terminal membrane-spanning domain (H. influenzea IgA protease). The MT Type II membrane protein contained the N-terminal signal peptide/membrane anchoring domain of neutral endopeptidase (PEP) fused to the C-terminus of the MT sequence. Fractionation of transfected cells showed that all fusion proteins were present in plasma membrane fraction. Transfected cells showed a 5–7-fold higher 99mOT binding than the control. We expect the described approach to evolve into a useful strategy for “tagging” transfected cells with 99mTc-OT and thus assessing efficiency of gene expression in vivo. In conclusion, the MT cell surface expression eliminates the need for the imaging drug to penetrate in the cytoplasm, gene insert has a size of less than 2 kb, 99mTc pertechnetate and planar imaging systems are widely available.
Abstract ID: 154
Maximilian Bockhorn Lei Xu Rakesh K. Jain Dai Fukumura
Radiation Oncology, Massachusetts General Hospital
Cancer
Abstract ID: 155
P. L. Choyke J. Yao M. V. Knopp
Radiology, NIH
Cancer
In addition to standard displays, we have implemented the following tools: Pure Amplitude and Kep maps: The Brix model generates combined color encoded Amplitude and Kep parameters. Pure Amplitude and Kep allow separate evaluation of these complementary parameters. Cluster/histogram analysis: Large regions of interest can generate scatter plots and histograms of Amplitude and Kep. This technique allows the detection of statistically significant shifts of parameters over the treatment period based on the large number of points. 3-D Volumes of Interest (VOI): This method allows the entire 3-D volume of tumor to be evaluated using a semiautomatic segmentation method. This permits direct comparison to PET scans and other volumetric imaging techniques. Transfer to PACS: Generated images are converted to DICOM format and transferred to the PACS system. This facilitates communication with and education of clinicians regarding DEMRI.
Abstract ID: 156
W. K. Moon Y. Lin R. Weissleder C. H. Tung, et al.
Radiology, CMIR/MGH
Cancer
Folate receptor (FR) is overexpressed in 80–90% of ovarian carcinomas and some cervix, brain, colorectal, renal, nasopharyngeal, and breast cancers. A novel optical probe consisting of a near-infrared fluorochrome (NIR-2, emission at 686 nm) and folic acid was designed for in vivo tumor detection. The uptake of the probe was tested with several FR-positive and FR-negative cells lines. Fluorescence microscopy showed punctate cellular uptake of the NIR-2-folate agent into FR-positive cells but not into FR-negative cells. Using the home-built fluorescence reflectance imaging system, nude mice with implanted tumors were imaged up to 48 hr after intravenous injection of 2 nmol NIR-2-folate conjugate or free NIR-2 compound. Tumor could be detected as early as 1 hr after administration of the conjugate and tumor signal-to-background ratio reached a peak at 24 hr. In tumors of equal size, there was a 2.4-fold higher fluorescence signal in the FR-positive tumors (870 arbitrary units ± 78) compared with the control tumors (366 ± 41).
Abstract ID: 157
K. Reijnders S. J. English M. C. Krishna Y. Zhang A. L. Sowers C. Menard P. L. Choyke K. C. P. Li J. B. Mitchell
Radiology, National Institutes of Health
Cancer
Tumor hypoxia is the result of and also drives many physiological and pathological processes that are important for understanding tumor biology, progression, and response to radiotherapy. BOLD MRI using Carbogen (95% O2 and 5% CO2) provides a relative measure of tumor hypoxia and may be a useful noninvasive tool for spatial mapping of hypoxic regions in vivo. These hypoxic regions may then be specifically targeted for tissue acquisition and genomic or proteomic profiling. In small animal MRI experiments, the animal's ability to regulate body temperature is compromised by anesthesia. Despite use of circulating water blanket or heated gas during imaging, body temperatures of anesthetized animals are not necessarily carefully maintained at or near normal physiological level. Using Carbogen breathing, BOLD MRI signals in 1 cm diameter mouse SCC tumors were determined for animals maintained at 30°C and then reimaged after the animal's temperature was adjusted to 37°C. Experiments were also performed with the order of temperature settings reversed. In both cases, a greater BOLD enhancement was observed for animals maintained at 30°C. Parallel studies, where tumor oxygenation status was measured using Eppendorf oxygen electrodes, confirmed a higher hypoxic tumor fraction for animals maintained at 30°C. The results of the present study serve to stress the importance of proper temperature maintenance of tumor-bearing animals undergoing BOLD MRI.
Abstract ID: 159
E. E. Graves A. Petrovsky D. Yessayan R. Weissleder V. Ntziachristos
Radiology, CMIR/MGH
Cancer
With the advent of fluorescent reporters for a number of disease-related biomolecular processes, there is increasing interest in moving from qualitative two-dimensional fluorescence reflectance imaging (FRI) to quantitative three-dimensional fluorescence-mediated tomography (FMT). We have constructed a chamber for the acquisition of high-resolution FRI and FMT data from living mice in a single examination. The performance of this instrument in imaging a Cy 5.5-based fluorescent probe sensitive to the activity of cathepsin B has been evaluated in murine tumor models. Subcutaneous tumors were implanted in the flanks and mammary fat pads of nude mice. Tumors were also implanted by direct injection into the lungs of nude mice. When the tumors reached sizes of approximately 5 mm, the mice were anesthetized with ketamine and xylazine, and the cathepsin B probe was injected intravenously in the tail vein. Twenty-four hours later, the mice were anesthetized again and FRI and FMT were performed. The subject was positioned so that the region of interest identified by FRI was centered in the approximately 2 × 2 cm FMT field-of-view. The total examination time was approximately 20 min. Qualitative FRI results demonstrated congruence with the reconstructed FMT distributions. Resolution of both the subcutaneous and lung tumors was possible with FMT, and returned estimates of in vivo fluorochrome concentrations that were in concordance with levels reported in other studies of this probe and tumor models. This technology has significant potential for quantitative biological studies of disease models using novel fluorescent reporters.
Abstract ID: 160
F. E. Diehn N. G. Costouros M. S. Miller K. C. P. Li S. K. Libutti, et al.
Department of Diagnostic Radiology, NIH
Cancer
Whole-body optical imaging in animals has emerged as a powerful tool for assaying molecular and cellular processes as they occur in vivo. To take advantage of this technology, we have assembled a noninvasive imaging station that has the capability of detecting both bioluminescent and fluorescent markers in the living animal. Our portable system consists of a CCD camera, a light-tight imaging chamber, and a computer for data collection and image analysis. The ability of the system to detect bioluminescence was demonstrated through a model of in vivo tumor-targeted gene expression. Tumor-bearing mice were injected with a luciferase-expressing vaccinia virus that had previously been shown to have strong tumor tropism. Four days after injection, bioluminescent imaging revealed high levels of luciferase activity specifically at the sites of tumors. In order to demonstrate the fluorescent imaging capabilities of the system, we utilized a recombinant, green fluorescent protein-expressing (GFP) mouse colon cancer cell line. When these cells were injected subcutaneously into mice and monitored for fluorescence over time, we observed consistent, reproducible detection of increasing GFP signal. This progressive increase in fluorescence strongly correlated with growth in tumor volume, as determined by caliper measurements (R2 = .99). Treatment of the tumors with one of several antineoplastic agents revealed varying levels of decrease in tumor-derived GFP signal compared to saline-treated controls. These signal changes were more sensitive than conventional tumor volume measurements and reflected a more direct measure of viable tumor cells. We have thus designed a novel in vivo imaging system that is versatile in both its ability to image multiple optical markers and its potential to model a variety of biological processes.
Abstract ID: 161
A. Brown1 >U. Mahmood2 >K. Leishear1 >H. Onda3, et al.
1Department of Neurology 2Department of Radiology, Massachusetts General Hospital 3Department of Internal Medicine, Brigham and Women's Hospital, Boston, MA
Neurology, Massachusetts General Hospital East
Cancer
Tuberous sclerosis is an autosomal dominant genetic disorder characterized by the development of benign hamartomas in multiple organs, believed to be caused by mutation or loss of tumor suppressor genes with resultant dysregulated growth. Clinically, central nervous system lesions have been associated with hydrocephalus (subependymal giant cell astrocytomas), seizures, and mental retardation (cortical tubers and subependymal nodules). The renal lesions (angiomyolipomas and renal cystadenomas) can be multifocal and at times can severely compromise renal function, with ~15% lifetime risk of progression to malignant renal cell carcinoma. We are developing a gene therapy approach to address these clinical lesions, which are often difficult to manage and problematic to address surgically. Our current project focuses on exploring the efficiency of in vivo gene delivery to lesions in a transgenic knockout mouse model of tuberous sclerosis (TSC2), using vehicle endothelial cells. Briefly, the protocol involves the evaluation of lesion models by magnetic resonance imaging and immunohistochemical assessment of cell-mediated gene delivery to the lesions. Over 20 TSC+/- heterozygous mice at 12–18 months of age have been imaged. High-resolution in vivo MR imaging appears to be more sensitive than traditional histopathological methods which do not involve whole-organ serial sectioning as, for example, analyses using the latter indicated a 5% frequency of renal carcinoma in these animals, while by MRI more than 20% of the animals imaged so far have this pathology. Subsequent pathological analysis confirmed the presence of papillary renal cell carcinoma in these animals, and hemangiosarcomas were also present, as predicted by MRI. These data confirm the potential for noninvasive sequential imaging and establish that spontaneous renal and liver lesions arise frequently in these heterozygous knockout animals.
Abstract ID: 162
D. Artemov N. Mori R. Ravi Z. M. Bhujwalla
Radiology, Johns Hopkins University
Cancer
The Her-2/neu cell surface receptor is overexpressed in approximately 25% of breast cancers and is important in cancer prognosis, and as a target for immunotherapy with humanized monoclonal antibodies (mAb). We developed a two-component Gd-based contrast agent for in vivo MR imaging of Her-2/neu receptors. Receptors are prelabeled with biotinylated mAb recognized by Gd-labeled avidin. Initially, experiments were performed with a mouse cell line, NT-5, obtained from a spontaneous breast cancer in a Her-2/neu transgenic mouse. Preliminary studies were also performed with a panel of human breast cancer cell lines expressing Her-2/neu receptors. Expression of the receptors in cells and tumors was established with Western blot, immunofluorescence, and FACS. Purified avidin was conjugated with DTPA and was labeled with gadolinium (labeling efficiency of ~9 GdDTPA per avidin). Efficiency of in vivo delivery was determined with fluorescence-labeled avidin and mAb. In vivo MR studies were performed with NT-5 and control EMT-6 subcutaneous tumor models. Animals received biotinylated mAb, followed with avidin-GdDTPA administered intravenously. T1 images were acquired at 4.7 T. A significant positive T1 contrast was detected in cell samples and in vivo in tumors prelabeled with the specific mAb. The relatively small molecular weight of both components of the targeted contrast agent results in an efficient delivery to the tumor interstitium. The estimated detection limit of the method is in the range of 105–106 receptors per cell. The method can be used to assess Her-2/neu status of tumors and to monitor efficiency of anti-Her-2/neu immunotherapy.
Abstract ID: 163
Samira Guccione1 >Ron J. Homer1 >Yi-Shan Yang1 >Steven Chang2 >Griff R. Harsh2 >Scott W. Atlas1 >Mark D. Bednarski1
Radiology, Stanford University
Cancer
GBM is a primary brain tumor with poor prognosis, and a median survival of 12–18 months. It has been pathologically characterized as a heterogeneous tumor often with areas of necrosis, surrounded by a distinct contrast-enhanced rim surrounded by edema. We hypothesized that MRI-directed stereotaxic guidance of tumor sampling would permit identification of genes, differentially expressed in E and NE areas, whose proteins might prove to be diagnostically or therapeutically useful. Preoperatively, E and NE regions of tumor were identified for surgical sampling on Gd-DTPA contrast-enhanced MR images of untreated GBM. Tissue harvested during stereotactic craniotomy was snap-frozen in the operating room. Total RNA from the samples was used for oligonucleotide microarray analysis. The gene expression profiles in E and NE regions were compared in a search of evidence for production of proteins of tumorigenic interest or therapeutic potential. The presence and distribution of these proteins were evaluated by immunohistochemical staining (IH). Striking differences are observed between the enhancing and adjacent nonenhancing regions. Some distinct classes of genes are highly up-regulated including profilins, apoptosis-related proteins, heat shock proteins, calcium binding proteins. The most interesting genes up-regulated in our current study is a small extracellular matrix protein, MGP. This protein is related to a class of proteins in the clotting cascade that gets g-carboxylated on glutamic acid residues using glutamic acid g-carboxylase. It is the only protein with this property in which the function is not yet determined. In our previous studies, MGP is up-regulated in areas of high contrast enhancement using MRI in a squamous cell carcinoma murine model. This raises an opportunity to correlate diagnostic clinical imaging with the molecular pathogenesis of human cancers. Further evaluation of the biological role of MGP in tumors is underway.
Abstract ID: 164
Samira Guccione Yi-Shan Yang Mark Bednarski
Radiology, Stanford University
Cancer
Gene and protein expression profiles in tumors can vary with temporal progression and spatial distribution. MRI is a powerful tool for the diagnosis of a variety of malignancies and is capable of visualizing changes in tissue contrast with micron spatial resolution and millisecond temporal resolution. We hypothesize that MRI can be used to determine temporal changes in tumor progression and reveal spatial heterogeneity within the tumor at different stages of growth for genomic analysis. Combination of imaging and microarray analysis can be used to validate potential molecular targets for new molecular imaging agents useful for cancer diagnosis and therapy. C3H/Km mice were transplanted with murine squamous cell carcinoma VII (SCCVII) cells. Tumors were imaged sequentially starting at a diameter of ~6 mm using a clinical 3 T MR Scanner. Standard spin-echo (T1-wt) and fast spin-echo (T2-wt) scan protocols were used. Postcontrast T1-wt image was obtained after an injection of gadolinium-DTPA. MRI contrast enhancing and nonenhancing regions were spatially marked at different stages of tumor growth, and the tumor tissue was surgically harvested for genomic analysis. We have found four distinct stages of tumor progression in the SCCVII murine tumor model based on MR imaging. We observed differences in gene expression profiles between each of these four stages of tumor growth and have identified several interesting gene products for each stage that are potential candidates as molecular targets for cancer imaging and therapeutics. We conclude that tissue sampling of tumors for genomic analysis using MRI-guided biopsy is sensitive to spatial and temporal changes in tumors. Clinical imaging can therefore serve as a powerful tool for discerning regions of distinct patterns of gene expression.
Abstract ID: 165
Mansoureh Sameni Julie Dosescu Bonnie F. Sloane
Pharmacology Wayne State University
Cancer
MMPs, serine proteases, and the cysteine protease cathepsin B have been linked to progression of human breast and colon cancers. The underlying basement membrane is disrupted during the transition from ductal carcinoma in situ to breast carcinoma and from adenoma to colon carcinoma. We have established a 3-D model with living human breast (BT20) and colon cancer cells (HCT 116 and Hkh-2) that allows us to image degradation of a quenched fluorescent-derivative of the basement membrane protein type IV collagen (DQ-collagen IV). Proteolysis of DQ-collagen IV by HCT 116 and Hkh-2 cells was primarily intracellular. In contrast, proteolysis of DQ-collagen IV by BT20 cells was primarily pericellular. The cytoskeleton contributes to cellular interactions, enzyme secretion, tissue remodeling, tumor invasion, and angiogenesis. Morphology of cells that are grown in two-dimensional monolayers differs from that of cells in vivo. Therefore, we have examined the actin cytoskeleton of cells grown in 2-D or 3-D matrices. Cell viability in both systems was 96–98%. In the 3-D cultures, cells were bipolar and lacked actin stress fibers. In vivo, stromal elements often contribute to the spectrum of proteolytic activities associated with tumors. The intracellular degradation of type IV collagen by living breast and colon cancer cells might reflect an absence of stromal cell proteases. Therefore, we examined the contribution of fibroblasts and macrophages to degradation of DQ-collagen IV. Fibroblasts themselves exhibited only modest pericellular degradation of type IV collagen. The pericellular degradation was markedly increased in cocultures as compared with cancer cells alone or fibroblasts alone. On the other hand, intracellular degradation of type IV collagen was markedly increased in macrophages cocultured with tumor cells. Pericellular degradation of DQ-collagen IV by the macrophages could not be detected. Coculture of tumor cells, fibroblasts, and macrophages increased both pericellular and intracellular degradation of DQ-collagen IV. The present findings suggest that both pericellular and intracellular degradation of basement membrane proteins by tumor cells and tumor-associated stromal cells contribute to the invasiveness of colon and breast cancer. (Supported by CA 36481 and 56586.)
Abstract ID: 166
T. D. Harris S. Kalogeropoulos T. Nguyen D. Onthank, et al.
Discovery R & D, Bristol Myers Squibb Medical Imaging
Cancer
Cell–cell and cell–matrix interactions are essential to the processes of angiogenesis and tumor metastasis. The integrin avb3 (vitronectin receptor) has been shown to play a key role in these processes. This receptor is highly up-regulated on endothelial cells during neovascularization and on the tumor cells of many human cancer cell lines, making it an attractive target in anticancer research (Miller et al., Drug Discovery Today 2000, 5, 397–408). As part of our continuing program of research on integrin-directed radiopharmaceuticals, we have synthesized a series of peptidomimetic avb3 antagonists based on the quinolone carboxamide core. These peptidomimetics were derivatized with the macrocyclic chelator DOTA for labeling with In-111 (imaging) or Y-90 (therapy) and were evaluated in animal tumor models. Tumor uptake and biodistribution properties were optimized by structural modifications that included tethering of the DOTA chelator at different positions on the quinolone peptidomimetic, the use of polar pharmacokinetic modifiers, and dimerization. All compounds synthesized displayed high affinity for integrin avb3, and good selectivity relative to integrin aIIbb3. The majority of compounds showed excellent tumor uptake in the c-Neu Oncomouse model (tumor uptake as high as 14% ID/g at 24 hr PI), rapid blood clearance, and predominantly renal excretion. This article will present an overview of the synthesis and structure–activity relationships of this very promising class of antitumor agents.
Abstract ID: 167
M. G. Swanson J. Kurhanewicz D. B. Vigneron R. E. Hurd, et al.
Radiology, UCSF
Cancer
There is a great need to identify novel molecular markers which better correlate with prostate cancer presence, aggressiveness, and response to therapy. To this end, we used nondestructive high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to obtain full metabolic profiles of human postsurgical prostate tissues (n = 57) and primary and metastatic tumors (n = 25) obtained from Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Human prostate cancer tissues demonstrated significantly higher choline/creatine, (GPC + PC)/creatine, total choline/creatine, and taurine/creatine ratios, and significantly lower citrate/creatine and spermine/creatine ratios versus benign glandular and stromal tissues.
Abstract ID: 168
S. A. Soghomonyan M. Doubrovin J. Pike D. Bermudes J. Gelovani Tjuvajev R. Blasberg
Neurology and Radiology, Memorial Sloan-Kettering Cancer Center
Cancer
Abstract ID: 169
G. D. Luker V. Sharma C. M. Pica J. L. Prior W. Li D. Piwnica-Worms
Mallinckrodt Institute of Radiology, Washington University School of Medicine
Cancer
To enable molecular imaging of protein–protein interactions in vivo by PET and fluorescence imaging, we have used microPET and fluorescence imaging to detect interactions between p53 tumor suppressor and large T antigen (TAg) of SV 40 virus in a tetracycline-inducible two-hybrid system. To further validate this technology, we investigated if expression of the reporter gene, comprised of a mutant HSV1-TK fused to green fluorescent protein, could quantify relative differences in amounts of interacting hybrid proteins. Mice bearing HeLa cell xenografts stably transfected with reporter gene and interacting (p53-TAg) or noninteracting (p53-CP) hybrid proteins were treated with serial doses of doxycycline (60 μg/g every 12 hr) for 12, 24, and 48 hr before microPET imaging or biodistribution studies with the HSV1-TK substrate 18F-FHBG. Treatment with doxycycline produced time-dependent increases in hybrid proteins as determined by Western blots of tumor specimens. Both imaging and biodistribution studies showed proportional increases in accumulation of radiotracer over time only in p53-TAg tumors, while content of 18F-FHBG did not change in p53-CP tumors. In response to increasing doses of doxycycline (10–60 μg/g every 8 hr for 48 hr), dose-dependent increases in hybrid proteins were observed by Western blot. Expression of hybrid proteins resulted in enhanced accumulation of 18F-FHBG by tracer analysis only in p53-TAg tumors. Without doxycycline, no increases in protein or radiotracer accumulation above background levels could be detected in either tumor. These data demonstrate that the imaging two-hybrid system responds in a proportional fashion to increasing amounts of interacting proteins in vivo.
Abstract ID: 170
E. Ackerstaff D. Artemov R. J. Gillies Z. M. Bhujwalla
Radiology, Johns Hopkins University School of Medicine
Cancer
Endothelial cells and endothelial cell injury play an important role in cancer invasion and metastasis. Understanding the interaction between cancer cells and endothelial cells in cancer invasion, under conditions of hypoxia which exist in solid tumors, is important to prevent invasion and metastasis. To understand the role of the physiological environment in invasion, we recently developed and characterized an invasion assay which dynamically tracks the invasion of cancer cells and simultaneously measures oxygen tensions and metabolic parameters, using magnetic resonance imaging and spectroscopy. We have further modified this assay to understand the role of cancer cell–endothelial cell interaction in invasion under hypoxic conditions found in solid tumors. In this study, we present data obtained for the human prostate cancer cell line PC-3 under oxygenated and hypoxic conditions, in the presence or absence of human umbilical vein endothelial cells (HUVECs). Hypoxia resulted in a significant decrease in the Invasion Index of PC-3, but the presence of HUVECs on the surface of Matrigel resulted in an attenuation of this decrease indicating that the presence of HUVECs provided an advantage in invasion under hypoxic conditions. However, the presence of HUVECs did not alter invasiveness under well-oxygenated conditions. These results suggest that endothelial cells confer an advantage in invasion of cancer cells in hypoxic regions of the tumor. While in vivo, the presence of hypoxia in the vicinity of endothelial cells may not be anticipated, this can occur in tumors due to intermittent vascular collapse or, following radiation therapy, during reoxygenation.
Abstract ID: 171
G. Tsarfaty G. F. Vande Woude I. Tsarfaty
Molecular Oncology, Van Andel Institute
Cancer
Met activation via autocrine, paracrine, or mutational mechanisms leads to tumorigenesis and metastasis. We recently demonstrated functional molecular imaging of Met receptor activity in tumors expressing high levels of Met. Organs and tumors expressing Met showed substantial alteration in blood oxygenation levels as measured by blood oxygenation level dependent MRI (BOLD-MRI), Doppler, and contrast media ultrasound. No significant alteration was observed in tumors or organs that do not express Met. The extent of MRI signal alteration correlated with HGF/SF doses. In an autocrine model in which the tumor secretes high levels of HGF/SF, the blood flow in the tumor is greatly increased as compared to tumors that do not express HGF/SF. Moreover, the kidney and liver of the mice bearing these tumors demonstrate increased blood flow proportional to the tumor size. We compared HGF/SF-induced alteration of hemodynamic in three different inherited activated Met mutations. HGF/SF-induced alteration of hemodynamic was high in all the tumors that harbor activated Met and correlated with the degree of receptor activation. In highly activated mutation, the increased alteration of hemodynamic was significantly higher than in nonactivated Met mutation. These results show that the novel functional Met molecular imaging modality can serve as a powerful tool in understanding the metabolic activity induced by this signal transduction. Moreover, Met functional molecular imaging can serve as a tool for the detection, analysis, and prognosis of a wide spectrum of tumors.
Abstract ID: 172
Y. Pu M. Mintun F. Dehdashti J. Lewis M. Welch
Radiology, Division of Nuclear Medicine, Washington University Medical School
Cancer
Abstract ID: 173
Chrit Moonen
Magnetic Resonance of Biological Systems, CNRS/University of Bordeaux 2
Cancer
Abstract ID: 174
Y. R. Kim M. D. Savellano C. H. Tung A. Bogdanov R. Weissleder
Radiology, CMIR/HMS
Cancer
Understanding tumor vascular physiology is critically important in developing noninvasive, molecularly targeted imaging diagnostics and therapies. In this murine tumor xenograft study using three different human prostate cancer cell lines (MDA PCa 2b, PC-3, and LNCaP), structural and physiological parameters of neoplastic vasculature were explored with a widely available magnetic resonance imaging (MRI) pulse sequence (3-D SPGR: spoiled gradient echo). Using a dual injection technique employing two T1 contrast agents of different molecular masses, steady-state (SS) MRI measurements and dynamic contrast agent enhancement (DCE) MRI measurements were simultaneously acquired and analyzed using a two-compartment model for calculating tumor vascular parameters. In particular, interstitial volume and vascular permeability were independently quantified using these two different MRI techniques. Relative vascular water exchange rate, calculated by the flip angle dependence of measured blood volume via the SS technique, and permeability surface product (PS), extrapolated from DCE MRI, were quantitatively compared. It was found that the SS and DCE techniques were comparable in terms of yielding similar qualitative results for vascular structure (interstitial volume) and permeability (water exchange rate and PS). These MRI studies were then utilized to interpret optical imaging results obtained with enzyme-specific fluorogenic probes designed for the detection of tumor-associated protease activity. Qualitative comparison of the optical data with the MRI studies indicated that fluorogenic probe contrast was highly dependent on the structural and physiological characteristics of vascular/extravascular spaces, suggesting that accurate quantification of tumor-associated protease activity should take into account local tissue variability and probe accumulation.
Abstract ID: 175
A. Babsky S. K. Hekmatyar N. Bansal
Radiology, University of Pennsylvania
Cancer
A transmembrane sodium gradient is critically important for survival and normal functions of cells. A better understanding of Na+ physiology and its relationship with energy metabolism and intracellular pH (pHi) may help in designating more effective cancer therapy. We have investigated the effect of hyperthermia (HT, 45°C, 30 min) on intracellular sodium (Nai+), pHi, and cellular energy status in perfused RIF-1 tumor cells by 31P and 23Na nuclear magnetic resonance. HT produced a 50–60% increase in [Nai], which returned back to the baseline value after HT. In the presence of 3 μM 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a potent and specific inhibitor of Na+/H+ antiporter, the increase in Nai+ during hyperthermia was completely abolished suggesting that the heat-induced increase in Nai+ was due to an increase in Na+/H+ antiporter activity The pHi initially decreased by 0.1 units and then increased during hyperthermia. Hyperthermia did not produce significant changes in cellular energy status as measured from ATP/Pi. In the presence of 1 μM Monensin, an Na ionophore, [Nai+] increased to the same level as during HT alone. However, [Nai+] did not return back to the baseline value after HT and it increased even further 80–90 min after HT. The changes in pHi during hyperthermia were identical with or without Monensin. However, the pH increased further up to 20 min after HT and then returned back to the baseline value. Monensin did not change the ATP/P during and after HT. Monensin alone also increased [Nai+], but the increase was only 20–25% compared to the baseline value. Clonogenic assays showed more than an order of magnitude increase in the thermosensitivity of RIF-1 cells with Monensin. These results indicate that some of the HT-induced ionic changes are mediated by activation of the Na entrance into the cells without large changes in bioenergetic status, and Monensin can increase the efficiency of HT treatment substantially.
Abstract ID: 176
S. K. Hekmatyar A. Babsky N. Bansal Radiology
University of Pennsylvania
Cancer
There is a great interest in developing novel paramagnetic lanthanide contrast agents for molecular imaging by MRI. The lanthanide ion in these complexes can induce very large chemical shifts, which show strong dependence on temperature. Thus, these complexes can also be used for noninvasive thermometry, especially for hyperthermia applications where high temperature resolution is necessary. We have examined the temperature dependence of the proton chemical shifts of Pr, Eu, Yb, Tb, Dy, and Tm complexes of DOTA (1,4,7,10-tetra azacyclododecane-N,N′,N″,N‴-tetraacetic acid) to determine which complex is optimum for thermometry. GdDOTA is widely used as an MRI contrast agent and these DOTA complexes are nontoxic. The temperature dependencies of the proton chemical shifts (CT, unit ppm/°C) for the six protons in each complex were linear over 22–55°C. The CT values were larger for the H4 protons of Tm, Dy, Tb, Yb, and Eu complexes and the H5 proton of the Pr complex. The ratio CT/full width at half maximum (CT/FWHM) was used as a criterion to select the best resonance for MR thermometry. This ratio was largest for the H5 proton of Tm and Pr complexes and H4 of Dy, Tb, Yb, and Eu complexes. Out of all the complexes examined, H4 proton resonance of DyDOTA showed the largest chemical shift (-442.1 ppm at 35°C), the largest CT (2.56 ppm/°C) and the largest CT/FWHM value (1.56). Considering the advantages such as, high temperature sensitivity, relatively narrow line width, and low toxicity, DyDOTA appears to be a promising probe for MR thermometry.
Abstract ID: 177
Barentsz, MD, PhD W. Deserno, MD, MSc M. Harisinghani, MD R. Heesakkers, MD G. Jager, MD, PhD, J. Witjes, MD, PhD J. de la Rosette, MD, PhD C. Hulsbergen van der Kaa, MD, PhD M. Taupitz, MD
Radiology, UMC Nijmegen
Cancer
Abstract ID: 178
V. Ponomarev M. Doubrovin I. Serganova L. Ageyeva E. Vider T. Beresten S. Soghomonian A. Shavrin J. Balatoni R. Finn R. Blasberg J. Gelovani Tjuvajev
Radiology and Neurology, Memorial Sloan Kettering Cancer Center
Cancer
The objective of this work was to develop a nonimmunogenic reporter gene for imaging different molecular-genetic processes in humans using PET. Human thymidine kinase type 2, cDNA was truncated at the N-terminal to abolish the mitochondrial localization signal (dTK2) and was fused with green fluorescent protein (GFP) cDNA. The dTK2GFP fusion gene was cloned into a retrovirus and was used to transduce the U87 glioma cells. A mixed population of transduced dTK2GFP/U87 cells was obtained by FACS. The level of hTK2GFP enzymatic activity was assessed in vitro using the 14C-FIAU and 3H-gancyclovir (GCV) accumulation assays. In vitro sensitivity to AraC was performed as well. The dTK2GFP/U87 cells exhibited an intensive pancellular green fluorescence as visualized by microscopy and measured by FACS (460 rU). The Ki of 14C-FIAU was 0.068 ± 0.020 and 0.007 ± 0.002 ml/min/g in dTK2GFP/U87 and U87 cells, respectively (transduced/nontransduced ratio of 10). The Ki of 3H-GCV was 0.001 ± 0.001 and 0.001 ± 0.001 ml/min/g in dTK2GFP/U87 and U87 cells, respectively. The ID50 for AraC in vitro was 0.04 and 0.120 μmol in dTK2GFP/U87 and U87 cells, respectively. The dTK2GFP/U87 and U87 subcutaneous xenografts were established in nude mice. MicroPET imaging was performed at 2 and 24 hr after intravenous injection of 124I-FIAU (150 μCi/animal) followed by tissue sampling for gamma spectroscopy. MicroPET images revealed a highly specific localization of radioactivity to the dTK2GFP/U87 xenografts, with only background levels of radioactivity in the wild-type U87 xenografts. Tissue sampling at 24 hr postinjection yielded values of 0.24 ± 0.09 and 0.038 ± 0.002% dose/g 124I-FIAU in dTK2GFP/U87 and U87 xenografts, respectively. The dTK2GFP/U87 xenograft/plasma and xenograft/muscle ratios were 87 ± 17 and 27 ± 11, respectively. In conclusion, the nonimmunogenic dTK2 reporter gene could be used for long-term monitoring of different molecular-genetic processes in human subjects using 124I-FIAU and PET.
Abstract ID: 179
A. Ivanova V. Ponomarev L. Ageyeva M. Doubrovin I. Serganova E. Vider S. Soghomonian J. Balatoni R. Finn R. Blasberg J. Gelovani Tjuvajev
Radiology and Neurology, Memorial Sloan Kettering Cancer Center
Cancer
Studies on directed differentiation of adoptively transferred stem cells are very important as they can improve the results of treatment of different degenerative or genetic disorders. Another important application of adoptive stem cell transfer is for reconstitution of hematopoiesis after high dose chemo- or radiation therapy. Several recent studies have demonstrated a strong tumor tropism of the transferred stem cells. Based on the latter observations, we suggest that stem cells can be used as gene delivery vehicles for therapy of various malignances. We generated three types of retroviral vectors, in which the HSV1-tk/GFP fusion gene expression was controlled by the LTR (vector ST8G), CMV (vector SCT8G), or PGK (vector SPT8G) promoters. Undifferentiated mouse embryonic stem (ES) cells were transfected with these vectors and the expression levels of the HSV1-tk/GFP reporter gene were assessed in vitro. FACS analysis demonstrated 87%, 94%, and 97% GFP-positive cells in ESST8G, ESSCT8G and ESSPT8G bulk populations of transduced cells, respectively. However, the expression of HSV1-tk/GFP from LTR gradually diminished and was silenced by Day 15 both in embryonic bodies (EBs) and sparsely growing ES cells. In contrast, HSV1-tk/GFP expression from the CMV promoter was attenuated only by 20% and, in the case of the PGK promoter, remained unchanged over an extended period of observation. Similar studies in transduced neuronal progenitor cells and mesenchymal or endothelial precursors demonstrated incomplete silencing of HSV1-tk/GFP expression from LTR and stable expression from both, the CMV and PGK promoters. Preliminary in vivo [124I]FIAU PET imaging studies in normal and subcutaneous tumor-bearing mice injected intravenously with different types of stem (or progenitor) are underway. The results of PET imaging will be presented and discussed.
Abstract ID: 180
A. Ivanova V. Ponomarev M. Dubrovin L. Ageyeva I. Serganova E. Vider S. Soghomonian J. Balatoni R. Finn R. Blasberg J. Gelovani Tjuvajev
Radiology and Neurology, Memorial Sloan Kettering Cancer Center
Cancer
Multimodality imaging approaches allow for different imaging technologies to be combined in the same study. For example, planar bioluminescence imaging (BLI) of luciferase reporter gene expression could be used for initial screening and monitoring of the status and location of genetically labeled cells, tumors, or other transfected tissues, or assessment of the molecular-genetic reporter system activity in murine models. Once the critical time points or conditions are determined, one could follow up with a study involving PET imaging of TKGFP expression. An important advantage of BLI is that there is virtually no nonspecific background in the images. Therefore, we developed a retroviral vector (L89gCluc) in which the expression of TKGFP fusion reporter gene is driven by the retroviral LTR and the expression of luciferase gene by the CMV promoter. The L98gCluc vector was used to transduce RG2 rat glioma cells (LT9gCluc/RG2). The LT9gCluc/RG2 cells exhibited highly intensive GFP-derived fluorescence (>103 RLU by FACS) and a high accumulation rate of [14C]FIAU (0.1 ml/g/min) in vitro. BLI of the LT9gCluc/RG2 cells in vitro revealed a highly intensive signal after addition of D-luciferine to the culture medium. The LT9gCluc/RG2 and wild-type RG2 cells were used to establish xenografts in nude mice (both in the same animal). When the tumors grew to approximately 0.5 cm in diameter, we performed sequential BLI, whole-body GFP-fluorescence, and PET with [124I]FIAU imaging studies in the same animals. The subcutaneous RG2/LT8gCluc xenografts could be easily detected with 2-D BLI and whole-body fluorescence imaging. MicroPET images were acquired in 2 hr postadministration of [124I]FIAU (100 μCi/animal) and demonstrated similar localization of transduced RG2/LT8gCluc xenografts as obtained with BLI of luciferase expression and whole-body imaging of TKGFP expression. In conclusion, the LT9gCluc retroviral vector can be used for multimodality imaging of various molecular processes.
Abstract ID: 181
T. Lahoutte J. Mertens C. Joos A. Bossuyt
Nuclear Medicine, AZVUB Brussels
Cancer
Besides increased glucose uptake, tumor cells also show an increased amino acid metabolism. An appropriate radioiodinated amino acid analog can allow us to visualize the metabolic state of tumors with SPECT. 123I-IMT is used for the study of cerebral tumors, but intense renal accumulation limits its use in extracerebral malignancies. Therefore, we developed the new radioiodinated neutral amino acid
Abstract ID: 182
L. Zhou D. Blessington C. Tung1 R. Weissleder1 B. Chance
1Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, Biochemistry/Biophysics, University of Pennsylvania
Cancer
Proteases are involved in the invasion and metastasis of tumor cells. Cathepsin B overexpression has been shown in some neoplastic tissues. This study assesses the expression of Cathepsin B in the human breast cancer (DU4475) and the human fibrosarcoma (HT1080) in the mouse model by near-infrared (NIR) imaging. The nude mice were intravenously injected “a stealth probe“—an activable Cathepsin B sensing near-infrared fluorescence (NIRF) probe (24 hr before sacrifice) and indocyanine green (ICG) (30 sec before sacrifice). The animals were freeze-trapped and NIR images were obtained by low-temperature NIR scanner at the following excitation–emission wavelength pairs: 366, 450 nm (NADH); 436, 520 nm (FAD); 670, 695 nm (Cathepsin B probe); and 780, 830 nm (ICG). After imaging, the samples were submitted for histopathological evaluation. The tumor redox ratio NADH/(NADH + FAD) increased significantly because of the hypoxic state of tumor tissue. A comparison of the redox ratio, Cathepsin B probe, ICG, and histopathology images clearly demonstrates coregistration. The Cathepsin B probe was uniformly distributed throughout the tumor. The delivery efficiency of the Cathepsin B probe was calculated according to the blood ICG concentration and ICG image. This study indicated the efficient usage of the Cathepsin B probe in the molecular imaging for the detection of the early stage tumors.
Abstract ID: 183
H. Karacay G. L. Griffiths W. McBride A. Sheerin R. M. Sharkey H. Hansen D. M. Goldenberg
Garden State Cancer Center
Cancer
Pretargeting using a bispecific antibody (bsMAb) and a peptide labeled with Ga-67 was examined as a method for tumor imaging, with the goal of potentially applying this procedure with the positron emitter, Ga-68. The bispecific antibody was a chemical conjugate of Fab0 fragments of hMN-14, an anti-CEA monoclonal antibody, and m679, an antihistamine–succinyl–glycine (HSG). The peptide, IMP 241, is composed of two HSGs for recognition by the bsMAb and DOTA for binding Ga-67. In nude mice, the peptide was cleared very rapidly from the blood (e.g., % ID/g at 1 hr = 0.11 ± 0.05) with the urinary excretion as the primary route of elimination. No appreciable normal tissue or tumor uptake was observed. For pretargeting, nude mice bearing human colonic xenografts were given 125I-bsMAb (6 mCi, 15 mg) followed 1 day later with 67Ga-IMP 241 (0.6 mCi, 1.5 × 10−11 mol). Tumor uptake was observed as early as 1 hr (13.17 ± 5.77% ID/g) postinjection and was maintained for at least 6 hr (16.05 ± 8.75% ID/g), which was nearly 50 times higher than that seen with the 67Ga-peptide alone. Tumor/nontumor ratios for the pretargeted 67Ga-peptide were 8.5, 2.5, and 13 for the blood, kidney, and liver, respectively, compared with ratios in these same organs of 5, 0.075, and 1.4 for the 67Ga-IMP-241 alone. These data show excellent targeting and tumor/nontumor ratios at very early times that suggest this procedure would be highly suitable for immuno-PET imaging. (Supported in part by grant from DOE DE-FG02-95ER62028.)
Abstract ID: 184
E. O. Aboagye
PET Oncology, Imperial College Science, Technology and Medicine, Hammersmith Hospital, London, UK
Cancer
Drugs labeled with long-lived isotopes such as carbon-14 have been used in the past to provide toxi-cokinetic information on new drugs in humans. Such studies are based almost exclusively on analysis of blood and urine samples. Isotopic substitution with a positron emitting atom enables the pharmacokinetics in tumors and normal tissues to be studied by positron emission tomography (PET). PET is a sensitive and specific noninvasive technique that uses external detectors to measure the three-dimensional (3-D) distribution and kinetics of injected compounds that have been labeled with short-lived positron-emitting radioisotopes. Using this imaging technology, we can now ask questions about the kinetics and magnitude of drug delivery to tumors and normal tissues and calculate blood–tissue exchange rate constants. The opportunity to select and develop promising drugs in a safe, timely, and cost-effective manner forms the strategic objective of incorporating PET pharmacokinetic studies in anticancer drug development. PET pharmacokinetic studies may be performed preclinically in animals or humans prior to conventional Phase I studies. In addition, a few studies have now been performed during Phase II trials to study schedule dependence. Depending on the trial design, the information obtained from PET studies can provide scientific feedback on the mechanism of action of new compounds. Together with information from target modulation, these studies may provide an early indication of whether a drug may be ineffective in tumors or predict for normal tissue toxicity.
Abstract ID: 185
S. Jambawalikar H. Li T. M. Button
Radiology, SUNY Stony Brook
Cancer
Abstract ID: 186
W. Huang A. Tudorica K. Dulaimy P. Fisher, et al.
Radiology, SUNY Stony Brook
Cancer
Abstract ID: 187
T. M. Button, PhD H. Li, PhD K. Dulaimy, MD S. Li, MD, et al.
Radiology, SUNY Stony Brook
Cancer
Abstract ID: 188
T. M. Button P. Fisher R. Rosenblatt M. J. Janicek, et al.
Radiology, SUNY Stony Brook
Cancer
Abstract ID: 189
K. Dulaimy H. Li T. M. Button
Radiology, SUNY Stony Brook
Cancer
Abstract ID: 190
H. Li, PhD T. M. Button, PhD
Radiology, SUNY Stony Brook
Cancer
Abstract ID: 191
E. Sahai C. Marshall
Cell and Molecular Biology, Institute of Cancer Research
Cancer
Metastatic tumor cells are able to migrate through the extracellular matrix and cross tissue boundaries. By using confocal microscopy to reconstruct immunofluorescence images of colon carcinoma and melanoma cells migrating through a 3-D matrix, we have identified two distinct ways in which tumor cells can migrate. We have also performed time-lapse 3-D confocal microscopy on GFP expressing tumor cells. Some tumor cells migrate as very elongated cells similar to mesenchymal cells. In contrast, other tumor cells migrate as very rounded cells with small dynamic bleb-like structures. We have further characterized the requirement for Rho family GTPases for the two types of movement. The elongated mode of migration does not require the Rho-effector kinase ROCK, whereas the rounded mode of movement does. We hope to extend these studies to analyze tumor cells migrating in “in vivo” models.
Abstract ID: 192
J. D. Hazle R. J. Stafford C. J. Rosser R. E. Price M. Tonaka N. Tonaka L. Pisters
Imaging Physics, UT M.D. Anderson Cancer Center
Cancer
Abstract ID: 193
H. Karacay G. L. Griffiths W. J. McBride A. Sheerin R. M. Sharkey H. J. Hansen D. M. Goldenberg
Research, Garden State Cancer Center
Cancer
Pretargeting using a bispecific antibody (bsMAb) and a peptide labeled with Ga-67 was examined as a method for tumor imaging, with the goal of potentially applying this procedure with the positron emitter, Ga-68. The bispecific antibody was a chemical conjugate of Fab fragments of hMN-14, an anti-CEA monoclonal antibody, and m679, an antihistamine–succinyl–glycine (HSG). The peptide, IMP 241, is composed of two HSGs for recognition by the bsMAb and DOTA for binding Ga-67. In nude mice, the peptide was cleared very rapidly from the blood (e.g., % ID/g at 1 hr = 0.11 ± 0.05) with the urinary excretion as the primary route of elimination. No appreciable normal tissue or tumor uptake was observed. For pretargeting, nude mice bearing human colonic xenografts were given 125I-bsMAb (6 mCi, 15 mg) followed 1 day later with 67Ga-IMP 241 (0.6 mCi, 1.5 × 10−11 mol). Tumor uptake was observed as early as 1 hr (13.17 ± 5.77% ID/g) postinjection and was maintained for at least 6 hr (16.05 ± 8.75% ID/g), which was nearly 50 times higher than that seen with the 67Ga-peptide alone. Tumor/nontumor ratios for the pretargeted 67Ga-peptide were 8.5, 2.5, and 13 for the blood, kidney, and liver, respectively, compared with ratios in these same organs of 5, 0.075, and 1.4 for the 67Ga-IMP-241 alone. These data show excellent targeting and tumor/nontumor ratios at very early times that suggest this procedure would be highly suitable for immuno-PET imaging. (Supported in part by grant from DOE DE-FG02-95ER62028.)
Abstract ID: 194
M. Seiden
Medicine, Massachusetts General Hospital
Cancer
Current therapy for epithelial ovarian cancer typically is effective at leaving women with minimal residual disease, which is below the level of detection by all standard radiologic procedures. Unfortunately, the majority of these women will eventually suffer from recurrent disease, which will ultimately prove fatal. The intelligent delivery of biologically targeted therapies to women with minimal residual tumor will require the development of novel imaging techniques to both identify small volume tumor and ideally define key biologic processes within the tumor. Maximizing the utility of this technology will also require the development of imaging probes that will allow intratumoral pharmacodynamics to be monitored in real time in situ. Optimizing this technology will lead to benefits for patients, their treating physicians, and pharmaceutical companies interested in streamlining drug development strategies. This presentation will attempt to define key issues in building a molecular imaging program directed to imaging the peritoneal cavity in women with epithelial ovarian cancer.
Abstract ID: 195
D. E. Hall A. Rehemtulla L. D. Stegman M. S. Bhojani, et al.
Radiology, University of Michigan
Cancer
Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer chemosensitization gene therapy by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (CD) along with an optical reporter gene (luciferase). Following intratumoral injection of the vector into orthotopic 9L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates, an increase in mean tumor diffusion values and spatial heterogeneity in tumor diffusion associated with the CD/5-fluorocytosine (5FC) gene therapy. In addition, in vivo bioluminescence imaging (BLI) detected luciferase gene expression, which was found to decrease over time during administration of the prodrug, providing a surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the CD/5FC strategy for the treatment of brain tumors and reveals the feasibility of using multimodality molecular and functional imaging for assessment of gene expression and therapeutic efficacy.
Abstract ID: 196
Brian D. Ross
Radiology, University of Michigan
Cancer
Recent advances in both hardware and software have resulted in significant improvements in noninvasive imaging methods used for studying animal tumor models. This overview will focus on the approaches and results using MRI/S for studying rodent brain tumors. Serial images of intracerebral tumors using MRI over time following therapeutic intervention can be used to quantitate cell kill in vivo. In fact, increases in animal survival elicited by therapy are not solely attributable to the fraction of tumor cells killed but are a function of cell kill and altered tumor cell repopulation kinetics. Methods allowing earlier and more accurate quantitation of therapeutic response in individual patients are still needed. Because diffusion MRI is sensitive to tissue structure, this technique has the potential to detect important quantitative information about tumor cellular changes that occur following successful therapeutic intervention. Examples on the use of diffusion MRI to quantitate heterogeneous changes within tumor tissue following therapeutic interventions will be presented. Finally, the ability of MRS to distinguish signals from chemically distinct compounds offers the potential to measure the expression of transgenes encoding enzymes that catalyze therapeutic reactions. The use of MRI/S for quantitative noninvasive evaluation of expression of a therapeutic transgene in experimental tumors will be presented for the yeast cytosine deaminase gene therapy paradigm. In conclusion, the use of noninvasive imaging modalities for quantitation of therapeutic outcome, gene delivery, and monitoring of cellular events is emerging as an important approach for translating findings from the lab into animal and humans.
Abstract ID: 197
I. Serganova V. Ponomarev L. Ageyeva M. Doubrovin T. Beresten E. Vider S. Soghomonian J. Balatoni R. Finn R. Blasberg N. Rosen J. Gelovani Tjuvajev
Radiology and Neurology, Memorial Sloan Kettering Cancer Center
Cancer
Protein kinase B or Akt is a serine/threonine kinase, which is frequently constitutively active in many types of human cancer. Akt phosphorylates and regulates the function of many cellular proteins involved in processes that include metabolism, apoptosis, and proliferation, including different members of the forkhead family of transcription factors. The activation mechanism is mediated by PI3 kinase, which is essential for the translocation of Akt to the plasma membrane where it is phosphorylated by PDK1. Constitutive Akt activation can occur due to amplification of Akt genes or mutations in components of the signaling pathway that activates Akt, which contributes to cancer progression by promoting proliferation and increased cell survival. To noninvasively image Akt pathway activity, we developed a series of retroviral reporter vectors which encoded for: (1) AFX forkhead factor fusion to DsRed2 (RedAFX) expressed under LTR and neoR gene under CMV promoter; (2) HSV1-tkl/GFP fusion reporter gene (TKGFP) under control of a newly designed artificial forkhead-specific enhancer element; (3) the combination of expression cassettes 1 and 2 in one vector (but no neoR). Different tumor cell lines were transfected with each of these vectors and selected by G418 or FACS. Down-regulation of Akt activity in cells in vitro by starvation or exposure to the ZD1839 (EGFR inhibitor), LY294002 (PI3 kinase inhibitor), or AAG17 (hsp90 inhibitor), resulted in nuclear translocation of RedAFX transcription factor (by dynamic fluorescence microscopy imaging) and different degrees of TKGFP reporter gene expression (by microscopy and FACS). The degree of Akt-reporter activity correlated with other independent measurements (RT-PCR, Western blot, ELISA) of Akt-activity (phospho-Akt) and the expression of endogenous forkhead factor-dependent genes. Although still under optimization, the RedAFX/TKGFP reporter system should allow for noninvasive PET imaging of Akt activity in vivo.
Abstract ID: 198
G. R. Reddy T. L. Chenevert D. E. Hall B. A. Moffat M. S. Bhojani A. Rehemtulla B. D. Ross
Research and Development, Molecular Therapeutics
Cancer
We here describe the development of a nanoparticle contrast agent, which could be used for both detection of tumors as well as for delivery of therapeutic agents. Superparamagnetic iron oxide crystals (10 nm average size) have been encapsulated into a polyacrylamide polymer matrix. The resulting nanoparticle with an average size of 80 nm also contains reactive groups for attachment of various bioactive and therapeutic agents. The experimental R1 and R2 values of the resulting nanoparticles were 29 and 281, respectively. Nanoparticles at 300 mg/kg were injected into rats bearing an experimental 9L brain tumor followed by analysis using dynamic MR imaging. This revealed a plasma half-life of a few minutes for “naked” nanoparticles as opposed to several hours for particles that were “Pegylated.” T2-weighted MR imaging was performed pre-, 0.5, and 20 hr postadministration using a 7-T MR system. At 0.5 hr following administration, the contrast in the tumor, but not in the contralateral brain tissue, was dramatically altered by the presence of the nanoparticles as evidenced by the shortening of the T2 relaxation time resulting in a decrease in the MR T2 signal intensity. The selectivity of entrance into the tumor tissue is due to the disrupted blood–brain barrier within the tumor mass. Future studies will focus on targeting of these particles through the attachment of specific molecular moieties to the surface. For example, targeting to endothelial cells, which express avβ3 integrins (angiogenic vasculature) can be accomplished using a surface-localized RGD-peptide.
Abstract ID: 199
T. Lahoutte J. Mertens C. Joos A. Bossuyt
Nuclear Medicine, Academic Hospital Free University Brussels
Cancer
Besides increased glucose uptake, tumor cells also show an increased amino acid metabolism. An appropriate radioiodinated amino acid analog can allow us to visualize the metabolic state of tumors with SPECT. 123I-IMT is used for the study of cerebral tumors, but intense renal accumulation limits its use in extracerebral malignancies. Therefore, we developed the new radioiodinated neutral amino acid
Abstract ID: 201
J. D. Hazle1 >R. J. S. Heckl >K. Braun >R. Pipkorn >W. Waldeck, et al.
Oncological Diagnostics and Therapy, German Cancer Research Center
Cancer
A clear distinction between tumor and normal cells has not yet been possible due to a lack of cell specificity in contrast agents. The different oncogene expression can be used as a distinguishing feature. The examined tumor cells (human prostate cancer DU145-, human melanoma Sk-Mel-, and the HeLa cervix carcinoma cells) are characterized by the overexpression of the c-myc gene. Our aim was to enable passage of the gadolinium (Gd)-complex into the cytoplasm and to differentiate between c-myc expressing DU145, Sk-Mel, and HeLa cells as well as nontumor cells (lymphocytes). A transmembrane transport system of human origin was developed which was, firstly, coupled to a Gd-complex and, secondly, to an antisense sequence targeted at c-myc mRNA.
Abstract ID: 202
G. D. Luker V. Sharma C. M. Pica J. L. Prior W. Li D. Piwnica-Worms
Radiology, Washington University School of Medicine
Cancer
To enable molecular imaging of protein–protein interactions in vivo by PET and fluorescence imaging, we have used microPET and fluorescence imaging to detect interactions between p53 tumor suppressor and large T antigen (TAg) of SV 40 virus in a tetracycline-inducible two-hybrid system. To further validate this technology, we investigated if expression of the reporter gene, comprised of a mutant HSV1-TK fused to green fluorescent protein, could quantify relative differences in amounts of interacting hybrid proteins. Mice bearing HeLa cell xenografts stably transfected with reporter gene and interacting (p53-TAg) or noninteracting (p53-CP) hybrid proteins were treated with serial doses of doxycycline (60 mg/g every 12 hr) for 12, 24, and 48 hr before microPET imaging or biodistribution studies with the HSV1-TK substrate 18F-FHBG. Treatment with doxycycline produced time-dependent increases in hybrid proteins as determined by Western blots of tumor specimens. Both imaging and biodistribution studies showed proportional increases in accumulation of radiotracer over time only in p53-TAg tumors, while content of 18F-FHBG did not change in p53-CP tumors. In response to increasing doses of doxycycline (10–60 mg/g every 8 hr for 48 hr), dose-dependent increases in hybrid proteins were observed by Western blot. Expression of hybrid proteins resulted in enhanced accumulation of 18F-FHBG by tracer analysis only in p53-TAg tumors. Without doxycycline, no increases in protein or radiotracer accumulation above background levels could be detected in either tumor. These data demonstrate that the imaging two-hybrid system responds in a proportional fashion to increasing amounts of interacting proteins in vivo.
Abstract ID: 203
F. A. Jaffer C. H. Tung A. K. Houng T. J. O'Loughlin, et al.
Center for Molecular Imaging Research/Cardiology, Massachusetts General Hospital
Cardiology
Activated coagulation factor XIII (F13a), a transglutaminase, cross-links alpha2-antiplasmin (A2AP) and fibrin chains to form mechanically and proteolytically stable thrombi. Imaging of F13 activity could therefore provide important insight into thrombus formation and aging, as well as help assess anti-F13a therapies. The purpose of this study was to evaluate a new F13a-targeted MRI probe for imaging F13a activity. This probe (F13-CLIO) consists of a dextran-coated caged iron oxide particle (CLIO) conjugated to an A2AP peptide fragment. A control probe (C13-CLIO) was similarly synthesized except for a single amino acid substitution in the peptide. Human plasma clots were incubated for 2 hr with 3% volume of either: F13-CLIO, C13-CLIO, CLIO, or normal saline (NS). All CLIO probes had the identical iron concentration of 1.7 mg/ml. MRI of clots was then performed on a 1.5-T MR system, and clots were enzymatically digested and subsequently analyzed by gel electrophoresis. Clots incubated with F13-CLIO had a dramatic increase in uptake compared to the other probes, due to increased iron content as determined by electron microscopy. At a 20-msec echo time, the buffer/clot signal ratio was: F13-CLIO: 27:1; C13-CLIO: 2.1:1; CLIO: 1.3:1; NS: 1.0:1. Gel electrophoresis confirmed that the A2AP peptide moiety of only F13-CLIO probe was cross-linked by F13a to fibrin. These data indicate that F13-CLIO is a novel targeted MRI probe that specifically images F13a activity through covalent cross-linking into thrombi. As CLIO agents are biologically well tolerated, the probe could provide a new method to study F13 activity in vivo.
Abstract ID: 204
F. A. Jaffer C. H. Tung R. E. Gerszten R. Weissleder
Center for Molecular Imaging Research/Cardiology, Massachusetts General Hospital
Cardiology
Thrombin, a serine protease, is critically involved in thrombosis as well as other cellular processes. Imaging of thrombin activity in vivo could provide new insight into thrombin's effects in various homeostatic and pathological conditions. We have therefore recently developed a biocompatible, specific thrombin-activatable near-infrared (NIR) molecular probe. The probe consists of a thrombin peptide substrate that contains an N-terminal NIR fluorochrome, Cy 5.5. At baseline, the probe is optically quenched, but after thrombin cleavage, quenching is eliminated and a large NIR signal is generated. Two in vivo murine thrombosis models were studied. In a hematoma model, mice underwent transection of the distal tail vein following thrombin probe injection. Fluorescence reflectance imaging (FRI) was performed using a custom-built optical imaging system. In a model of intravascular thrombosis, 10% topical ferric chloride was applied to exposed femoral vessels in mice. After thrombus formation, the thrombin probe (n = 5) or free Cy 5.5 fluorochrome (n = 3) was systemically injected, followed by FRI using an epifluorescence microscope. In the hematoma model, the hematoma NIR signal nearly doubled over the adjacent tissue (n = 5; 94 ± 108% increase, p = .039). In the intravascular thrombosis model, all mice injected with the thrombin probe demonstrated focal NIR fluorescence in multiple areas of venous microthrombi. Mice injected with free Cy 5.5 fluorochrome, however, revealed minimal focal fluorescence within thrombi.
Abstract ID: 205
P. M. Winter S. D. Caruthers A. H. Schmieder T. D. Harris L. Chinen T. Williams M. P. Watkins J. S. Allen S. A. Wickline G. M. Lanza
Cardiovascular Division, Washington University
Cardiology
Angiogenesis is integral to the development and progression of atherosclerotic disease, which is characterized by the expression of avB3 integrins.
Abstract ID: 206
A. S. Perez M. T.Johnstone S. Witte J. Laredo W. Quist P. B. Graham R. M. Weisskoff J. Barett W. J. Manning R. M. Botnar
Cardiology, Harvard Medical School
Cardiology
Abstract ID: 207
John V. Frangioni
Hematology/Oncology, Beth Israel Deaconess Medical Center
Cardiology
Recent data suggest similarities between bone growth and pathologic vascular calcification. Hydroxylapatite (HA), the mineral content of the bone, is the most stable form of calcium in the body and is a common component of both bone and calcified vessels. We have previously described a near-infrared (NIR) fluorescent bisphosphonate derivative that has specific binding to HA both in vitro and in vivo. We now demonstrate NIR in vivo imaging of pathologic vascular calcification in the living mouse. The mgp -/- knockout mouse, deficient in the GLA protein, develops widespread vascular calcification by age 3 weeks, and dies by age 4–5 weeks from vascular hemorrhage. We have developed an intraoperative NIR fluorescence imaging system that permits simultaneous acquisition of color and NIR fluorescent images. For vessels of only 200 μm in diameter, vascular calcification could be imaged with exposure times as short as 30 msec, while still achieving signal-to-background ratios of *1.7. Pathologic calcification in the coronary vessels, aorta, renal arteries, mesentery, stomach, and peripheral vessels could be detected with the described combination of NIR fluorescent probe and imaging hardware, with results correlated to conventional histology and Von Kossa calcium salt staining. This imaging technology may be useful for the sensitive and specific detection of in vivo pathological calcification as might occur in coronary atherosclerosis, peripheral vascular disease, tympanosclerosis, and other HA-mediated processes.
Abstract ID: 208
S. Achilefu
Radiology, Washington University School of Medicine
Chemistry and Probes
Site-specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effects of drugs and enhances the contrast between normal and pathologic tissues. One approach to achieve selectivity is to target overexpressed receptors on the membranes of tumor cells and to visualize the tumors by a noninvasive optical imaging method. The successful use of radiolabeled small synthetic somatostatin analogs to image neuroendocrine tumors in humans have attracted considerable interest in nuclear medicine. Somatostatin receptor (SSTR) is up-regulated in a variety of human cancer cells with high receptor density and reliable small animal tumor models that have good correlation with clinical data are available. Since the SSTR is a well-understood system, it can serve as a good model for the development of receptor-based imaging agents. Accordingly, we conjugated visible and near-infrared fluorescent optical probes to SSTR-avid peptides, and examined their receptor-binding affinities in vitro and selective uptake in SSTR-positive tumor-bearing rats. We also prepared potential dual imaging probes consisting of a bioactive peptide for tumor targeting, a biocompatible dye for optical imaging, and a radioactive or paramagnetic metal chelator for scintigraphic or magnetic resonance imaging of tumors. We found that the bioconjugates retained high receptor binding affinity and this feature correlated with selective uptake of the agents in SSTR-positive tumor in vivo. These findings indicate that, despite their small size, conjugation of dyes to truncated somatostatin peptide analogs could be a useful strategy to develop tissue-specific optical imaging agents for clinical applications.
Abstract ID: 209
Paul A. Clemons
Molecular Target Laboratory, Harvard Institute of Chemistry and Cell Biology, Harvard University
Chemistry and Probes
Recent advances in chemical technology (library formatting) make possible the systematic annotation of complex and diverse libraries, prepared by diversity-oriented organic synthesis (DOS), with data sets delineating their biological performance. This is analogous to acquiring analytical chemistry data on every compound in such a collection, and represents an important first step in understanding the rules of engagement between chemical structure and biological function. By drawing on principles of genomic analysis, we are able to make broad comparisons both among many compounds in a particular biological assay, and among multiple assays performed on a common compound collection.
Abstract ID: 210
J. M. Perez T. O'loughlin L. Josephson R. Weissleder
Center for Molecular Imaging Research, MGH-Harvard Medical School
Chemistry and Probes
Sensitive and highly selective probes are required for the high throughput detection of various biomolecules such as DNA, RNA, and proteins in unmodified biological samples and turbid media. Herein, we report the development of biocompatible magnetic nanoparticles (50 nm) capable of detecting specific biomolecular interactions upon self-assembly of the nanoparticles into stable clusters (100–200 nm). This target-induced assembly occurs with a concomitant change in the spin–spin relaxation times (T2) of neighboring water molecules, rending these nanoparticles as magnetic relaxation switches (MRS) capable of sensing the presence of a biomolecule as a function of changes in water T2 relaxation times. We have shown that these magnetic nanoparticles can be used to detect complementary DNA/RNA sequences and specific proteins in complex media. The developed system could be useful in a variety of applications such as rapid magnetic resonance imaging of molecular interaction in array format, as probes for magnetic force microscopy and potentially for in vivo imaging.
Abstract ID: 211
Ching H. Tung
Center for Molecular Imaging Research, Massachusetts General Hospital
Chemistry and Probes
Proteases are known to play important roles in various diseases, such as cancer, vascular diseases, infection, degeneration, and autoimmune disorders. Specific information of those proteases may allow earlier detection and characterization of disease, direct molecular assessment of treatment effects, as well as a more fundamental understanding of the disease process. Recently, we have developed a series of near-infrared fluorescent molecular probes for the in vivo detection of specific protease activity. These probes are designed to have minimal fluorescence signal in their initial circulating state, until they are recognized and activated by designated enzymes. The technology has been applied to study cancer, tumor metastasis, arteriosclerosis, arthritis, thrombosis, inflammation, and protease inhibition.
Abstract ID: 212
Wellington Pham Ralph Weissleder Ching-Hsuan Tung
Radiology-Center for Molecular Imaging Research, Harvard Medical School
Chemistry and Probes
Caspases play important roles in the process of apoptosis. Detection of caspase activity can provide useful information about the physiological roles of the enzyme. Molecules absorbing wavelengths in the near-infrared (NIR) region have great applications in molecular imaging, since light of longer wavelengths penetrates tissue more efficiently than light in the visible region. Fluorescence resonance energy transfer (FRET) has been used in the detection of proteolysis and nucleic acid hybridization. Several NIR probes have been developed and used widely in analytical and clinical studies, but only a limited number of quencher have been developed. In this study, we report the synthesis and application of azulenyl squaraine analogs as novel NIR quenchers. Azulenyl squaraine dyes 1 and 2 were synthesized from seven-step and two-step experiments, respectively. The electron-donating isopropyl moiety on guaiazulene stabilizes the positive charge, and as a consequence, the absorbance of two shifts to 750 nm compared with 700 nm in the case of 2-methyl azulene of 1. A caspase-3 substrate was synthesized by SPPS containing an N-terminal glycine for the attachment of an azulenyl squaraine quencher, and a C-terminal cysteine for conjugation to an Alexa Fluor. The absorption wavelength of 2 overlaps the emission spectrum of the Alexa Fluor (713 nm), leading to a quenching of fluorescence. Cleavage the probe with caspase-3 showed a four-fold increase in the fluorescent intensity of the Alexa Fluor. In the first evaluation as a quencher in a FRET detection system for proteolysis, azuelenyl squaraine dyes have been found to be promising energy acceptors. This probe is to be used in an in vivo study of caspase-3 activity.
Abstract ID: 213
K. N. Raymond
Chemistry, University of California, Berkeley
Chemistry and Probes
Magnetic resonance imaging (MRI) has revolutionized diagnostic medicine. About one-third of MRI scans are now acquired using a paramagnetic contrast agent, which enhances the image quality. Gadolinium complexes are most widely used and these complexes currently are all based on a poly(amino-carboxylate) ligand scaffold. While effective, the water proton relaxivity of these agents are only a few percent of that theoretically possible. Attachment of the agent to macromolecules, either synthetic or in vivo biomolecules, can increase the relaxivity by lowering the rotational correlation time, but the rate of water exchange from the gadolinium center then becomes the limiting factor. A new family of gadolinium complexes is based on a tris(bidentate) ligand scaffold. These complexes (composed of hydroxypyridonate, hydroxypyrimidonate, or catecholate) are stable and have substantially higher relaxivity than commercial poly(amino-carboxylate) complexes, due to a water exchange rate about two orders of magnitude higher. The fundamental chemical properties and relaxivity (explored in collaborative studies) will be reported. These second-generation agents portend new Gd MRI agents that enable relaxivity of one or two orders of magnitude greater than what is available today.
Abstract ID: 214
J. Grimm J. M. Perez L. Josephson R. Weissleder
Center for Molecular Imaging, Harvard Medical School-MGH
Chemistry and Probes
A number of “activatable” imaging probe technologies have been described that can be used to sense molecular targets and functions. These include optical approaches (e.g., FRET-based activation), paramagnetic chelates sensitive to their environment, and nanoparticulate magnetic relaxation switches (MRS). The latter technology is based on supramolecular assemblies that transition from single to oligomeric fluid phase states, accompanied by 5–10-fold R2 changes, easily detectable by MR imaging. This has been used to sense (and image) DNA hybridization, methylation, and cleavage. The goal of the current study was to evaluate various strategies to sense telomerase activity. In analogy to telomeric repeat amplification protocol (TRAP) assays, we investigated the telomerase activity in extracts from different tumor cell types. Our results show that telomeric repeats synthesized by telomerase can be sensed by MRS and potentially allow multiplexed detection of mRNA, protein, and telomerase activity at the same time.
Abstract ID: 215
M. E. Weber N. Torrey J. Kim J. A. Engelbach M. J. Welch,
Radiological Sciences, Washington University
Chemistry and Probes
The inherent importance of nitric oxide (NO) in biological systems is now well documented (Furchgott, et al. Nature. 1980; Nathan and Xie, J. Biol, Chem. 1994). In recent years, NO has been implicated in vasodilation, neurotransmission, angiogenesis, and immune regulation. Endogenous NO is produced by a family of enzymes, the nitric oxide synthases (NOS), from the conversion of
Abstract ID: 216
M. Alauddin A. Shahinian J. Fissekis P. Conti
Radiology, University of Southern California
Chemistry and Probes
Abstract ID: 217
W. A. Volkert G. L. Sieckman L. R. Forte T. J. Hoffman N. K. Owen D. G. Mazuru
Radiology, University of Missouri
Chemistry and Probes
It has been demonstrated that radiolabeled Escherichia coli ST[h]-peptide conjugates are capable of selective in vitro and in vivo targeting of guanylin/guanylate cyclase-C [GC-C] receptors that are expressed on colon cancer cells. Expression of ST[h]-cognate receptors expressed on noncolorectal cancer cells has not been previously reported. Studies herein reported show that Phe[19]-ST[h] (i.e., N-S-S-N-Y-C-C-E-L-C-C-N-P-A-C-T-G-C-F, where C(6) and C(11), C(7) and C(15), and C(10) and C(18) specifically form S—S bonds) and [111]In-DOTA-Phe[19]-ST[h] conjugates are capable of high-affinity specific binding to receptors expressed on several human ER[+] and ER[-] human breast cancer cell lines (e.g., T47D, MB-231, MB-468). Scatchard analysis of ST[h] analogs with these cell lines demonstrated Kd values ranging from 3.1 to 4.4 nM and ST[h] receptor densities on cancer cells ranging from 41,000 to 112,000 receptors per cell. In vivo studies with In-111-DOTA-Phe[19]-ST[h] in T47D cell derived tumor xenografts on SCID mice demonstrate rapid blood clearance via the kidneys into the urine and moderate selective tumor uptake (i.e., 0.67 ± 23% ID/g) at 1 hr pi. Results from these studies indicate that radiolabeled ST[h] conjugates are potential candidates for further development as breast cancer specific imaging and/or therapeutic radiotracers.
Abstract ID: 218
Zheng-Rong Lu Dennis L. Parker K. Craig
Goodrich Pharmaceutics and Pharmaceutical Chemistry, University of Utah
Chemistry and Probes
The clinical application of macromolecular gadolinium complexes as MRI contrast agents is impeded by the slow excretion of Gd(III) complexes after MRI exams and resulted in long-term tissue accumulation of toxic gadolinium ions. We intend to design and develop biodegradable macromolecular Gd(III) complexes as safe, effective MRI blood pool contrast agents. These agents can provide effective contrast enhancement in blood pool MRI and then gradually degrade into smaller complexes removable by renal filtration.
We have designed and prepared a new class of [DPTA-Gd(III)]-disulfide copolymers, which can gradually be broken into smaller molecules by the endogenous thiols in the blood plasma via thiol-disulfide exchange reaction. The molecular weight of the macromolecular complexes is approximately 18,000 Da and the T1 relaxivity is 4.65 mM−1 sec−1 per complexed Gd ion at 400 mHz. The copolymer has low toxicity as shown in the preliminary study in mice. An in vitro degradation study was performed by the incubation of the copolymer with
Abstract ID: 219
C. R. Bertozzi
Chemistry, HHMI, University of California, Berkeley
Chemistry and Probes
A major lesson from eukaryotic genome sequencing projects is that the absolute number of genes an organism's genome encodes is not the best parameter for defining complexity of function. It appears that the complex functions associated with human health and disease are determined by combinatorial expansion of genomic information in the form of posttranslational modifications. Of these, the most complex and ubiquitous is glycosylation, highlighting the importance of glycobiology in the postgenomic era. This presentation will focus on new chemical approaches for perturbing and studying oligosaccharide function within the context of the cell surface.
Abstract ID: 220
G. Zheng M. Zhang H. Li D. Blessington L. L. Zhou S. Lund-Katz B. Chance J. Glickson
Radiology, University of Pennsylvania
Chemistry and Probes
Stable bacteriochlorophyll (BChl) analogs derived from R. sphaeroides are excellent candidates as NIR active photosensitizers for photodynamic therapy (PDT) and probes for fluorescence imaging because of their favorable photophysical properties (singlet oxygen yield >50%) and spectroscopic characteristics (labs 825 nm; lem 840 nm). Since tissue is relatively transparent to NIR light, such agents are capable of detecting and treating even subsurface cancers. A current limitation of both fluorescence imaging and PDT modalities is their lack of sufficient tumor-to-tissue contrast due to the nonspecific nature of delivering the dye to the tumor, which has led to false negatives for imaging and skin toxicity for PDT. Therefore, agents targeting the “cancer signatures” (i.e., molecules that accumulate in various types of cancer cells) are particular attractive. Our current programs focused on two of such signatures: the LDL receptor (LDLr) and the tumor-specific mRNAs. In the first approach, BChl cholesteryl oleate conjugates were synthesized and reconstituted into the LDL core. High-resolution 3- imaging of freeze-trapped mice showed that such agents were selectively internalized by LDLr overexpressing tumors. The PDT experiments were then carried out to validate their therapeutic potential. Our second approach is to develop BChl-based molecular beacons (hairpin antisense oligonucleotides) to target tumor-specific mRNAs. By developing these BChl beacons, our goal is to achieve a seamless transition between early cancer detection and treatment with a single, inexpensive, and noninvasive procedure—irradiating tumor at a single wavelength utilizing a diode laser with tunable laser power (low power for imaging and high power for PDT).
Abstract ID: 221
S. T. Gammon J. Prior V. Villalobos D. Piwnica-Worms
Radiology, Washington University
Chemistry and Probes
Permeation peptides and derivatives are under investigation as reagents for transducing proteins, oligonucleotides, drugs, fluorophores, iron oxide nanoparticles, and radiometals directly into the cell interior. To examine relationships between the amino-acid sequence and permeation peptide uptake into cells, a series of 42 different peptide conjugates containing a chelation moiety (C-terminus Lys–Gly–Cys tripeptide or N-terminus His–Gly) and proposed permeation sequences were synthesized by standard solid phase methods. Peptides were radiolabeled with 99mTcO by a transmetallation reaction or 99mTc(CO)3 and quantitative cell uptake experiments (20 min) were performed with human Jurkat cells. Cell-associated peptide content was determined by gamma counting. In addition, stability of two poly-Arg peptide conjugates was evaluated under cell uptake conditions using radio-TLC and radio-HPLC. It was determined that amino acid chirality of the permeation sequence, peptide length, metal chelation, Gln residue substitution in Tat basic domain peptide sequences, and peptide class significantly impacted net transduction, but neither chelate chirality nor N-terminal modification affected peptide uptake. Finally, the 3- to 13-fold enhanced uptake of peptides comprising
Abstract ID: 222
Alexei Bogdanov Jr. Wellington Pham Alexander Petrovsky Ralph Weissleder
Radiology, Center for Molecular Imaging Research
Chemistry and Probes
Tyrosinase is a copper monooxygenase involved in melanin biosynthesis. We have previously demonstrated that the incorporation of free paramagnetic cations (e.g., Fe, Mn) during melanin biosynthesis can be used to image tyrosinase expression indirectly in transfected cells. The goal of the current research was to develop paramagnetic (e.g., Gd) substrates for the direct MR imaging of tyrosinase activity. We hypothesized that the enzyme would amplify the R1 relaxivity when paramagnetic substrates of tyrosinase are oligomerized. Two substrates: tyramide-DOTA(Gd), I and hydroxytyra-mide-DOTA(Gd), II were synthesized, purified, and studied as potential substrates of tyrosinase in vitro and in a B16 model of murine melanoma. Both synthesized substrates were converted by tyrosinase but II demonstrated a twice faster kinetics than substrate I (pseudo-first-order kinetic constant k1 = 0.0002 sec−1). The oligomeric conversion product of II had a 2.5 times higher atomic R1 relaxivity of gadolinium. The latter relaxation effect is due to the decrease of tumbling rate of Gd atoms after the polymerization. The substrate II was polymerized by tyrosinase at a higher rate than by peroxidase, which induces polymerization of substrate, I with a very high efficiency (k1 = 0.01 sec−1). When B16 melanoma cells were incubated with substrate II, the measured MR signal intensity of these cells was 4-fold higher compared to control cells (1.5 T). These results indicate that tyrosinase catalyzes polymerization of paramagnetic oxyphenols resulting in increased R1 relaxivity and amplification of MR signal. This effect can be utilized for imaging of tyrosinase expression in vitro and in vivo.
Abstract ID: 223
J. W. M. Bulte H. A. Zywicke J. A. Frank T. Douglas, et al.
Radiology, Johns Hopkins University
Chemistry and Probes
Cowpea chlorotic mottle virus (CCMV) can undergo reversible structural changes allowing the formation of open or closed pores dependent on the pH of the microenvironment. This virus, measuring 28 nm in diameter, is ideally suited for the encapsulation of guest molecules with potential applications in drug delivery that depends on switchable access to the virus interior. CCMV also contains 180 metal binding sites that are normally occupied by calcium with a dissociation constant (Kd) of 1 mM. We have substituted Ca(II) with Gd(III) atoms, and probed the lanthanide binding using titration of Tb(III) and fluorescence resonance energy transfer (FRET) from tryptophan residues close to the metal binding sites. Using competitive binding, we measured a Kd of 1 uM for Gd(III). Variable field relaxometry of Gd-CCMV solutions yielded 1/T1 and 1/T2 curves that were dominated by the very long correlation time (tc) of the slowly tumbling virions. The T1 relaxivity ranged from 58 mM−1 sec−1 at 2 MHz (0.05 T) to 154 mM−1 sec−1 at the highest field (1.5 T) measured. The T2 relaxivities were even larger, ranging from 70 to 255 mM−1 sec−1. To the best of our knowledge, the Gd-CCMV relaxivities represent the highest values obtained for paramagnetic materials as of today. With 180 Gd atoms per virion, a total relaxivity of 2.7-4.6 × 104 mM−1 sec−1 per particle can be obtained. The observed high relaxivities justify further exploration of Gd-CCMV as potential drug container and MR contrast agent.
Abstract ID: 224
Xiangyun Wang Boli Liu
College of Chemistry and Molecular Engineering, Peking University
Chemistry and Probes
The geometry and total molecular energy of nine species in the system [Tc(CO) x (H2O)6-x]+ (x = 0–6) are calculated by using ab initio MO method at the MP2/CEP-121G level. Their formation energies are estimated in the order trans-[Tc(CO)2(H2O)4]+ > [Tc(H2O)6] + > trans-[Tc(CO)4(H2O)2]+ > mer-[Tc(CO)3(H2O)3]+ > cis-[Tc(CO)2(H2O)4]+ > cis-[Tc(CO)4(H2O)2]+ > [Tc(CO)5(H2O)]+ > fac-[Tc(CO)3(H2O)3]+ > [Tc(CO)6]+. The computation results of relative composition for various CO-to-H2O concentration ratios indicate that fac-[Tc(CO)3(H2O)3]+ is the dominate species when the pressure of CO is 0.1 MPa as experimentally observed, and preparation of other species is also possible in controlled conditions. The results maybe useful for labeling of receptors, ligands, peptides, and monoclonal antibodies with 99mTc(CO)3+.
Abstract ID: 225
M. F. Kircher L. Josephson R. Weissleder
CMIR/Radiology, MGH/Harvard Medical School
Chemistry and Probes
The aim was to develop an optical imaging technique which is able to measure enzyme activity in vivo independent of the accumulation of the probe. Two magneto-optical probes, an enzymatically activatable nanoparticle, denoted Cy5.5-R4-SC-CLIO (R4), and a nonactivatable nanoparticle, denoted Cy3.5-r4-SC-CLIO (r4), were synthesized. The two probes were used as a mixture (equimolar amounts of dye) and the ratio Cy5.5/Cy3.5 fluorescence was calculated. R4 and r4 have very similar sizes and chemical structures. Incubation with cathepsin B increased the NIR fluorescence of R4 by cleaving the basic arginine residues in the l-configuration, producing a 600% increase in fluorescence after 60 min. The Cy3.5 channel showed a constant fluorescence, indicating the presence of fluorophore on r4 but no activation by cathepsin B. When a mixture of R4 and r4 was incubated with different densities of macrophages—a model simulating different probe accumulation—it was shown that the fluorescence intensity from both R4 and r4 increased with higher cell density, whereas the ratio R4/r4 fluorescence was independent from the cell density. Thus, the dual wavelength ratio imaging method can differentiate between accumulation of nanoparticles and local enzyme activity. Experiments with inhibitors demonstrated the specificity of R4 for cathepsin B. R4/r4 ratio images, corrected for the accumulation of R4, were readily acquired from single macrophages as well as from mouse organs. We present a novel strategy for improved enzyme quantification termed dual wavelength ratio imaging. The technique enables noninvasive detection of enzyme activity that is corrected for differences in probe accumulation.
Abstract ID: 226
H. Bryant, Jr. L. Gutiérrez J. Bulte J. Frank
Laboratory of Diagnostic Radiology Research, National Institutes of Health
Chemistry and Probes
Dendrimer-based gadolinium chelates are a class of macromolecular MR contrast agents with relaxivities, at the clinical imaging field of 1.5 T, four times higher than the monomeric gadolinium chelates. In addition, dendrimers are used as cellular transfection agents for oligonucleotides and other biomolecules. Therefore, these agents are attractive candidates for use as markers of cellular uptake and delivery. We hypothesized that dendrimers with gadolinium chelates and rhodamine can be incorporated into cells. In this study, we report on the intracellular uptake of GdDOTA and fluorescent tetramethylrhodamine covalently attached to a high-generation dendrimer for optical and MR imaging.
Abstract ID: 227
J. A. Katzenellenbogen
Chemistry, University of Illinois
Chemistry and Probes
Nuclear hormone receptors, such as the estrogen receptor, regulate tissue responses to hormones. Endocrine-responsive cancers, such as breast and prostate cancers, often retain sensitivity to hormones and can be treated successfully and with minimal morbidity with hormone antagonists. Selection of patients who are likely to benefit from hormone therapy, however, requires an accurate determination of the receptor levels in tumors. This is typically done by biochemical or immunoassays on tumor biopsies, but this is an invasive procedure and is subject to tissue sampling errors. Assaying tumor receptor levels can be done noninvasively using radionuclide imaging, provided a suitable radiolabeled ligand can be prepared. We have developed agents for imaging the estrogen and progesterone receptor contents of breast tumors and the androgen receptor content of prostate tumors, by positron emission tomography, using fluorine-18 labeled steroids designed to retain high receptor binding affinity. Receptor-positive tumors can be imaged clearly in many cases. A limitation in this approach, however, is the limited availability of the short-lived fluorine-18 radionuclide. If it were possible to label ligands for nuclear hormone receptors with the inexpensive and widely available radionuclide technetium-99m, then such imaging could be done on a much wider basis. It is much more of a challenge, however, to label a nuclear hormone receptor ligand with the large radiometal Tc-99m than with the small radiohalogen F-18. We will discuss approaches that we have taken in this endeavor that utilize inorganic chelates and organometallic systems for labeling receptor ligands with technetium, either in a pendant or in an integrated mode. Receptor ligands with high affinity have been obtained, and these are being evaluated for their imaging properties. (Supported by grants from the Department of Energy and the National Institutes of Health.)
Abstract ID: 228
Nan-hui Ho Ralph Weissleder Ching-Hsuan Tung
Radiology, CMIR/MGH/HMS
Chemistry and Probes
Proteases play central roles in numerous biological processes. Development of highly sensitive fluorogenic probe to detect specific protease activity becomes a growing need in vitro and in vivo. We have designed a class of fluorogenic probe for sensitive protease detection. This class of probe is to incorporate a bifunctional fluorophore, 7-amino-4-carbamoylmethyl coumarin (ACC), into the peptide substrates using solid-phase peptide synthesis method. Upon proteolysis, the amino-conjugated substrate releases the free ACC resulting a high shift of emission spectrum. Due to the bifunctional nature of the fluorophore, the probe can be rapidly prepared by standard Fmoc-synthesis strategy. We report the synthesis, fluorescent properties, and in vitro enzyme detection of this probe. We also will report a facile method for the rapid preparation of functional cell-permeable peptide substrates.
Abstract ID: 229
M. Hasbach C. Olbrich M. Schirner
Ultrasound and New Modalities Research, Schering, CRBA DG & RP
Chemistry and Probes
In the last years, many studies showed the potential of target-specific ultrasound contrast agents as a potent diagnostic tool in the determination of disease-specific alterations in the body. When used with high-affinity targeting moieties like antibodies, it can be considered as one of the most sensitive agents in the field of molecular imaging, especially when used with highly sensitive ultrasound modes like stimulated acoustic emission (SAE), developed at the research laboratories of Schering. To assess the targeting properties of the target-specific ultrasound contrast agent, the determination of the amount of targeting moieties on the contrast agent is one prerequisite for systematic effectivity testing as well as for regulatory reasons. For this issue, a FACS assay, using Phycoerythrin (PE) labeled Protein A was developed. Using the Phycoerythrin-labeled Quantibrite beads (Beckton and Dickinson), it is possible to determine the amount of active molecules per contrast agent entity in one step. No titration is necessary and the amount can easily be determined by comparison with the Quantibrite beads, which are polymeric beads with different amounts of covalently bound Phycoerythrin on the surface, resulting in a calibration line. Experiments using this assay showed high reproducibility and is used as a standard tool to control the quality of our target-specific ultrasound contrast agents.
Abstract ID: 230
H. Bryant, Jr. L. Gutierrez J. Bulte J. Frank
Radiology, NIH
Chemistry and Probes
Dendrimer-based gadolinium chelates are a class of macromolecular MR contrast agents with relaxivities, at the clinical imaging field of 1.5 T, four times higher than the monomeric gadolinium chelates. In addition, dendrimers are used as cellular transfection agents for oligonucleotides and other biomolecules. Therefore, these agents are attractive candidates for use as markers of cellular uptake and delivery. We hypothesized that dendrimers with gadolinium chelates and rhodamine can be incorporated into cells. In this study, we report on the intracellular uptake of GdDOTA and fluorescent tetramethylrhodamine covalently attached to a high-generation dendrimer for optical and MR imaging.
Abstract ID: 231
M. Zhao L. Josephson Y. Tang R. Weissleder
Radiology, CMIR MGH/HMS
Chemistry and Probes
Proteases are involved in a wide spectrum of biological functions, including general metabolism, signal transduction, cell proliferation and apoptosis, immune functions, and disease progression. The goal of the current study is to design and investigate NMR-based assays for the measurements of posttranslational regulatory protease/inhibitor biosystems. This research may not only benefit the discovery and evaluation of protease-targeted pharmaceuticals in vitro, but may also have the potential of developing paramagnetic probes for the noninvasive detection of protease activities in vivo. Given the fact that the formation of aggregates among superparamagnetic nanoparticles causes increased susceptibility effect and T2 relaxivity, we have designed protease assay systems where iron oxide nanoparticles are held together by peptidergic substrates. Peptide substrate conjugated to paramagnetic nanoparticles provides a solution phase reaction condition. Upon protease digestion, the nanoparticles dissociate and disperse, resulting in detectable changes in T2, which can be measured using either NMR spectroscopic methods or MR imaging. With T2-weighted MR imaging, we have shown that this effect could be readily extended to high throughput assay systems, where protease reactions were carried out in microtiter plates. The MR-based methods possess sensitivity at the subnanomolar range of several investigated proteases. The use of magnetic readout systems has the advantage in that it is suitable for turbid environments where light absorbance and scattering render fluorescent signals unreliable.
Abstract ID: 232
Yong Taik Lim Sungjee Kim Moungi G. Bawendi John V. Frangioni
Hematology/Oncology, Beth Israel Deaconess Medical Center
Chemistry and Probes
Compared to conventional fluorophores, quantum dots have a number of attractive characteristics including broad absorption bands, relatively narrow and symmetric luminescence bands, and high resistance to photodegradation. The broad absorption bands, in particular, are believed to be a major advantage for in vivo imaging and biosensing applications since absorption cross-section increases in a near exponential fashion as the excitation wavelength is decreased far below the emission band. In this study, we have developed a simple mathematical model of photon propagation through tissue that includes quantum dot properties. Using this model, we are able to predict the net effect of absorption and scatter on quantum dot fluorescence emission in different biological tissues. The model suggests that if tissue attenuation is greater than a wavelength-dependent threshold, essentially all of the potential advantage of broad quantum dot absorption is negated by photon losses, and that the emission wavelengths of quantum dots for in vivo applications fall within a relatively narrow band of wavelengths. These model predications are validated using both phantom measurements and measurements on living tissue, and recommendations regarding quantum dot design are discussed.
Abstract ID: 233
Atif Zaheer Thomas E. Wheat John V. Frangioni
Hematology/Oncology, Beth Israel Deaconess Medical Center
Chemistry and Probes
The detection of human malignancies by near-infrared (NIR) fluorescence will require the conjugation of cancer-specific ligands to NIR fluorophores that have optimal photoproperties and pharmacokinetics. IRDye78, a poly-sulphonated heptamethine indocyanine NIR fluorophore, meets most of the criteria for an in vivo imaging agent, and is available as an N-hydroxysuccinimide ester for conjugation to low-molecular-weight ligands. However, IRDye78 has a high charge-to-mass ratio, complicating purification of conjugates. It also has a potentially labile linkage between fluorophore and ligand. We have developed an ion-pairing purification strategy for IRDye78 that can be performed with a standard C18 column under neutral conditions, thus preserving the stability of fluorophore, ligand, and conjugate. By employing parallel evaporative light scatter and absorbance detectors, all reactants and products are identified, and conjugate purity is maximized. We describe reversible and irreversible conversions of IRDye78 that can occur during sample purification, and describe methods for preserving conjugate stability. Using seven ligands, spanning several classes of small molecules and peptides (neutral, charged, and/or hydro-phobic), we illustrate the robustness of these methods, and confirm that IRDye78 conjugates so purified retain bioactivity and permit NIR fluorescence imaging of specific targets.
Abstract ID: 234
V. Sharma J. Prior C. M. Pica D. Piwnica-Worms
Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University Medical School
Chemistry and Probes
Functional imaging of MDR1 P-glycoprotein (Pgp)-mediated transport activity would enable noninvasive assessment of chemotherapeutic resistance as well as MDR1 gene therapy in vivo. Therefore, organic scaffolds capable of accommodating PET isotopes to generate stable radiopharmaceuticals recognized by Pgp are desired. We have synthesized and structurally characterized several Schiff-base and amine-phenol gallium(III) complexes, and a lead MDR imaging compound has been identified. Radiolabeled 67Ga- (for SPECT) or 68Ga-complexes (for PET) have been obtained through transmetallation reactions. The lead compound, namely Ga-ENBDMPI [(bis(3-ethoxy-2-hydroxy benzylidene)-N,N′-bis(2,2-di-methyl-3-amino-propyl)ethylene-diamine)gallium(III)] was stable in physiologic buffer at 37°C for 72 hr. Further analyses of this compound extracted from the heart, liver, and kidney of mice 60 min postinjection indicated no evidence of metabolism. In KB3-1(Pgp-) and KB8-5 (Pgp+) cells, the radiopharmaceutical demonstrated net cellular uptake profiles inversely proportional to Pgp expression. In KB8-5 cells, GF120918, PSC 833 and LY 335979, potent and selective MDR modulators, fully reversed tracer uptake. Like 99mTc-sestamibi, 67Ga-ENBDMPI demonstrated modest cross-reactivity with MRP1. However, no significant evidence of cross-reactivity with MRP2–6 was observed with this radiotracer. To further demonstrate utility in vivo, quantitative pharmacokinetic analysis was performed in wild type (WT) and mdr1a/1b(-/-) gene disrupted (KO) mice with 67Ga-ENBDMPI. Compared to the WT control, the tracer demonstrated markedly increased initial penetration and 17-fold enhanced retention in brains of KO mice, consistent with permeation through the known Pgp-mediated transport barrier normally present in brain capillaries in vivo. The evidence suggests that 67/68Ga-ENBDMPI may be suitable for PET imaging of Pgp transport activity in vivo.
Abstract ID: 235
Fred Reynolds Lee Josephson
CMIR, MGH/CMIR
Chemistry and Probes
Attaching a sequence of positively charged amino acids, like the tat peptide, to magnetic nanoparticles (CLIO), imbues the nanoparticle with a membrane translocating ability and permits the labeling and tracking of cells by MR. To further understand the behavior and metabolism of these important reagents for MR imaging research, we synthesized three new positively charged peptides and attached them to CLIO. The peptides were GRRRRGRRRRGYK(FITC)C-NH2, (FITC)GrrrrGrrrrGYC-NH2 and (FITC)GRRRRGRRRRGYC-NH2. The previously described tat peptide was also employed: GRKKRRQRRRGYK(FITC)C-NH2. Each peptide was attached to a Cy3.5-labeled CLIO through a disulfide or a thioether linkage, to yield a total of eight dual fluorochrome labeled peptide–nanoparticle conjugates. In vitro studies demonstrated that among the eight peptide–nanoparticles were conjugates where the two fluorochromes could be separated only by trypsin, only by DTT, by DTT or trypsin, or by neither agent. Fluorescence microscopy showed the fate of the nanoparticle (Cy3.5 fluorescence) while fluorescein fluorescence could be used to follow the attached peptide.
Abstract ID: 236
H.-W. Kang L. Josephson A. Petrovsky R. Weissleder A. Bogdanov, Jr.
Radiology, Massachusetts General Hospital
Chemistry and Probes
We prepared and tested covalent conjugates of the cross-linked iron oxide nanoparticles (CLIO) and high-affinity anti-human E-selectin F(abȼ)2 fragments and tested them in vitro and in vivo to establish the feasibility of proinflammatory marker MR imaging. The conjugates were obtained by using thiol–disulfide exchange reaction between 3-(2-pyridyl)propionyl-CLIO and S-acetylthioacetate-modified F(abȼ)2 fragments. The purified CLIO–F(abȼ)2 conjugates were used in experiments with human endothelial umbilical vein cells (HUVEC) in vitro. Cells treated with IL-1b expressed E-selectin and showed a 100–200 times higher binding of CLIO particles per cell than control cells. The binding resulted in a high superparamagnetism of HUVEC with the transverse water proton relaxation time (T2) decrease to 30–40 msec in cell precipitates. Cells did not bind control CLIO–F(abȼ)2 conjugates prepared using a nonspecific antibody fragment or nonconjugated iron oxide particles before or after treatment with IL-1b. MR imaging of cells showed a highly specific T2-weighted signal of cells treated with IL-1b followed by the incubation with anti-E selectin. Further, we performed an in vivo experiment in mice implanted with HUVECs into Matrigel plugs. After 10–20 days of implantation, CLIO–F(abȼ)2 (2 mg/kg) was injected to the mice following intraperitoneal injection of IL-1b. T2-weighted signal change was observed in the area implanted with HUVECs after IL-1b injection. In mice implanted either with Matrigel alone, or without IL-1b treatment, no signal intensity change could be observed. The initial experience with MR imaging of E-selectin expression justifies further development of MR-targeted agents for monitoring tumor vascular endothelial proliferation, angiogenesis, and atherosclerosis.
Abstract ID: 237
B. Moasser B. Grimmond E. E. Uzgiris K. M. Fish, et al.
Polymer and Specialty Chemicals, GE Global Research Center
Chemistry and Probes
Angiogenesis (new blood vessel growth) is critical to the development and metastasis of tumors and is currently an active and promising area of medicinal and pharmacological research. We have found elongated polymeric conjugates of gadolinium chelates to be effective contrast agents for the MR imaging of tumors in rat models. The extended structure of the polymers correlated with their selectivity for tumor penetration and, in turn, the extended structures of the polymers were found to be a function of the degree of conjugation. It is hypothesized that the extended polymer conjugates selectivity permeate the microstructure of tumor endothelium by the mechanism of reptation. In the absence of high conjugation, the molecules form coiled structures that do not have the ability to reptate. Exploration of these ideas required access to a collection of this class of polymeric agents. To this end, we have developed synthetic methods for the construction of a variety of Gd(chelate) polymer conjugates having very high degrees of conjugation (90–97%). We have assessed the scope and utility of the synthesis with respect to the structure of the polymer, its molecular weight, type of chelate, and synthetic scale. Additionally, we have developed purification schemes that are critical to our studies. The effect of variables such as polymer size, backbone structure, chelate structure, and chelate charge on relaxivity and tumor selectivity was studied and provided a preliminary structure–activity relationship which we use as for understanding the mechanism of tumor penetration by macromolecular constructs.
Abstract ID: 238
Meixiang Yu Anna-Liisa Brownell
Radiology, Massachusetts General Hospital
Chemistry and Probes
Abstract ID: 239
Terece O'Loughlin J. Manuel Perez Ralph Weissleder Lee Josephson
Radiology, CMIR, MGH
Chemistry and Probes
Proteomics studies are yielding many new biomarkers potentially useful in cancer screening and diagnosis. We previously developed magnetic relaxation switch (MRS) biosensors capable of forming reversible nano-assemblies upon protein detection. In this study, we compare MRS biosensors to the standard enzyme-linked immunosorbent assay (ELISA) for protein detection with the cancer antigen CA125. MRS probes consist of an iron oxide core surface passivated with cross-linked dextran (CLIO). We then conjugated the probes to Protein G (CLIO-PG), which binds the Fc region of IgG antibodies, facilitating noncovalent site directed attachment of active mouse monoclonal antibodies directed at CA125. The CA125 MRS biosensor was able to specifically and to quantitatively detect CA125 in the femtomole range, comparable to ELISA methods. Since MRS biosensors are nontoxic and biocompatible, they may be used for in vivo detection of protein expression.
Abstract ID: 240
K. Bullok M. Dyszlewski J. Prior C. Pica V. Sharma D. Piwnica-Worms
Radiology, Washington University School of Medicine
Chemistry and Probes
Tat basic domain, RKKRRQRRR, and its derivatives are known to translocate through cell membranes and have been conjugated to proteins, oligonucleotides, drugs, fluorophores, and radiometal chelators as membrane transduction reagents. [99mTc] is one commonly utilized radiometal since it is readily available, has a short half-life (6.02 hr), and is coordinated by a variety of chelation moieties, including recent coordination of [99mTc(CO)3] by aromatic amines such as histidine. [99mTc]tricarbonyl has shown stability over a broad pH range, making it an ideal candidate for labeling Tat basic domain for in vitro and in vivo analysis. To explore [99mTc(CO)3] radiolabeling, the Tat basic domain sequence, HGRKKRRQRRRGC, was synthesized by standard solid-phase methods and the C-terminus labeled with fluorescein-5-maleimide (Tat-FM). His–Gly metal coordination was then carried out utilizing [99mTc(CO)3(H2O)3]+ or [Re(CO)3Br3][NEt4]2 to obtain the dual-labeled Tat-peptide [99mTc(CO)3]-Tat-FM and the highly analogous [Re(CO)3]-Tat-FM. Quantitative transport assays were accomplished using [99mTc(CO)3]-Tat-FM and human Jurkat leukemia cells. Data analysis indicated a decrease in [99mTc(CO)3]-Tat-FM uptake (93–94 pM/(mg uMo)) compared with [99mTc(CO)3]-Tat-peptide lacking FM (628 pM/(mg uMo)). However, [99mTc(CO)3]-Tat-FM membrane translocation was directly confirmed by fluorescent microscopy utilizing the fluorescent analog labeled with [Re(CO)3]. Fluorescent microscopy of [Re(CO)3]-Tat-FM revealed diffuse cytoplasmic staining and nucleolar localization as previously seen with Tat-FM. Thus, [Re(CO)3]-Tat-FM localized in a manner similar to Tat-FM. We have demonstrated the utility of a dual-labeled Tat basic domain peptide for direct comparison of radiometric and fluorescence imaging analysis. This strategy could be generally applied to cell membrane–permeant peptides.
Abstract ID: 241
X. Chen R. Laforest T. Sharp C. Anderson, et al.
Authors at Washington University, Radiology, University of Southern California
Chemistry and Probes
Abstract ID: 242
Q. G. He Y. B. Miao H. M. Jia B. L. Liu
Beijing Normal University
Chemistry and Probes
In our previous work, we found the linear relationship between SAFv and R, where SAFv is the solid angle factor of the vacancy trans to the Tcȼõ(O) core of complex, and R is to be an indicator for relative ability of brain retention of Tc-complex. This relationship indicated that proper instability of Tcȼõ(O) complex for brain imaging agent may be valuable to their long brain retention. Considering that the SAS value is a better indicator for describing the stability of Tc-complexes, we used the SAS value instead of SAFv. In this article, we also found the inverse relationship between the SAS and R. The new suggestion demonstrated that Tc-complex with the SAS value between 0.090 and 0.91 was highly retained in the brain. Therefore, based on the average bond length and van der Waals radius, we designed a new type of brain perfusion imaging agent-99mTc-MPBDA. Its SAS value was calculated to be 0.914. Correspondingly, its R value was fallen in the range of 0.80–0.90. This derivation is verified by our experiments. 99mTc-MPBDA (N-(2-mercapto-propyl)-1,2-benzenediamine) was prepared in high yield (>95%) by stannous reduction of 99mTcO4- using NS2 ligand of MPBDA and cyclodextrin. In mice model, 99mTc-MPBDA showed a rapid blood clearance and a high initial brain uptake of 2.49 ± 0.46% ID/organ(6.79 ± 1.00% ID/g organ) at 2 min postinjection. Moreover, it presented a long retention time in the brain. The ratio of brain/blood was 2.44 at 1 hr after injection. SPECT studies in monkey also demonstrated that 99mTc-MPBDA was a potential cerebral perfusion imaging agent.
Abstract ID: 243
J. Balatoni L. Agayeva M. Doubrovin S. Soghomonian T. Miyagawa A. Shavrin R. Finn J. Gelovani Tjuvajev R. Blasberg
Neurology and Radiology, Memorial Sloan-Kettering Cancer Center
Chemistry and Probes
The 124I-labeled pyrimidine nucleoside FIAU has been shown to be a better probe (more sensitive and greater dynamic range) than the 18F-labeled acycloguanosine probes (e.g., FHBG and FHPG) for imaging HSV1-tk reporter gene expression. Since 18F is a better radiolabel than 124I for repetitive PET imaging studies over short time intervals in the same subject, we synthesized and tested an 18F-labeled analog of FIAU, [18F]FFEAU. The results of our initial in vitro uptake and in vivo imaging studies are presented and compared to FIAU. Paired [18F]FFEAU, [14C]FIAU, [3H]TdR uptake studies in HSV1-tk transduced (RG2TK+) and nontransduced (RG2) cells were performed. The FFEAU and FIAU uptake (clearance) constants in RG2TK+ cells were similar: 0.285 ± 0.035 and 0.296 ± 0.041 ml/min/g, respectively. In contrast, FFEAU uptake was significantly lower in wild-type RG2 cells compared to FIAU: 0.0005 ± 0.0003 and 0.042 ± 0.009 ml/min/g, respectively.
Abstract ID: 244
T. M. Corneillie P. A. Whetstone A. J. Fisher C. F. Meares
Chemistry, University of California, Davis
Chemistry and Probes
Attachment of a probe receptor molecule to a tissue of interest, followed by binding of the probe, provides a general approach to targeted imaging with very high target/nontarget discrimination. This is generally accomplished by conjugation of the probe receptor to a targeting molecule such as an antitumor antibody. We have found a probe receptor with the potential for wide applicability: an unusual antibody that binds DOTA complexes of all the rare-earth ions with comparable affinities. The photon emissions from such probes are suitable for PET and other scintigraphic techniques, or for visible or near-IR imaging; their paramagnetic properties may be useful for magnetic resonance imaging; and their particle emissions are useful for accompanying therapy. We describe the X-ray crystal structures of two complexes of the antibody with probe molecules: derivatives of yttrium(III)-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (Y-DOTA), and gadolinium-DOTA. Structural properties and relative binding affinities between the various trivalent rare earths will be presented, in addition to preliminary results describing how the antibody may be useful for MR and luminescence imaging.
Abstract ID: 245
T. Haradahira T. Suhara T. Okauchi J. Maeda, et al.
Medical Imaging, National Institute of Radiological Sciences
Chemistry and Probes
In vivo molecular imaging of N-methyl-
Abstract ID: 246
H. Alfke M. Béhé J. T. Heverhagen E. Wagner, et al.
Department of Radiology, Philipps University Marburg
Chemistry and Probes
Abstract ID: 247
B. McBride H. Karacay H. J. Hansen K. Chang E. Rossi R. M. Sharkey G. L. Griffiths D. M. Goldenberg
Research, Immunomedics
Chemistry and Probes
The delivery of substituted bis-(hapten)-containing peptides to tumors pretargeted with bispecific antibodies (bsAb) containing anti-CEA Fab’ chemically linked to an anti-indium DTPA, or to an antihistamine–succinyl–glycine (HSG) Fab', has been investigated. Bispecific diabodies reactive with both CEA and HSG have also been produced by recombinant DNA methods and evaluated for their efficacy as pretargeting agents. Mice bearing the human colonic tumor xenograft, GW-39, were injected with 1.5 × 10−10 mol of the bispecific antibody and were allowed to clear for an appropriate time (8 hr to 7 days, depending on the size of the bsAb used, which ranged from 50 to 200 kDa) prior to the injection of 1.5 × 10−11 mol of the peptide. The following peptides were examined in this model and typical tumor localization results obtained for the peptides with chemically linked bsAb (Fab’ × Fab', 100 kDa) are shown below. The bsAb was injected 24 hr prior to the peptide injection and the animals were sacrificed 3 hr after the injection of the peptide. Tc-99m IMP 192 Ac-K(In-DTPA)-Y-K(In-DTPA)-K(Tc-99m Tscg-C)-NH2 12.6% ID/g tumor/blood (T/B) 7.3; Re-188 IMP 192 Ac-K(In-DTPA)-Y-K(In-DTPA)-K(Re-188 Tscg-C)-NH2 16.9% ID/g, T/B 5.6; In-111 IMP 241 DOTA-F-K(HSG)-Yd-K(HSG)-NH2 11.3% ID/g, 8.1 T/B; Ga-67 IMP 241 DOTA-F-K(HSG)-Yd-K(HSG)-NH2 8.3% ID/g, 8.5 T/B; Tc-99m IMP 245 In-DOTA-F-K(HSG)-Yd-K(HSG)-K(Tscg-C)-NH2 14.2% ID/g, 6.3 T/B. Similar targeting results were obtained with bispecific diabodies when In-111-IMP 241 was injected 8 hr following the bispecific diabodies. This system of delivering a bispecific antibody followed by the administration of a bivalent hapten has proven to be highly versatile, allowing the site-specific delivery of many isotopes useful for molecular imaging.
Abstract ID: 248
Anthony W. Czarnik
R&D, Sensors for Medicine and Science
Chemistry and Probes
We are all familiar with pH indicators; as the [H+] increases, the color changes. Such an indicator is an example of a “chemosensor“—a molecule that transduces the presence of energy or matter into an observable signal. Like catalysts, chemosensors are not destroyed in the process of functioning and therefore provide continuous readout based on reversible binding to the analyte. In this talk, I will survey the relatively new field of “Fluorescent Chemosensors” and will highlight our company's research in the real-time measurement of subcutaneous glucose.
Abstract ID: 249
Darryl J. Bornhop Charles Manning Tim Goebel Rex Moats Reid Thompson Brent Bell
Massoud Motamedi, Chemistry and Biochemistry, Texas Tech University
Chemistry and Probes
In this article, we will describe a relatively new class of molecular imaging agents. It will be shown that these agents have bright luminescence, will provide MRI contrast, have a long emission lifetime for detection in the zero noise regime, can be used as a molecular-scale temperature or pH sensor, and can be tuned structurally for specific and sensitive molecular recognition. These new and unique molecular imaging agents are complexes based on polyazamacrocyclic lanthanide chelates and have been found to be nontoxic and potentially useful for in vitro diagnostics and in vivo disease detection. Furthermore, the lanthanide chelates described should be useful in bimodal imaging and therapy tracking. Capitalizing on the observations that human glial tumors display a high density of peripheral benzodiazepine receptor (PBR) binding sites, it will be shown that when the PBR ligand PK-11195 is conjugated to our lanthanide chelate, the compound (Ln-PK-11195) will act as a molecular signaling agent of PBR. Since PBR is up-regulated by human glioma cells making up glioblastoma, it has been possible to selectively label this disease ex vivo. The potential of studying CNS disease with this new MI agent will be introduced, as will the potential to the further study mitochondrial function and its relationship to cancer cell proliferation. We will also demonstrate the potential of using Ln-PK-11195 as a bimodal molecular imaging agent allowing anatomic scale in vivo brain cancer registration by MRI and subsequent cellular level characterization by fluorescence. The prospect of using lanthanide chelate MIs as dual imaging agents and for simultaneously monitoring and delivering therapeutic doses will be discussed.
Abstract ID: 250
P. Dubey H. Su N. Adonai S. Du, et. al.
Microbiology and Immunology, UCLA
Immunology
We assessed the use of positron emission tomography as a tool to repeatedly and noninvasively image the migration and localization of immune T lymphocytes to the tumor site. T cell localization is generally monitored by measurement of tumor volume, macroscopic tumor progression or regression, or detection of cytolytic activity in vitro. Kinetic measurements require the sacrifice of multiple experimental animals. We used a virally induced murine rhabdomyosarcoma cancer model to optimize methods for marking tumors and tumor-specific CTL with PET reporter genes as well as the technical aspects of imaging the tumors and T cells. Immunocompetent mice are challenged with the MSV/MLV virus complex. The challenge is rejected by a strong CTL response. Immune splenic T cells are isolated, retrovirally infected with the sr39TK PET reporter gene then adoptively transferred into immunodeficient tumor-bearing mice. Beginning at 3 days and up to 13 days after T cell transfer, animals are injected with the TK substrate, 18F-FHBG, and imaged in a microPET scanner. A strong 18F-FHBG signal is detected at the antigen-positive tumor, but not an antigen-negative control tumor. Naïve T cells do not home to the tumor, indicating that preimmunization is required. The same animals are imaged multiple times. Immunohistochemistry of tumor sections using anti-CD3 antibody showed more T cells in the antigen-positive tumor, corroborating the microPET data. This methodology will be useful in clinical practice for repeated monitoring of the patient to detect localization of transferred T cells to the tumor. Positron emission tomography is a sensitive method that can be used to track the migration and localization of antigen-specific T cells.
Abstract ID: 251
M. Doubrovin E. Doubrovina A. Ivanova V. Ponomarev L. Ageyeva J. Balatoni T. Beresten R. Finn S. Larson M. Sadelain R. Blasberg R O'Reilly J. Gelovani Tjuvajev
Radiology and Neurology, Memorial Sloan Kettering Cancer Center
Immunology
Adoptive cell therapy requires in vivo monitoring of the fate of transfused cells, which cannot be done using the ex vivo radiolabeling of the cells. A well-established hsv1-tk/gfp fusion (TKGFP) reporter gene was used to image the cytotoxic T-cell trafficking in vivo. Anti-EBV HLA-A0201 restricted cytotoxic T-cells were retrovirally transfected yielding 17% TKGFP-positive cells (TKGFP-CTL), which were expanded after FACS. A new multi-tumor mouse model containing four human lymphoid tumors varying in HLA-type and EBV antigen presentation was established. T-cells were administered at a dose of 5 × 107 cells per mouse on Day 21 after tumor inoculation. PET imaging of TKGFP-CTL with [124I]FIAU was started at Day 1 and repeated weekly for 3 weeks postinfusion of “tracer” T-cells. Fluorescent microscopy of cryosections of different tumors and lymphoid organs was performed to obtain a morphological confirmation of TKGFP-CTL localization (by GFP-fluorescence microscopy) in target tissues. The initial splenic accumulation (Day 1) was followed by redistribution of TKFGP-CTL to the autologous and HLA-A0201 allele matched, EBV-positive tumors. No specific [124I]FIAU accumulation was observed in the HLA-A0201 mismatched/EBV-positive and HLA-A0201 matched/EBV-negative tumors. A strong correlation was observed between the magnitude of cytotoxic TKGFP-CTL targetings of the autologous and HLA-A0201 matched EBV expressing tumors (% dose/g [124I]FIAU accumulation) and the efficacy of eradication of these tumors. Administration of [124I]FIAU did not suppress the specific cytotoxicity of TKGFP-CTL neither in vitro nor in vivo. PET imaging could significantly aid in the development, optimization, and clinical implementation of different adoptive T-cell therapies by defining the temporal and spatial dynamics of migration and by targeting of the adoptively transferred T-cells.
Abstract ID: 252
M. Doubrovin V. Ponomarev R. Schiro A. Ivanova E. Vider M. Van den Brink J. Gelovani Tjuvajev
Radiology and Neurology, Memorial Sloan Kettering Cancer Center
Immunology
GVHD is a common complication of allogenic BMT. Introduction of noninvasive imaging techniques in the studies of GVHD may considerably improve the understanding of the mechanisms of this severe complication by defining the temporal and spatial dynamics of migratory patterns of T-cells contributing to GVHD. When applied in the clinical setting, noninvasive imaging could provide the means for monitoring the development and treatment of GVHD. We used a well-established mouse model of GVHD in B6 recipients. T-cells were separated from the allogeneic donor bone marrow and transfected with a retrovirus, bearing multiple reporter genes: (a) hsv1-tk/gfp fusion (TKGFP), and (b) luciferase (Luc) under control of retroviral LTR and CMV, respectively. 1–2 × 106 cells per mouse of mixed T-lymphocyte population, containing 5% transfected “tracer” cells (T-LTG), were infused to the donor animals together with the bone marrow graft. Bioluminescence imaging was performed 10 min postinjection of
Abstract ID: 253
A. Moore J. Grimm L. Josephson R. Weissleder, et al.
Radiology, Massachusetts General Hospital
Immunology
A chronic mononuclear cell infiltration of the pancreatic islets (insulitis) represents the first pathological hallmark of Type 1 diabetes, and precedes the clinical manifestation of the disease for many years. The studies on pancreatic tissues are limited to autopsy, and non-invasive methods currently do not exist. We introduce a new superparamagnetic probe allowing for highly efficient labeling of diabetogenic CD8+ T-cells. We synthesized a superparamagnetic probe bearing an antigenic peptide (NRP-V7) that is recognized by a large fraction of diabetogenic CD8+ T lymphocytes in the context of H-2Kd Class I molecules in the NOD mouse. Such complexes have biotinylated C-terminal sequence of H-2Kd molecule, which we used for multimerization with CLIO-avidin. This probe is therefore exclusively recognized by a large fraction of diabetogenic CD8+ T lymphocytes. In this study, we first synthesized and tested CLIO-avidin, and then used it for coupling with biotinylated NRP-V7/H-2Kd complex. The resulting probe (CLIO-NRP-V7) specifically labels CD8+ T cells from NOD mice expressing a transgenic, highly diabetogenic, NRP-reactive T-cell receptor but not T-cells from nontransgenic NOD mice. The probe proved to be nontoxic. As expected, an activation of the cells upon labeling was detectable, but not significant, if compared to unlabeled cells. Studies are currently performed to use the CLIO-NRP-V7 probe for MR imaging of insulitis in an adoptive transfer mode, transferring CLIO-NRP-V7 labeled CD8+ cells from transgenic diabetic NOD mice into healthy NOD mice in vivo. The current study is expected to greatly aid and simplify pharmacological intervention of this disease.
Abstract ID: 254
S. M. Burns H. Zhao M. H. Bachmann J. Hardy D. K. Stevenson C. H. Contag
Pediatrics, Stanford
Immunology
Host defenses that protect the neonate from infection are varied, complex, and interactive, requiring that individual mechanisms be evaluated in the presence of the contextual influences of the intact living organism. The ability to noninvasively monitor and evaluate disease in whole living biological systems offers new opportunities for understanding disease process and developing effective therapeutics. The objective of this study was to reveal the innate immune mechanisms that are protective in adults but possibly lacking in neonates, and understand the maturation steps involving key molecules that are likely to mediate innate host responses. The age-related differences in disease progression were revealed using in vivo bioluminescent imaging (BLI), and these data were used to guide tissue sampling for microarray analyses to assess the differences in host response to infection. BLI revealed the age-related differences in Balb/c mice after oral inoculation with a bioluminescent engineered Salmonella typhimurium strain. Time points and tissues were selected based on BLI data for microarray analysis. These studies indicated several potential targets that may mediate the host response. Several potential targets have been previously selected as likely mediators of age-related susceptibility and these include Nramp1 (natural resistance associated macrophage protein), IFNg, iNOS (inducible nitric oxide synthase), Granzyme B, and HO-1 (heme oxygenase 1). We have created reporter mice that are designed to reveal expression profiles for selected genes. Patterns of expression will be assessed over the full disease course using whole body imaging of these transgenic reporter mice. The BLI data from these reporter mice will serve to validate the microarray analyses and indicate other time points and tissues that may be critical to the innate immune response to infection.
Abstract ID: 255
G. D. Luker J. P. Bardil J. L. Prior C. M. Pica D. Piwnica-Worms D. A. Leib
Radiology, Washington University School of Medicine
Infection
Herpes simplex virus-1 (HSV-1) is a widespread human pathogen that infects approximately 80% of all persons by adulthood and can cause morbidity and some mortality from diseases such as blinding keratitis and encephalitis. Conventional methods to study the pathogenesis of HSV-1 in mouse models typically require sacrifice of infected animals to determine the full extent of viral spread and replication. To develop a noninvasive method for investigating HSV-1 in living mice, we used a recombinant strain KOS virus that expresses firefly and Renilla luciferases and monitored infection with bioluminescence imaging. Viral infection in mouse foot pads, peritoneal cavity, brain, and eyes could be detected by bioluminescence imaging of firefly luciferase. Activity of Renilla luciferase could be imaged after direct administration of substrate to infected eyes but not following intravenous delivery of substrate. In vivo measurements of bioluminescence from firefly luciferase correlated directly with input titers of recombinant virus used for ocular infection of mice. Treatment of infected mice with valacyclovir, an effective inhibitor of HSV-1 replication, produced dose-dependent decreases in firefly luciferase that correlated with changes in titers of virus measured in tear films. These data demonstrate that bioluminescence imaging can be used for noninvasive, real-time monitoring of HSV-1 infection and therapy in living mice.
Abstract ID: 256
D. A. Benaron I. H. Parachikov Y. T. Talmi
Spectros
Instrumentation
We have previously developed at Stanford optical imaging systems for noninvasive tomographic human brain structural and functional imaging, and have codeveloped the original method and system for planar luciferase imaging in vivo in animals. We now set out to develop a flexible system for imaging the distribution and localization of targeted fluorescent agents in humans under room light surgical operating room conditions. A filtered, gated, intensified CCD system with a low-f custom lens was constructed and characterized. Images of model systems for lymphatic localization and margin detection were collected. We imaged lymph node models down to 5 cm depth with 1-sec image updates, and detected residual populations of 1000 cells at tumor resection margins. The system will be made available for general research use in early 2003. We conclude that this system will function in the operating room environment, and may have applications in bone scans and postsurgical follow-up as well.
Abstract ID: 257
Simon R. Cherry
Biomedical Engineering, UC Davis
Instrumentation
Positron emission tomography (PET) is one of the modalities being developed for a broad range of molecular imaging applications. High-resolution PET systems for in vivo imaging animal imaging have been developed by a number of groups and more than 30 institutions now have access to this technology. Significant progress has been made with an order of magnitude improvement in volumetric resolution of these devices over the past 5 years. Systems with a resolution approaching 1 mm are now a reality. In this talk, the latest developments in small animal PET instrumentation will be reviewed, and the challenges and opportunities that lie ahead will be discussed. The prospect of multimodality systems in which PET is integrated with X-ray computed tomography and magnetic resonance imaging will also be presented.
Abstract ID: 258
H. H. Barrett Z. Liu L. Furenlid G. A. Kastis, et al.
Radiology Department and Optical Sciences Center, University of Arizona
Instrumentation
The Center for Gamma-Ray Imaging (CGRI) and the Southwest Animal Imaging Resource (SWAIR) at the University of Arizona are developing new SPECT imaging systems for small animals. Two distinct detector technologies are employed: modular scintillator cameras and semiconductor detector arrays. Image formation uses either ultrahigh-resolution parallel-hole collimators, submillimeter pinholes, or the new approach of synthetic collimators. Two different animal SPECT systems have been completed and a third is under construction. The two completed systems are FASTSPECT II, which uses 16 modular scintillation cameras, and a dual-modality CT/SPECT system using a 64 × 64 cadmium zinc telluride (CZT) array. Under construction is SemiSPECT, which will initially use eight of the CZT arrays and be upgradeable to 16. Spatial resolution is about 1 mm in all three dimensions with FASTSPECT II and is expected to be about 0.5 mm for SemiSPECT. Applications currently being investigated include studies of myocardial infarction in rats, multidrug resistance in human xenograft breast tumors in mice, apoptosis in tumors following chemotherapy and gene transfer studies in mice. This talk will present a survey of the technologies of the new detectors and the SPECT instruments that use them, as well as representative results from the ongoing animal studies.
Abstract ID: 259
J. R. Stickel Y. C. Tai A. F. Chatziioannou Y. F. Yang, et al.
Biomedical Engineering, University of California at Davis
Instrumentation
MicroPET II is a second-generation PET scanner designed for in vivo molecular imaging applications in small animals. The system is composed of 90 high-resolution gamma-ray detector modules arranged in three rings, with 30 modules per ring. Each detector module contains a 14 × 14 array of 0.975 × 0.975 × 12.5 mm3 lutetium oxyorthosilicate (LSO) scintillator crystals coupled to a Hamamatsu H7546 multichannel photomultiplier tube via a 5-cm-long optical fiber bundle. The bore size of microPET II is 14.3 cm and the axial field of view is 4.8 cm. The first data sets from this new system provide preliminary estimates of reconstructed image resolution of 1.2 mm at the center of the field of view using a Fourier rebinning algorithm followed by 2-D filtered backprojection. At a 4-cm radial offset, the resolution degrades to 1.7 mm due to depth of interaction effects. The peak sensitivity of the system was measured as 2.5% in the center of FOV. High-quality images from phantoms and from mice have further demonstrated the capabilities of this new PET scanner. These initial results demonstrate a 3.4-fold improvement in volumetric resolution and a five-fold improvement in sensitivity compared with the initial prototype microPET developed in our laboratory in 1996. It is therefore anticipated that microPET II will significantly expand the range of possible applications for PET in molecular imaging studies, particularly in the mouse where spatial resolution is often a limiting factor in PET.
Abstract ID: 260
Y. Chen L. Zhou C. Mu X. Intes, et al.
Biophysics/Biochemistry, University of Pennsylvania
Instrumentation
Recent developments in receptor-specific targeting fluorescent contrast agents can give tumor/tissue contrasts in the excess of 10, and are capable of selectively labeling various precancer/cancer signatures to enhance the sensitivity and specificity of cancer detection [ref: Weissleder et al., Achilefu et al.]. In order to meet the need of portable, handheld breast and lymph node cancer detection, we have developed a novel imaging system for fast and accurate localization of fluorescent contrast agent labeled tumor embedded several centimeters inside the highly scattering medium mimicking breast tissue using the phased cancellation configuration (0° and 180° source-pair) with a goniometric probe. The instrumentation performance on the phantom test can detect 3 mm diameter sphere filled with 1 nM fluorescent dye, Indocyanine Green (ICG), and 3 cm deep inside the scattering medium with similar optical properties as human breast tissue with the localization error of 1 mm. MMTV mammary tumor mouse model immersed in appropriate scattering/absorbing medium is used for animal test. Intratumor injection of ICG demonstrates the localization of the tumor (5 mm in diameter) submerged 3 cm deep inside the highly scattering medium with 2 mm position error. Results with somatostatin subtype 2 (sst2) receptor-positive fluorescent contrast agent, cytate, on the AR42J tumor-bearing nude mouse are also presented with 3 mm localization error. This system is capable of in vivo localization of mouse tumor labeled with fluorescent contrast agents about 3.5 cm deep. The accuracy of the localization suggests that this system would be helpful to guide the clinical fine-needle biopsy for early detection of breast cancer.
Abstract ID: 261
D. Cody, PhD E. Johnson, BS
Diagnostic Imaging Physics, UTMD Anderson Cancer Center
Instrumentation
A micro-CT scanner manufactured by Enhanced Visions Systems (EVS, Toronto, CA) was installed at our institution in October 2001. This instrument utilizes cone-beam CT geometry, a micro-focus X-ray tube, and a CCD camera. The EVS Hybrid system has three modes of operation: 25, 45, and 90 μm (isotropic voxel size). It was selected specifically to accommodate live mice and rats during CT scanning. This presentation will describe our initial experience with the scanner, and will include a description of the advantages and disadvantages inherent to this system design. Several bone tissue studies have been successfully completed to date. This scanner is specifically calibrated to bone mineral density. Specimens such as the hind legs of mice and rats, and mice skulls, have been evaluated with this instrument. Results have been obtained that were unavailable through more conventional methods. We have had some success applying this scanner to mice in vivo. Gas anesthesia was used for sedation, and the thorax was examined using the 90 μm mode. Although no attempt was made to regulate breathing or to trigger the scanner based on respiration, the resulting images were not affected by respiratory motion artifact. Intravenous contrast materials have been used to selectively enhance vasculature, but the relatively long scan time (20 min) makes this goal a challenge. The development of long-lasting contrast materials is underway. Sample images and quantitative analysis results will be presented, which reflect typical outcomes achieved by this micro-CT scanner.
Abstract ID: 262
K. Iwata L. R. MacDonald J. Li S. P. Williams, et al.
Radiology, University of California, San Francisco
Instrumentation
Dual-isotope SPECT imaging is beneficial for in vivo studies that require two biological processes to be monitored. While dual-isotope studies in humans are compromised by down scatter from the higher energy gamma rays into the lower energy window, imaging of small animals is facilitated by the small size of a mouse that reduces scatter contamination. In vivo imaging studies were performed with a radionuclide animal imager (A-SPECT, Gamma Medica) integrated with a microCT system. Radionuclide images were acquired with a pixellated NaI(Tl) scintillator coupled to position sensitive photomultiplier tubes. The microCT system used a 5 × 5-cm2 CMOS detector coupled to a gadolinium oxysulfide phosphor having 50-μm pixels in a 1024 × 1024 format, and an X-ray tube rated at 50 kV and 1.0 mA. Coregistered tomograms were acquired by rotating the animal vertically without removing it from the system. Since the geometry of these two modalities is known, the acquired tomograms were coregistered by a predetermined spatial transformation, independent of the imaged object. An I-125 labeled radiopharmaceutical was infused through a catheter into a live animal 3 days prior to the imaging, while Tc-99m MDP was injected 6 hr prior to the imaging. The animal was scanned under anesthesia for 64 min with simultaneous dual-isotope SPECT and for 5 min with CT. The resulting I-125 and Tc-99m SPECT images were clearly separated without any visible down scatter. The coregistered CT images improved anatomical localization of radionuclide uptake. (Authors acknowledge support from NIH NCI#1R01CA91771, NIH NCRR#1R43RR16393, and DOE#DE-FG-0399ER82854.)
Abstract ID: 263
D. Schellingerhout R. Accorsi D. J. Wagenaar J. Idoine R. C. Lanza R. Weissleder
Radiology, Massachusetts General Hospital
Instrumentation
We introduce and demonstrate coded aperture (CA) nuclear scintigraphy for small animals, a new imaging technique well suited for imaging research in this field. CA imaging uses multiple pinholes in a patterned mask mounted on a conventional gamma camera (MCAM, Siemens). System performance was assessed using point sources and phantoms, while multiple animal experiments were performed to demonstrate the usefulness of the imaging system in vivo, utilizing commonly used radiopharmaceuticals. The sensitivity of the CA system was 15.2 cps/Bq or 9400 cpm/mCi of 99mTc, compared to 1.6 cps/Bq or 990 cpm/mCi for a conventional collimator system, with a system resolution of 1.7 mm, as compared to 4.6 mm for the conventional imaging system (high-sensitivity low-energy collimator). Animal imaging demonstrated superior resolution and image quality compared to conventional collimator images in multiple cases. We conclude that: (A) CA imaging is a useful new nuclear imaging technique for small animal imaging, with greatly improved signal-to-noise ratios compared to traditional imaging methods. This advantage can be traded to achieve higher resolution, decreased dose, or reduced imaging time. (B) CA imaging works best for images where activity is highly concentrated, such as bone scans. The signal-to-noise benefit is less apparent where activity is spread diffusely, and a low count outline may be better demonstrated using conventional collimator imaging. Thus, CA imaging should be viewed as a complementary technique, able to supplement and augment traditional nuclear imaging methods. (C) CA can be readily adapted to existing gamma cameras, making their implementation a relatively inexpensive retrofit to most systems.
Abstract ID: 264
D. Piston F. Carroll M. Rizzo J. Rocheleau
Molecular Physiology and Biophysics, Vanderbilt University
Instrumentation
The convergence of newly developed instrumentation and optical probes allows us to examine quantitatively dynamic processes within ever more complicated biological systems. These new tools are affecting how almost all biological optical microscopy experiments are done. Studies of living cells, for instance, have dramatically changed since the introduction of the Green Fluorescent Protein (GFP). By using various quantitative fluorescence imaging methods of multicolored GFPs fused to the glucose sensing enzyme glucokinase (GK), we discovered that both the location and the activity of beta cell GK are acutely regulated by glucose. As molecular imaging methods have been perfected in single cells, they have also been rapidly applied to noninvasive imaging of intact tissues and organisms. We have used two-photon excitation microscopy to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. Further, we can use the intrinsic optical resolution to delineate signals from the cytoplasm and mitochondria, and show that the metabolic response of these two compartments can be differentially stimulated. Two-photon excitation coupled with GFP-labeled transgenes allows for subcellular resolution deep into intact animals. Finally, we have built a stand-alone tabletop X-ray source designed specifically for monochromatic X-ray imaging of animals and human clinical applications. This monochromatic CT offers the potential to be a competitive alternative to MRI, PET, and ultrasound for many noninvasive in vivo imaging applications. Monochromaticity allows the choice of X-ray wavelengths where the largest signal contrast is obtained. For example, mammograms performed with this source will yield the same information with only 2% of the total dose to the patient. Because of the tunability of the instrument, molecular X-ray absorbers can also be used to specifically label sites within the body.
Abstract ID: 265
T. Schaeffter G. Mens S. Weiss H. Eggers J. J. van Vaals
Philips, Philips Research Hamburg
Instrumentation
There is an increasing need for local delivery of high-risk pharmaceutical treatments in order to avoid side effects. Real-time interactive MRI is an attractive tool to guide such local injections of drugs or cells. In comparison with other imaging modalities, MRI offers a superior soft tissue contrast and allows a wide range of contrast manipulations. For guidance of interventional procedures, advanced capabilities are required such as real-time imaging, interactive scan control, and devices tracking (e.g., needles, catheters). We have extended a clinical MR-scanner (Philips Intera) to perform interactive MR fluoroscopy with simultaneous tracking of invasive devices. An interactive user interface allows the change of several MR parameters during continuous scanning including the slice position and orientation, contrast manipulations and tracking of the imaging slice to the position of the invasive device. A sliding window reconstruction technique allows real-time reconstruction and viewing of up to 20 images/sec for all kinds of MR sequences (Cartesian, Radial, Spiral MRI). For device tracking, a micro-coil has been integrated on the tip of a 5F-catheter (Cordis). The received MR-signal of the micro-coil is used to track the imaging slice to the position of the catheter tip. Catheter tracking can be used to guide an injection to the region of interest that is visualized simultaneously by real-time imaging. Interactive contrast manipulation can be used to visualize injections of drugs or cells labeled with contrast agents. MRI can furthermore be used after the injection to monitor the pharmacokinetics and the effect of the treatment.
Abstract ID: 266
E. E. Graves J. Ripoll R. Weissleder V. Ntziachristos
Radiology, CMIR/MGH
Instrumentation
We have constructed a novel parallel-plate imaging system capable of acquiring fluorescence reflectance imaging (FRI) and fluorescence-mediated tomography (FMT) data sets from a living mouse in a single examination. The subject is placed between a glass window and a movable compression plate containing a grid of 32 fiber-optic laser sources. A CCD camera and a bandpass filter are used to image the glass window at a wavelength band of interest. FMT data are acquired in a transillumination geometry by imaging the glass window with each of the sources active sequentially. FRI is performed through the imaging window while expanding a laser source at the appropriate wavelength to illuminate the subject. This allows the acquisition of a conventional surface-weighted FRI image, which can be used to direct an FMT measurement to resolve fluorochrome concentrations in three dimensions. The signal-to-noise of the instrument has been measured to be 35 dB for a 0.08 cm3 volume of 2 μM Cy 5.5 dye, suggesting an approximate in vivo threshold of detection of 50 nM for a similarly sized object. Measurements of 2 and 5 mm tubes titrated with concentrations of Cy 5.5 from 100 to 1000 nM demonstrate the linearity and accuracy of the reconstructed FMT concentrations. This technology allows absolute determination of fluorochrome concentrations in turbid media, and will be a valuable tool for the quantitative study of murine disease models when used in conjunction with fluorescent molecular reporters.
Abstract ID: 267
C. P. Lin J. M. Runnels X. Wei A. Bogdanov
Wellman Laboratories of Photomedicine, Massachusetts General Hospital
Instrumentation
Leukocyte–endothelial interaction is an essential step in the regulation of immune function. Leukocyte rolling, adhesion, and migration in tissue can be visualized by the powerful method of intravital microscopy. A shortcoming of the method, however, is the inability to identify specific leukocyte populations involved in the immune response, since commonly used fluorescent probes (such as aridine orange or rhodamine 6G) are taken up nonspecifically by all leukocytes. We have constructed a scanning laser confocal microscope that is capable of real-time image acquisition (30 frames per second) and allows simultaneous one- and two-photon fluorescence detection. The latter modality uses a femtosecond laser source to generate nonlinear excitation at a specific depth in tissue (up to ~200 μm) and produces a confocal-like optical section. We have successfully performed in vivo immunofluorescence microscopy through the intact skin of live mice, by systemic administration of fluorescent antibodies directed against specific cell surface markers (e.g., CD4, CD45, and CD31 (PECAM-1)). Circulating leukocytes and vascular endothelial cells can be imaged after a single injection of the antibodies at doses as low as 0.5 mg/kg body weight. Labeled cells are visible for 4 days after a single injection.
Abstract ID: 268
Akira Nakayama Federica del Monte Roger J. Hajjar John V. Frangioni
Hematology/Oncology, Beth Israel Deaconess Medical Center
Instrumentation
For centuries, the human eye and visible light have been the tools most used by surgeons to image the human body. We have exploited the invisibility of near-infrared (NIR) fluorescent light to permit the surgeon to “see,” by means of an exogenously administered NIR fluorophore, any additional function within the surgical field. We describe a portable NIR fluorescence intraoperative imaging system, and demonstrate the use of this system during surgery in small and large animals. The system is composed of two wavelength-isolated excitation sources, one generating 400–700 nm “white” light, and the other simultaneously generating 5 mW/cm2 of 725–775 nm light over a 15-cm diameter field of view. Photon collection is achieved with custom-designed optics that maintains separation of the white light and NIR fluorescence (790 nm) channels. After computer-controlled camera acquisition, anatomic (white light) and functional (NIR fluorescent light) images can be displayed separately or merged, depending on the application. The entire apparatus is suspended on an articulated arm over the surgical field, thus permitting noninvasive and nonintrusive imaging. As proof of principle, two NIR fluorophores, one a vascular agent, and one a perfusion agent, have been used in rat and pig models of cardiovascular disease to optimize system components. Future studies are aimed at oncologic surgery, where such a system could provide real-time assessment of surgical margins, detect occult metastases within the surgical field, and facilitate image-guided gene therapy.
Abstract ID: 269
W. Warren P. Tian D. Keusters
Chemistry/Radiology, Princeton/Penn
Instrumentation
Two-photon excitation with fluorescence detection has become an important technique in modern molecular imaging. As compared with conventional (one-photon) imaging, it has the advantage of better spatial localization and reduced scattering artifacts due to the intensity-squared dependence of the absorption. However, many biologically important molecules (such as NAD+) generate little fluorescence; in addition, fluorescence reabsorption contributes to the depth limitations of the technique. We have recently demonstrated that it is possible to measure two-photon absorption by observing the extremely small light loss at the fundamental (one-photon) wavelength, even with very modest peak powers, and even in the presence of strong scattering. Our techniques use modern developments in femtosecond laser pulse shaping to produce specific spectral signatures on the exciting optical field; these signatures permit sensitive detection of two-photon effects, even in the presence of substantial residual scattered light at the fundamental. We have demonstrated that we can measure accurate two-photon absorption cross-sections, using laser pulses with only 1/100,000 of the energy used in previously reported methods. At the shot noise limit for our current approach, we should be able to resolve two-photon absorptions as small as 1 part in 107 of the transmitted light, and this very low value enables the use of very weak pulses (picojoule pulse energies).
Abstract ID: 270
K. Hynynen N. McDannold L. Josephson N. Vykhodtseva, et al.
Radiology, Brigham and Women's Hospital
Instrumentation
The Blood–Brain Barrier (BBB) is a major limitation for utilizing large molecular markers for brain imaging. Recently, it was demonstrated that ultrasound can be focused through human skulls and that focused ultrasound beams can induce focal opening of the BBB. In this study, we have explored if large particles suitable for molecular imaging could pass through a BBB defect induced by ultrasound. The brains of New Zealand white rabbits were sonicated using a focused transducer moved within a clinical MR scanner (1.5 T, General Electric) by a positioning device. Four or six locations were sonicated with the focus aimed 10 mm deep in the brain at different peak acoustic power levels ranging from 0.2 to 4 W. The sonications were pulsed with a burst length of 100 msec and a repetition frequency of 1 Hz. The duration of the whole sonication was 20 sec. Approximately 10 sec prior to the start of the sonication, a bolus of ultrasound contrast agent (Optison, Mallinckrodt, St. Louis, MO) containing micro-bubbles was injected in the ear vein. Several minutes before or shortly after the sonications, the MR contrast agent MION (hydrodynamic radius 20 nm) was administered (5 mg/kg). Delivery of the agent through the BBB was confirmed by a signal intensity (SI) reduction at the ~1–2 mm diameter focal locations. This study demonstrates for the first time that MRI-guided focal BBB opening allows large particles to enter into the brain. This finding can have significant potential in molecular imaging of brain and delivery of therapeutic agents.
Abstract ID: 271
P. J. Gareau C. Heyn A. Alejski B. K. Rutt
Medical Biophysics, Robarts Research Institute
Instrumentation
Magnetic resonance microscopy (MRM) is conventionally performed at high field. Recently, we have developed new tools that enable low-field MRM. These include: (i) a custom-built, insertable, high-powered imaging gradient set, (ii) customized radio-frequency coils, and (iii) optimized pulse sequences. This approach has produced images with high spatial resolution, SNR and CNR, which have allowed us to visualize single iron loaded cells. THP-1 cells were loaded with SPIO nanoparticles (SHU 555A, Schering) and fluorescently labeled with a lipophilic carbocyanine dye DiI. Samples were prepared in one of two ways: (i) uniform gel suspensions (4% w/w gelatin) were prepared with a cellular concentration of 2000 cells/ml and placed in 5 mm NMR tubes, and (ii) a monolayer of cells sandwiched between two layers of 8% gelatin was prepared in optically transparent plastic wells to facilitate both fluorescence imaging and MRI. MRI of cells was performed on a 1.5-T GE CV/i MR with a custom-built gradient coil (inner diameter 24 cm, peak slew rate 1200 mT/m/sec, maximum gradient strength 300 mT/m) and customized radio-frequency coils. Three pulse sequences were optimized for cell imaging: a 2-D T2W FSE, a 3-D GRE, and a 3-D SSFP (FIESTA). FIESTA images (TR/TE 7.5/3.6 msec) acquired in 6 min with 100 mm isotropic resolution show regions of low signal intensity due to the presence of SPIO particles. Individual cells were not easily visualized with FSE or GRE images at equivalent spatial resolutions. Good correlation was attained between individual cells visualized using optical microscopy and patterns of low signal intensity in FIESTA images.
Abstract ID: 272
Matthew R. Palmer Jonathan B. Kruskal Robert E. Lenkinski
Radiology, Beth Israel Deaconess Medical Center
Instrumentation
An optical imaging system has been developed for in vivo imaging of small animals with near-infrared (NIR) fluorescent probes. The system is designed to acquire quantitative measurements of the shallow distributions of fluorescent compounds in animals during molecular imaging experiments. The basic configuration is a conventional epi-illumination setup, with a broadband illumination source followed by interchangeable excitation wavelength selection filters. The stage is a 10-cm2 move-able platform designed to support the animal and provide controlled movement during imaging. Emission light is optically filtered then collected by a high-resolution, high quantum efficiency, cooled CCD camera, which is attachedto acomputer. The computer acquires images in real time and supervises the sequencing and control of the experiment. A combination of multiple wavelength excitation and stage rotation is employed in order to uncouple depth and density to yield quantitative estimates of fluorophore concentration. The system has been employed in a number of in vivo applications including the whole body imaging of cell trafficking and the imaging of osteoblastic activity. This presentation will cover the system design, capabilities, and molecular imaging applications.
Abstract ID: 273
Susie Y. Huang Yung-Ya Lin
Chemistry and Biochemistry, University of California, Los Angeles
Instrumentation
Magnetic resonance (MR) stands out among existing imaging technologies for its high spatial resolution and its ability to probe a wide array of physiologic parameters. These characteristics of MR imaging make it ideally suited for molecular imaging to study such events as metabolic activity in tumors at the molecular level. Extracting such information from MR imaging requires that sufficient contrast be generated. Recent studies of spin dynamics in solutions reveal the existence of spin chaos under the strong magnetic fields used in high-resolution MR microscopy [1,2]. The striking phenomena observed originate from the nonlinearity triggered by the joint action of radiation damping and the distant dipolar field, both present in modern MR experiments. Radiation damping is a macroscopic reaction field, which is fed back to the spins through the free induction decay-induced current in the receiver coil. The dipolar field in liquids arises from the residual dipolar interaction, which has withstood the spatial and temporal averaging and is long-ranged in nature. The extreme sensitivity of the chaotic dynamics to its initial experimental conditions provides a fundamentally new mechanism for contrast enhancement. Such chaotic MR microscopy would generate molecular imaging sensitive to spatial variation of spin density, relaxation times, and motions and therefore allow the detection of low concentrations of target biomolecules. Further contrast enhancement can be achieved by hardware modification to electronically feedback the amplified radiation damping signals through the decoupling network of the spectrometer. Preliminary results will be demonstrated and compared to the conventional relaxation-weighted imaging.
Abstract ID: 274
T. Dixon D. Blezek P. Dhawale M. Harisinghani
Imaging Technologies/Visualization and Computer Vision, GE Global Research Center
Instrumentation
Abstract ID: 275
Barry Lai Zhonghou Cai Jorg Maser Dan Legnini Petr Ilinski Stefan Vogt Francesco DeCarlo
Advanced Photon Source, Argonne National Laboratory
Instrumentation
Two new X-ray imaging techniques with unique capabilities have been developed: (1) phase contrast imaging and (2) X-ray fluorescence microscopy. While traditional radiography relies on absorption contrast, it has now become possible to implement phase contrast imaging with partially coherent X-ray beams. The benefits are much enhanced contrast, improved resolution on the order of several microns, and the potential of reduced radiation dose. During imaging of a mouse, even soft tissues such as hairs and internal organs were readily observed together with hard tissues. Acquisition speed close to video frame rate can be achieved with a fast CCD, which may be useful for studying live mouse. The second technique, based on a scanning X-ray fluorescence microprobe, allows trace quantity of many elements to be imaged at the subcellular level. Benefits include: (1) it enables any element above silicon, including most metals, to be directly imaged without any staining or enhancing agent. In fact, this technique had been used for studying natural bacteria in a hydrated state. (2) For most transition and heavy metals, trace concentrations of <1 ppm are detectable, while more than 10 different elements can be mapped simultaneously. (3) The demonstrated spatial resolution is 200 nm. (4) It can also reveal the chemical states, such as Cr(VI) versus Cr(III), Pt(IV) versus Pt(II). This is particularly useful for imaging small metal complexes such as cisplatin, chromium complexes, metal-containing nonsteroidal anti-inflammatory drugs (e.g., copper-indomethacin), where any attachment of biomarkers to the complexes may lead to possible alternation of cellular metabolism. Initial applications of this technique included determining the intracellular biotransformation products of chromium carcinogens and metal-containing drugs on individual cells. The high elemental sensitivity of this technique allowed these studies to be performed at naturally occurring concentrations.
Abstract ID: 276
T. Wang M. Mandella C. Contag G. Kino
Pediatrics, Stanford University
Instrumentation
Conventional confocal microscopes employ high numerical aperture (NA) lenses, and their in vivo use is limited by short working distances. We have developed a novel confocal microscope that uses a dual-angle-axis architecture such that collection of fluorescence is off-axis, thus reducing noise from scattered light. This new design uses low NA lenses that facilitate miniaturization of the overall system and result in long working distances, features important for in vivo imaging. The tabletop prototype has two lenses (NA = 0.16) arranged with their optical axes crossed at 30° from the midline. Excitation light at 488 nm is delivered through an optical fiber coupled to the first objective, and the specimen is scanned on a three-axis translational stage. Fluorescence collected by the second objective is spatially filtered with a 3 μm core single-mode optical fiber, and is detected by a photomultiplier tube. Images are collected in 25 sec with a field of view of 200 μm and a working distance of 15 mm. With this prototype, the transverse and axial resolutions were 1.9 and 2.1 μm, respectively. With a laser power of 500 mW, fluorescence images collected from HEK cells expressing GFP in culture demonstrated a signal-to-noise ratio of 3 ± 1. No evidence of photobleaching was observed. Ex vivo images of sections of mouse brain and liver expressing GFP were collected and demonstrated an imaging depth of 50 μm. These results show that near-micron resolution fluorescence images can be collected with a novel dual-angle-axis confocal prototype that offers the advantage of miniaturization. This system will have applications for in vivo cellular and molecular imaging.
Abstract ID: 277
Richard Levenson
Biomedical Systems, CRI
Instrumentation
The ability to sense more than one fluorescent color at a time is at a premium because of the explosion of probes made available by the genetics revolution. Traditional approaches to multicolor imaging generally employ filter wheels, which lack spectral finesse and, in addition, can exhibit slow switching, mechanical complexity, image shift due to changes in the optical path, and vibration. True spectral imaging can generate precise optical spectra at every pixel. Furthermore, once complete optical spectra are known for a particular sample, an efficient subset of wavelengths can be identified for use in subsequent imaging. A convenient approach uses liquid crystal tunable filter devices that can transmit in a number of wavelength ranges (e.g., 400–720 or 850–1800 nm, with bandwidths as narrow as 5 nm). Recently, CRI has released a C-mount version in the visible range for imaging on standard microscopes. Hyperspectral software tools exist to analyze the resultant data, including simple spectral similarity mapping, principal component and independent component analysis, and automated clustering algorithms in n-dimensions. Spectral imaging has proved useful for multicolor FISH, and in particular, for resolving multiple species of GFP with overlapping emission spectra. It is helpful for the identification and elimination of interfering autofluorescence. The excellent match between LCTF technology and the new quantum-dot-based fluorescent labels is emphasized. Spectral imaging in brightfield can also be performed: It is possible to detect and quantitate the simultaneous presence of at least three overlapping chromogens (deposited as part of immunohistochemical or in situ hybridization procedures).
Abstract ID: 278
T. Mueggler M. Rausch D. Baumann M. Rudin
CT/Analytical and Imaging Sciences, Novartis Pharma
Neurobiology
APP23 transgenic mice overexpressing amyloid precursor protein (APP751) reproduce neuropathological changes associated with Alzheimer's disease. Functional magnetic resonance imaging (fMRI) was applied to characterize brain functionality in 24-month-old mice using a peripheral stimulation paradigm. Hindpaw stimulation led to CBV changes in the expected contralateral somatosensory cortical region. Maximal CBV increases of 11 ± 3%, 16 ± 4%, and 19 ± 3% of baseline values for current strengths of 0.5, 1, and 2 mA, respectively, have been observed in control animals. In-between stimulation CBV returned to levels of the order of 1–3% higher than baseline. In huAPP23 transgenic mice CBV did not depend on the current strength. Maximal CBV increases were 11 ± 2%, 11.5 ± 2%, and 8.3 ± 2% for electrical current strengths of 0.5, 1, and 2 mA, respectively. The reduced somatosensory response in APP23 mice can be explained by a compromised cerebrovascular reactivity as revealed by the reduced responsiveness to vasodilatory stimulation. In wild-type mice CBV increases of the order of 28 ± 2% have been measured 6 min after injection of acetazolamide followed by a slower increase until the end of the experiment. The corresponding response in huAPP23 showed a significantly smaller increase of 18 ± 4%. The compromised response to peripheral sensory input in huAPP23 parallels findings following GABAA inhibition using systemic administration of bicuculline. In these experiments, the CBV response was reduced by 50% in transgenic animals as compared to wild-type littermates. These studies illustrate the potential of fMRI for phenotyping of transgenic mice developed as models for neuropathologies.
Abstract ID: 279
Karel Svoboda
Cold Spring Harbor Laboratory, HHMI
Neurobiology
Do synapses turn over in the adult neocortex? Do synapses form to encode novel experience? We used two-photon laser scanning microscopy in mice expressing fluorescent proteins in a subset of cortical neurons to chronically (up to 3 weeks) image individual synapses. For ultrastructural analysis, previously imaged dendrites and synapses were reconstructed in 3-D using electron micrographs from serial sections. We find that a subset of synapses turns over rapidly in the adult brain and that the rate of synapse formation and elimination increases after novel sensory experience. The resulting experience-dependent changes in cortical connectivity occur sufficiently rapidly to account for the physiological remodeling of sensory maps.
Abstract ID: 280
Bradley T. Hyman, MD, PhD
Massachusetts General Hospital/Harvard Medical School, Neurology/Alzheimer Unit
Neurobiology
Alzheimer's disease is characterized by the development of extracellular amyloid plaques. Transgenic mouse models replicate many features of this pathophysiology. Recent advances in multiphoton microscopy provide the ability to image microscopic plaques, neurons and their processes, and functional information such as calcium levels or free radical generation in vivo essentially in real time. Longitudinal studies of the pathophysiology over days to months are possible. This new technology allows insight into the natural history of amyloid plaques, and provides a means for evaluation of therapeutic approaches.
Abstract ID: 281
F. Pain L. Besret L. Zimmer R. Mastrippolito L. Pinot W. Hassoun F. Vaufrey L. Valentin V. Leviel P. Hantraye P. Laniece
Interfaces Physique-Biologie, Institut de Physique Nucléaire
Neurobiology
Abstract ID: 282
J. W. M. Bulte T. Ben-Hur B. Miller R. Mizrachi-Kol, et al.
Radiology, Johns Hopkins University
Neurobiology
Transplantation of myelinating cells is a new experimental mode of therapy that has proven effective in myelinating the CNS in animal models of (hereditary) dysmyelination and focal demyelination. The immediate dispersion, migratory capacity, and long-term survival of transplanted cells are critical factors in determining whether there will be a beneficial clinical effect. We have transplanted magnetically labeled neural precursor cell spheres, prepared from newborn rat striatal stem cells, into rats with acute experimental allergic encephalomyelitis (EAE). Cells were labeled with Hoechst 33342 or BrdU, and with either OX-26-MION-46L or magnetodendrimers (MD-100) as the magnetic probe. Approximately 1.0–1.5 E4 neurospheres were injected into the lateral ventricle of Lewis EAE rats (n = 8) at the peak of their disease, as well as into normal, nondiseased animals (n = 9). At 1 week following transplantation, the brains were imaged ex vivo at 100 or 50 mm resolution using 4.7 and 7.0 T imaging spectrometers, respectively. Histology revealed periventricular migration of cells into the brain parenchyma of EAE but not naïve rats. On the MR images, migration patterns of cells were found to occur in the white matter but not in the gray matter. The corpus callosum, internal capsule, external capsule, and fimbria contained periventricular patches of hypointense labeled cells. For the naïve (nondiseased) animals, the labeled cells appeared to stay mainly within the ventricles and no widespread migration into white matter structures was seen. We conclude that MR imaging appears well suited to evaluate cell transplantation protocols.
Abstract ID: 283
Khalid Shah1,2 Yi Tang1 Xandra Breakefield2 Ralph Weissleder1
1Center for Molecular Imaging
2Department of Neurology, Harvard Medical School
Neurobiology
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis selectively in tumor cells. Its controlled induction could thus be used for tumor therapy. The aims of the current study were: (1) to determine therapeutic effects of gliomas treated with HSV amplicon vectors containing TRAIL; and (2) to image vector-mediated protein expression and therapeutic efficacy by dual enzyme substrate (renilla/firefly) bioluminescence imaging. The cDNA for TRAIL was cloned into HSV amplicon and packaged into replication deficient HSV-virions. Viability assays (WST) were carried out 24 hr after infection of cultured human glioma cells (Gli36) with TRAIL amplicon vectors to quantitate cell death. TRAIL induced apoptosis in about 80% of glioma cells in culture when compared to the cells infected with the control vector. The efficacy of TRAIL delivered by HSV amplicon vectors in vivo was determined by bioluminescence imaging of firefly luciferase (Fluc) expressing gliomas with d-luciferin injections. Gliomas showed regression over a period of 3 weeks in response to TRAIL as compared to the control vector injections. In order to visualize both amplicon-mediated vector gene delivery and tumor reduction, the cDNA for renilla luciferase (Rluc) gene was cloned into HSV amplicons, packaged into replication deficient HSV-virions, and injected directly into gliomas. Gli36 cells, infected with Rluc-HSV amplicon vectors and transiently expressing Rluc, were located in the gliomas 36 hr later by injecting coelenterazine. These combined imaging methods provide a means to evaluate gene delivery and expression in brain tumors in vivo and to monitor changes in tumor volume caused by apoptotic and other therapeutic genes.
Abstract ID: 284
S. M. Kumar Y. Tang K. Shah M. Giovannini R. Weissleder X. O. Breakefield
Molecular Neurogenetics, MGH East
Neurobiology
Spontaneous schwannomas were detected by MR imaging in a transgenic murine model of NF2 expressing a dominant mutant form of merlin under the Schwann cell specific P0 promoter. Approximately 85% of the investigated mice showed putative tumors by 24 months of age. In 21% of mice, tumors were multiple and occurred in the following locations: intercostal muscle 21%, limb muscles 14%, spinal cord and spinal ganglia 7%, external ear 7%, abdominal muscles 14%, intestine 7%, and uterus 66% of the female mice. The tumors were isointense with muscle by T1-weighted MR imaging, showed strong enhancement following administration of gadolinium (Gd)-DTPA, and were markedly hyperintense by T2-weighted, all hallmarks of the clinical manifestation. Histology indicated that the tumors consisted of schwannomas and Schwann cell hyperplasias. The lesions stained positively for S-100 protein and a marker antigen for the mutated transgenic NF2 protein. Lesions were found to be infectable with a replication conditional recombinant HSV-1 vector, hrR3, bearing lacZ by double staining for beta-galactosidase and the schwannoma marker, S-100. In vivo imaging of gene delivery was observed by injection of lesions with a helper virus-free HSV amplicon vector containing luciferase and tail vein injection of luciferin. HSV amplicon vectors with tetracycline-regulated gene expression will be used to deliver apoptotic genes to reduce the size of these benign tumors. The ability to detect and track spontaneous schwannomas by MRI and to infect them with HSV vectors provides the tools for assessment of therapeutic intervention.
Abstract ID: 285
Y. Tang K. Shah X. Breakefield R. Weissleder
Radiology, Harvard Medical School, CMIR/Radiology, MGH
Neurobiology
The ability to noninvasively track the migration (and possibly proliferation) of neural progenitor cells would have significant clinical and research implications. The purpose of our study was to explore the time course of migration of such cells towards brain tumors (9L) following implantation into nude mice. We stably transfected C17.2 neural progenitor cells (obtained from Dr. E. Snyder) with firefly luciferase gene (Luc) and implanted them into the contralateral brain (2 × 106 cells), the intraperitoneal cavity (5 × 106 cells) or subcutaneously (2 × 106 cells). In vivo bioluminescence imaging was performed 1, 4, 9, 15, 22, 36 days after C17.2 implantation. For neural progenitor cells implanted into the brain, we observed migration across the corpus callosum towards the tumor at Day 9 and massive migration across the midline by 2–3 weeks of implantation. When injected peripherally, no detectable migration towards the gliomas was observed. These results provide important insight into the time course and mechanism of migration of neural progenitor cells.
Abstract ID: 286
A.-L. Brownell Y. I. Chen M. Yu B. G. Jenkins
Radiology, Massachusetts General Hospital
Neurobiology
3-Nitropropionic acid (3-NP), a succinate dehydrogenase inhibitor, is widely used as an experimental model to study Huntington's disease (HD), energy metabolism, and cell death. We used a rat model to investigate 3-NP-induced acute and prolonged neurotoxicity using in vivo imaging of cerebral glucose utilization (CGU) and dopamine receptor function by PET, neuroanatomy by MRI and neurochemicals by MRS. 3-NP was administered (male Sprague–Dawley) twice a day (10 mg/kg ip) until symptomatic or max of 5 days. PET studies of CGU were conducted daily using a super high resolution (1.3 × 1.3 × 1.8 mm3) in-house built PET device. MRI and MRS studies were conducted with a GE Omega 4.7-T imager. Studies of CGU showed significant interanimal variation in the acute response of toxin, similar to motor activity. The average decrease of CGU in the lesions a day after cessation of 3-NP was 31 ± 12%. Four weeks later, CGU was recovered to − 13 ± 5% and then in 3 months decreased again to 48 ± 10%. After 3-NP, dopamine D1 and D2 receptors showed progressively decreasing binding using 11C-SCH and 11C-raclopride, respectively. However, the binding of dopamine transporter imaged by 11C-CFT showed an early increase (1 week after 3-NP) followed by a progressive decrease. MRS showed elevated peaks of lactate and macromolecules as well as succinate immediately after 3-NP toxicity, which diminished in 4 months, indicating a reversible process. Choline peak increased and NAA peak decreased in 4 months indicating loss and damage of neurons. Postmortem histological studies confirmed the neural loss indicated by in vivo imaging.
Abstract ID: 287
R. J. Wong X. N. Nguyen H. Zhao H. J. Vreman, et al.
Pediatrics, Stanford University School of Medicine
Neurobiology
Heme oxygenase (HO) is the rate-limiting enzyme in heme degradation pathway. There are two well-described isozymes, the inducible HO-1 and the constitutive, HO-2. A third isozyme (HO-3) has been identified but its role is incompletely known. It is well known that HO-2 is the predominant isozyme in the adult rodent brain. However, through noninvasive in vivo HO-1 transcription detection, we found that high rates of HO-1 transcriptional activity are present in the newborn brain. We therefore hypothesized that this difference in HO-1 expression is developmentally regulated. The objective of the study was to characterize this developmental pattern of HO gene expression and enzyme activity. HO-1 transcription levels were assessed in HO-1-luc transgenic mice, where the transgene is comprised of the full-length HO-1 promoter driving expression of the luciferase (luc) reporter gene. Bioluminescent imaging of luciferase activity using a cooled CCD camera was used as an indicator of HO-1 transcriptional activity. Brain tissue (n = 3 for each group) was harvested and sonicated with buffer. HO-1 and HO-2 proteins were detected by Western blot analysis and were quantitated by densitometry. Total HO activity was quantitated via measurements of carbon monoxide by gas chromatography. All results are expressed as fold change from 35-day (adult) levels. Age in Days 1, 3, 7, 14, 21, 28, HO-1 Transcrip: 20.4 ± 13.4, 3.8 ± 1.7, 7.8 ± 9.2, 1.8 ± 1.7, 0.7 ± 0.4, 0.4 ± 0.3; HO-1 Protein: 10.2 ± 1.4, 8.1 ± 0.2, 6.0 ± 0.6, 4.8 ± 0.1, 1.8 ± 1.9, 1.1 ± 0.2; HO-2 Protein: 1.4 ± 0.0, 1.1 ± 0.3, 1.3 ± 0.1, 1.2 ± 0.1, 1.1 ± 0.1, 0.8 ± 0.1; HO Activity 1.3 ± 0.1, 0.7 ± 0.1, 1.1 ± 0.2, 1.1 ± 0.2, 1.2 ± 0.1, 1.1 ± 0.1. These results show that HO-1 transcription and HO-1 protein levels are elevated at birth and then decrease progressively to adult levels. HO-2 protein and HO activity levels, however, remain relatively stable throughout life. Therefore, we conclude that HO-1 gene expression is not only induced at birth, but also is developmentally regulated.
Abstract ID: 288
F. Xue M. Mintun D. Holtzman M. Welch H. Kung
Radiology, Washington University School of Medicine
Neurobiology
Evidence supports beta-amyloid deposition in the brain as an early and central event in the pathogenesis of Alzheimer's Disease (AD). Imaging beta-amyloid deposition in the brain, therefore, has the potential for providing a powerful method in AD diagnosis. IMPY, 6-iodo-2-(4′-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine, is a synthetic derivative of thioflavin that binds to beta-amyloid aggregates in vitro with high affinity. To further characterize this tracer, we have studied [125I]IMPY in a mouse model of AD. Biodistribution and kinetics of [125I]IMPY in the mouse brain were first evaluated in 12–18-month-old normal Swiss Webster mice (n = 24). Our data confirmed that [125I]IMPY had excellent early brain penetration after intravenous injection (5.03% injected dose per gram at 2 min for the hippocampus). This was followed by rapid washout (0.50% injected dose per gram at 60 min for the hippocampus). Uptake of [125I]IMPY was then studied in two strains of transgenic (Tg) mice that produce excessive amyloid plagues in the brain. We found up to 2.3-fold increased uptake of [125I]IMPY in the hippocampus and cortex of APPSWE (n = 9) and APPV717F (n = 12) Tg mice compared to age-matched control mice. Furthermore, there was a correlation between [125I]IMPY binding activity and beta-amyloid load in the mouse brain by anti-beta-amyloid antibody staining of brain sections. These results suggest that IMPY binds to beta-amyloid plaques in the mouse brain in vivo and may be used as a molecular imaging agent for assessing beta-amyloid load. We are currently investigating the potential for IMPY-based tracers for positron emission tomography (PET) in imaging beta-amyloid plaques in human.
Abstract ID: 289
J. Schenck A. Alyassin D. Alsop
Genomics and Molecular Imaging, General Electric
Neurobiology
It has long been known that mineralized iron particles associated with the iron storage proteins ferritin and hemosiderin are found in specific deep nuclei of the human brain. This effect is prominent in the basal ganglia and the dentate nucleus of the cerebellum and has been demonstrated classically by postmortem Perls staining. Although some controversy still exists, it is widely accepted that these particulate deposits provide an endogenous contrast mechanism in brain MRI leading to decreased T2 in these specific brain regions as first proposed by Drayer et al. It has not been demonstrated, however, that elevated iron concentrations are the only source of short T2 values in the brain. The associated signal hypointensity on T2-weighted MRI has been reported by several authors to be significantly increased at higher field strengths. We have shown that, through a combination of high field (3 T) imaging using thin slices and computer postprocessing, a large number of small brain nuclei and cortical regions with rapid transverse relaxation compared to surrounding regions can be consistently identified. Specially written software was used to analyze, T2E, the effective T2 distribution. This distribution has a tail at lower T2E values, which corresponds largely to known high iron regions within the basal ganglia. However, this technique also identifies a number of voxels in other brain regions, many of which correspond to the locations of smaller nuclei or nuclei of lower iron content. These regions include the subthalamic nuclei, the superior and inferior colliculi, the mammillary bodies, the pineal gland, the lateral geniculate bodies, and other regions. Inferior to the anterior commissure, a region of low T2E in the vicinity of, but perhaps not identical with, the nucleus basalis is consistently seen. Because of the importance of this nucleus in Alzheimer's disease, iron-related imaging of this region will be of great interest in these patients.
Abstract ID: 290
B. J. Korte
Division of Biomedical Imaging, NIBIB/NIH
Other
The National Institute of Biomedical Imaging and Bioengineering was established in December 2000 in an effort to increase the visibility and recognition of biomedical imaging and bioengineering within the National Institutes of Health, and to promote the fundamental discovery, design, and development of technological capabilities in these areas. A key opportunity identified for NIBIB was the need for funding opportunities in molecular imaging, a research area that requires the combined efforts of molecular and cell biologists, chemists, physicists, mathematicians, engineers, and computer and imaging scientists. Recognizing that discoveries in molecular and cell biology present extraordinary opportunities for biomedical imaging to play an important role in the early detection, diagnosis, and treatment of disease, the NIBIB has created initiatives to support research and technology development of devices, methods, and imaging agents and probes that enable the investigation of biological processes in vivo. These initiatives support discovery and development of cross-cutting technologies for molecular imaging that can be applied broadly to research on biological or disease processes.
Abstract ID: 291
John M. Hoffman
MD, Biomedical Imaging Program, National Cancer Institute
Other
Over the past several years, the National Cancer Institute (NCI) has been keenly aware of the potential power of molecular imaging techniques. The Biomedical Imaging Program (BIP) (http://cancer.gov/bip/default.htm) of the Division of Cancer Treatment and Diagnosis is responsible for the extramural grant portfolio and programs related to oncologic imaging. Imaging has been identified as an area of “Extraordinary Opportunity” in the past several “NCI Bypass Budgets” (http://plan.cancer.gov/scipri/imaging.htm). The “NCI Bypass Budget” (http://plan.cancer.gov/budget/2003.htm) is a public document produced annually by NCI to identify for the Administration and Congress those scientific priorities on which the budget appropriation will be spent. To assure that the initially defined goals for molecular imaging are met and completed in future years, the NCI has set forth in the 2003 Bypass Budgets specific priorities and initiatives. These include: Expand the number of In-Vivo Cellular and Molecular Imaging Centers (ICMIC), which will be accomplished by reissuing the Request for Applications for the In Vivo Cellular and Molecular Imaging Centers (http://grants.nih.gov/grants/guide/rfa-files/RFA-CA-01-014.html) again in 2003, and hopefully in 2004. Expand the Development of Clinical Imaging Drugs and Enhancers (DCIDE) program (http://cancer.gov/bip/dcide.htm), which will facilitate and promote preclinical development and validation of important imaging agents and ligands. NCI will, on a competitive basis, synthesize, test, and distribute probes that image the physiological and functional status of tumor tissue in the human body. Establish a publicly available database of agents available to the research community, together with information on their properties. Support the development of in vivo and molecular clinical imaging research tools (http://grants.nih.gov/grants/guide/pa-files/PA-01-030.html) and technology development (http://grants.nih.gov/grants/guide/pa-files/PAR-01-101.html) for assessing the biologic effect of cancer drugs on their intended target or pathway.
Abstract ID: 293
R. Weissleder
Center for Molecular Imaging, Massachusetts General Hospital
Other
The Society for Molecular Imaging is proud to hold its first annual meeting following several years of conceptualization and a year of planning and organizing. Our Society has grown from a kernel of founding members in 1999 to a sizable organization with hundreds of members from diverse backgrounds. The Society has many important functions and I will briefly highlight some of these as well as provide “behind-the-scenes” glimpses. The first annual meeting is a culmination of important research in this growing field. I will attempt to briefly summarize significant achievements although many more will be presented at this meeting. While programmatic funding will drive much of the future research, a road map of important research topics and challenges will be laid out with an eye towards rapid clinical translation.
Abstract ID: 294
Evan C. Unger, MD
ImaRx Therapeutics, Department of Radiology University of Arizona, ImaRx Therapeutics, Professor of Radiology and Bioengineering, University of Arizona
Other
Recently, ultrasound contrast agents based upon perfluorocarbon-filled microbubbles have been approved by the FDA for imaging. Versions of these contrast agents may be developed for therapy. In this abstract, we will cover some of the potential therapeutic applications, emphasizing gene delivery. Microbubble gene carriers were developed. Several different parameters were studied including: ultrasound-mediated DNA expression with and without microbubbles, various microbubble and perfluorocarbon compositions, targeted microbubbles bearing ligands, intensity and duration of ultrasound. Experiments were performed in tissue culture and in vivo in mice with gene expression being assayed. With or without microbubbles, ultrasound was found to increase gene expression. Micro-bubbles were synergistic with ultrasound to increase gene expression. Targeted microbubbles demonstrated enhanced effect. We conclude that ultrasound-enhanced gene expression may have clinical applications in gene therapy. Microbubbles carriers may also have applications in gene delivery.
Abstract ID: 295
G. D. Luker J. P. Bardil J. L. Prior C. M. Pica D. Piwnica-Worms D. A. Leib
Mallinckrodt Institutue of Radiology, Washington University School of Medicine
Other
Herpes simplex virus-1 (HSV-1) is a widespread human pathogen that infects approximately 80% of all persons by adulthood and can cause morbidity and some mortality from diseases such as blinding keratitis and encephalitis. Conventional methods to study pathogenesis of HSV-1 in mouse models typically require sacrifice of infected animals to determine the full extent of viral spread and replication. To develop a noninvasive method for investigating HSV-1 in living mice, we used a recombinant strain KOS virus that expresses firefly and Renilla luciferases and monitored infection with bioluminescence imaging. Viral infection in mouse foot pads, peritoneal cavity, brain, and eyes could be detected by bioluminescence imaging of firefly luciferase. Activity of Renilla luciferase could be imaged after direct administration of substrate to infected eyes, but not following intravenous delivery of substrate. In vivo measurements of bioluminescence from firefly luciferase correlated directly with input titers of recombinant virus used for ocular infection of mice. Treatment of infected mice with valacyclovir, an effective inhibitor of HSV-1 replication, produced dose-dependent decreases in firefly luciferase that correlated with changes in titers of virus measured in tear films. These data demonstrate that bioluminescence imaging can be used for noninvasive, real-time monitoring of HSV-1 infection and therapy in living mice.
Abstract ID: 296
M'hamed Bentourkia
Nuclear Medicine and Radiobiology, University of Sherbrooke
Other
Positron emission tomography (PET) is a quantitative tool having the capability of estimating physiological parameters in vivo. However, in order for these parameters to be accurate, the PET data need to be corrected for image degrading effects such as scatter. The amount of scatter and its axial and transaxial distribution in the images depend on the position of the emitting sites, on the scattering object, and on the collimators. Generally, scatter functions are determined from point sources, or by extrapolation of the radioactivity distribution from out of the object in the image, or by analytical estimation of single scatter based on emission, transmission, and photon detection. In this work, scatter is determined by Monte Carlo calculations based on PET images in humans and in rats measured with the CTI HR+ scanner and the Sherbrooke small animal scanner, respectively. Assuming the image slice is made of tissue only, the scatter fraction was estimated as a function of the number of Compton interactions in human (rat): no scatter: 52% (90%), single scatter: 31% (8%), double scatter (both photons scatter once): 6% (1.6%), multiple scatter: 11% (0.4%). Moreover, the distribution of each of these types of scatter has its characteristics. The single scatter shows high intensity at source emission and this intensity rapidly decreases with distance from the source. The double scatter shows a reverse pattern. It has low intensity at source location then slightly increases apart from the source. The subsequent types of scatter present a flat distribution irrespective of source position. In conclusion, scatter functions need to be determined as a function of the type of scatter in order to process an accurate scatter correction.
Abstract ID: 297
W. Lai C. Chang Y. Tang R. Weissleder, et al.
Center for Molecular Imaging Research, Massachusetts General Hospital/Harvard Medical School
Other
Osteoarthritis (OA) is usually diagnosed utilizing X-ray and MRI, which are based on morphology changes of tissue. Once the changes are shown on X-ray or the MRI, the tissue appears as an irreversible defect. The purpose of this research is to investigate early diagnosis at molecular basis for OA using cathepsin-B activated near-infrared fluorescent (NIRF) probes. Multiple fluorochromes (Cy5.5) were bound to a long circulating graft copolymer consisting of a poly-lysine (PL) backbone sterically shielded through methoxypolyethylene glycol side chains. After enzymatic cleavage of the backbone, the fluorochromes are released resulting in a bright fluorescence signal, which can be detected in vivo. Right knee joints of five male nude mice were intra-articularly injected with 10 ml solution of collagenase 1.0% (w/v). A collagen-induced arthritis was characterized by mild to moderate degenerative cartilage 1 month after injection. Signal measurements were performed on an optical imaging system using an excitation wavelength of 610–650 nm and an emission wavelength of 680–720 nm. NIRF imaging of all mice showed a significant difference of signal intensity between osteoarthritic joints and normal joints 24 hr after intravenous injection of the reporter probe. Immunohistochemical evaluation also revealed cathepsin-B expression in the arthritic lesion of femorotibia joints, but not in the contralateral knee joints. The cathepsin-B activated NIRF probe offers the possibility of early detection at molecular basis for OA.
Abstract ID: 298
R. H. Singer D. Fusco N. Accornero B. Lavoie, et al.
Anatomy and Structural Biology, Albert Einstein College of Medicine
Other
Numerous mRNA species localize within cells. In yeast, ASH1 mRNA localizes to the bud tip and is necessary for asymmetric gene expression between the mother and the bud, allowing mating type switching. A major question has been whether mRNA localization is motor driven or is a result of diffusion, or both. In order to address whether mRNA movement requires a motor(s) and whether motor-like movement is sequence dependent, a method for visualizing the movement of mRNAs within living cells was developed. ASH1 mRNA movements were observed in living yeast cells using a GFP reporter system fused to the RNA-binding protein MS2 and introduction of MS2 sequences into the reporter RNA (Bertrand et al., Mol. Cell 2:437, 1998). This study showed unequivocally that the mRNA formed a particle that moved on actin cables at the rate expected from a myosin V. An amplified MS2-GFP system was used in Cos cells to permit the sensitive visualization and analysis of the movement of single RNA molecules. The analysis demonstrated that the RNA exhibits four types of behaviors: static, diffusional, directed, or wandering. Double-labeling in living cells showed that these movements occur most frequently on microtubules. Notably, mRNA movements in Cos7 cells displayed sequence dependence. When the beta-actin zipcode was present, the mRNAs displayed a significantly greater proportion of persistent movements. We conclude that zipcode sequences increase the likelihood of association of an mRNA with a motor.
Abstract ID: 299
M. Fortin M. Lépine B. Massie P. Hugo A. M. Steff
Research and Development, PROCREA BioSciences
Other
Endometriosis is a gynecological disease characterized by the implantation and estrogen-dependent growth of endometrial tissue at extra-uterine sites. This disease occurs in 10–15% of women of reproductive age and is responsible for intense abdominal pain and infertility. Current therapies for endometriosis include surgical removal of endometriotic lesions or induction of a hypoestrogenic state by hormonal therapy. We have used an animal model for endometriosis, whereby human endometrial fragments are introduced either subcutaneously or intraperitoneally into immunodeficient nude mice. It has previously been shown that endometriotic lesions growing in these animals histologically and biochemically mimic the human disease. This model, although potentially powerful, has been of limited use because it is not suited for dynamic studies. Indeed, assessing the efficiency of a drug treatment requires the killing of a cohort of animal for each time point; such end-point analysis might not allow distinguishing variations in initial implantation rates from drug-induced lesion regression. In the present study, we have used an adenoviral vector encoding a highly fluorescent variant of the green fluorescent protein (GFP) to transduce whole endometrial tissue fragments. Following their transplantation into nude mice, these GFP-expressing fragments were able to form lesions that closely resemble endometric foci found in women suffering from endometriosis. More importantly, we demonstrate that a simple fluorescence-imaging device can be used to directly visualize the bright GFP fluorescence of lesions through the skin of living mice. This dynamic setup allows following implantation and evolution of endometrial tissue over time in the same animal. This noninvasive mouse model, besides helping to better understand the pathophysiology of the disease, will constitute a valuable tool for direct evaluation of treatment efficacy before their application to humans.
Abstract ID: 300
A. L. Klibanov J. Christiansen H. Leong-Poi A. Takalkar K. Ley S. Kaul J. R. Lindner
Internal Medicine (Cardiovascular Div), University of Virginia
Other
Ultrasound contrast materials are typically designed as gas-filled microbubbles stabilized with lipid, polymer, or protein shell. Current uses of ultrasound contrasts include delineation of blood pool and organs, and tissue perfusion assessment. The amount of contrast material needed for successful ultrasound contrast is exceedingly small, especially with the new imaging modalities, like ultraharmonic and pulse inversion imaging. Individual bubbles with a size of several microns and a mass of several picograms can be detected. With currently approved agents, several microliters of gas or submilligram quantity of contrast is sufficient to achieve robust blood pool enhancement. Microbubbles can be targeted to diseased tissue by attaching ligands, such as antibodies, peptides, or other molecules, to the microbubble shell using a chemical link, a PEG spacer and/or noncovalent streptavidin-mediated coupling. Microbubbles with targeting ligands on the shell can selectively and firmly adhere to the receptor-coated surfaces in vitro, including the micro-bubbles perfused through the parallel plate flow chambers and in the cell culture systems. Binding to the control surfaces lacking the receptor is minimal. Micro-bubbles attached to the receptor deposited on solid surfaces (coverage of as little as 0.5% of the surface area by microbubbles) can be easily detected by ultrasound imaging. This high sensitivity has permitted imaging of microbubbles targeted to the receptors up-regulated on the surface of endothelial cells in the areas of inflammation, ischemia, and angiogenesis in vivo. In addition to targeted imaging, microbubbles can be applied as carrier systems for ultrasound-activated tissue-selective drug and plasmid DNA delivery.
Abstract ID: 301
J. Kadurugamuwa L. Sin E. Albert J. White R. Kimura M. DeBoer J. Yu K. Francis T. Purchio P. R. Contag
Infectious Diseases, Xenogen
Other
Biofilm formation on medical implants is a common occurrence and represents a serious medical problem, as these bacteria are difficult to eradicate with antibiotics and lead to chronic infections. Rapid, direct, nondestructive, real-time quantitative monitoring methods that are adaptable to the clinical situation are needed to develop new preventive, therapeutic methods to combat biofilm-related infections. We have developed a rapid, innocuous method for real-time monitoring of biofilms in vitro and in a mouse infection model by establishing biofilms of bioluminescent bacteria on Teflon catheters. The capacity of the microbes to form biofilm on catheter material, their suitability for long-term experiments, and the metabolic state of biofilms were assessed in situ by monitoring the bioluminescence with an IVISô imaging system (Xenogen, Alameda, CA). Two important biofilm-forming bacterial pathogens, Staphylococcus aureus, and Pseudomonas aeruginosa, were made bioluminescent by insertion of a complete lux operon and produced significant bioluminescent signals for both in vitro studies and for the development of an in vivo model, allowing for effective real-time assessment of the physiological state of the biofilms. In vitro viable counts and light output were parallel, highly correlated (S. aureus r = .98, P. aeruginosa r = .99), and could be maintained for 10 days or longer, provided that growth medium was replenished every 24 hr. In the murine model, subcutaneous implantation of the catheters (precolonized or post-implant infected) was well tolerated. An infecting dose of 103–105 CFU/catheter for S. aureus and P. aeruginosa resulted in a reproducible, localized, infection surrounding the catheter that persisted until the termination of the experiment at Day 20. Recovery of the bacteria from the catheters of infected animals showed that the bioluminescent signal corresponded to the CFU and that lux constructs were highly stable even after many days in vivo.
Abstract ID: 302
Christopher H. Contag C. Garry Fathmann Robert S. Negrin
Pediatrics, Stanford University
Other
Biological light sources can be built into animal models of human physiology and disease such that they serve as indicator lights providing external readouts for studying biology. Although mammalian tissues are relatively opaque, light is transmitted through tissues at low levels that can be externally monitored with sensitive detection systems. To further enhance this imaging modality, we have developed a set of multifunctional reporter genes such that cells expressing these constructs can be followed in vivo, can be analyzed by flow cytometry or fluorescence microscopy, and can be sorted by FACS. The cell analyses can precede the in vivo imaging step such that labeled populations can be selected. Alternatively, the cell assay can be conducted post in vivo trafficking studies. These dual-function reporter genes have enabled the labeling of immune cell populations for sorting and then assessing the in vivo trafficking patterns. This approach has also been used to select viral vectors for in vivo gene transfer and to optimize the delivery of these vectors to lung tissue. Monitoring immune cell trafficking patterns will advance our knowledge of immune surveillance of neoplastic disease, autoimmune dysfunction, and accelerate the development of effective immune cell and DNA-based therapies. Spatio-temporal information about interactive physiological processes is generated rapidly and is obtained in the context of whole biological systems using these imaging strategies. Sensitive in vivo imaging assays, such as in vivo bioluminescent imaging, which are accessible and permit rapid analyses of experimental therapeutic interventions in vivo and the dual-function reporter genes can further accelerate the development of novel therapeutic strategies.
Abstract ID: 303
C. Billotey F. Gazeau P. Cattan M. Janier, et al.
Animage
Other
Abstract ID: 304
Caitlin E. O'Connell-Rodwell Dmitri M. Simanovskii Cameron McClure Josh T. Beckman Yu-an Cao Weisheng Zhang Michael H. Bachmann Jennifer A. Baran E. Duco Jansen Daniel Palanker H. Alan Schwettman Christopher H. Contag
Pediatrics, Stanford University
Other
We developed a model for thermal stress using the stress-inducible heat shock gene (Hsp70A1) and luciferase expression as a measure of endogenous Hsp70 proteins generated in living cells. A stable line was created using NIH 3T3 cells transfected with a plasmid containing the regulatory region from the murine heat shock gene promoter Hsp70A1 driving the expression of the reporter gene, luciferase. We applied thermal pulses to cultures of these cells and used the stress-inducible reporter gene as an indicator of cellular stress. Cells were treated at different heat regimes using heating blocks and a CO2 laser at durations ranging from minutes to milliseconds where cultures reached peak temperatures of 80°C. Maximum thermotolerance ranged between 67.5°C and 76°C and evidence of thermally induced cell death ranged between 76°C and 80°C in the millisecond treatments. Luciferase expression was assessed in live cells every 2 hr for the first 10 hr and at 24 hr after thermal stress treatments, using a cooled CCD camera. Fluorescence activated cell sorting (FACS) was used to determine cell viability. The results of these studies indicate that there is a narrow temporal window of Hsp70 gene expression across a thermal gradient and a fine line between thermal resistance and apoptosis. This reporter gene construct may be useful for the prediction of collateral thermal damage created during treatment of biological tissue with lasers and other sources of heat.
Abstract ID: 305
H. Alfke J. T. Heverhagen H. H. Hahn M. Wegmann, et al.
Department of Radiology, Philipps University Marburg
Other
Abstract ID: 306
M. Brindle M. H. Bachmann E. Skarsgard C. H. Contag
Pediatrics and Surgery, Stanford University
Other
The mouse model has great appeal in the development of pulmonary DNA-based therapies, however, determining the levels of gene expression in the lungs of a living animal is particularly difficult in a small animal model. In vivo bioluminescent imaging addresses this difficulty and was used to assess the levels of expression following delivery of a targeted recombinant adeno-associated viral vector (rAAV) into the lungs of mice using perfluorocarbon (PFC) as a delivery medium. Mice underwent tracheal delivery of either rAAV2 or rAAV5 serotypes, both containing a fusion gene encoding luciferase and yellow fluorescent protein (YFP). In half of each study group, delivery of virus was followed by PFC and the other half was left untreated. The light emitted from the reporter genes in animals was detected, quantified, and localized using a ultra-cooled charge-coupled device camera, in the presence of luciferin, at 5-day intervals, over a 95-day time course. Lung tissue was collected at intervals and analyzed for luciferase content and viral DNA, and confocal microscopy was performed to localize YFP within tissues. Relative levels of gene expression were determined using bioluminescent imaging of study animals. Levels of expression increased until achieving a plateau at 15 days. A 100-fold increase in luminescence was noted in animals treated with rAAV5 over animals treated with rAAV2 (p = .002). PFC-aided delivery significantly increased the luminescence detected over the lung zones for animals given the AAV5 serotype. The use of a recombinant adeno-associated virus that targets respiratory epithelium in combination with PFC as a delivery agent allowed for effective delivery of DNA-based therapeutics achieving wide distribution and long-term expression. The use of bioluminescent imaging allowed for in vivo assessment of the spatio-temporal pattern of gene expression in a mouse model and accelerated the optimization of this treatment regimen.
Abstract ID: 307
Jonathan Hardy Kevin Francis Karine Gibbs Christopher Contag
Pediatrics, Stanford University
Other
Listeria monocytogenes is a food-borne bacterial pathogen responsible for hundreds of deaths each year in the US, often attacking pregnant women and infants. The murine model of Listeriosis is one of the most complete in all of bacterial immunology, and has proven extremely valuable in the study of the induction of cellular immunity by bacteria. We have engineered Listeria monocytogenes for bioluminescence imaging (BLI), by tagging the genome with the lux operon of Photorhabdus luminescens, and have obtained the first in vivo bioluminescence images of Listerial infection. Listeria grows in the liver and spleen before becoming systemic, disseminating to other organs such as the brain. Upon oral infection with 10(9) bacteria, Balb/c mice became sick, but the vast majority of bacteria cleared within a few days, whereas intravenous inoculation with 10(4) bacteria is rapidly fatal. In both cases, signal is detected in the liver and spleen of the animals, as expected, but BLI also revealed a strong signal from the gall bladder, indicating the presence of metabolically active bacteria in this organ. Gall bladder signal can occur in the absence of detectable symptoms 7 days post, and may indicate a possible carrier state applicable to human transmission. Because this signal is transient and the oral model of infection displays considerable variability, the ability of BLI to follow individual animals over time will be valuable for the study of Listeriosis.
Abstract ID: 308
C. V. Bowen X. Zhang P. Gareau B. K. Rutt
Imaging, Robarts Research Institute
Other
Knowledge of the time course, spatial distribution, and magnitude of inflammatory response is critical for understanding inflammatory disease progression, yet such information is difficult to obtain from conventional histopathologic evaluation. The detection of macrophage inflammatory cells, in vivo, is possible through the use of superparamagnetic (SPM) iron oxide nanoparticles. The methods use SPM particles to image cellular uptake and trafficking with MRI by exploiting the ability of certain cell types to ingest small particles in culture and in vivo. Although such studies report sensitivity to the presence of iron oxide using T1-, T2-, or T2*-weighted imaging, methods quantifying the concentration of iron oxide in vivo, as a first step towards characterizing the degree of inflammatory cell infiltration, are still lacking. We have confirmed relaxation rate predictions of static dephasing (SD) regime theory as applied to quantifying the presence of iron oxide particles within cells. THP-1 cells were loaded with iron oxide particles by incubation in cell culture. Two cellular suspension groups were prepared with iron concentration varying through changes in either the cellular density or the iron mass per cell. 4-T measurements of R2 were sensitive to both iron concentration and the iron mass per cell, thus complicating efforts at quantification. For a given iron concentration, samples with a greater iron mass per cell have smaller R2 values, consistent with theoretical predictions by Jensen and Chandra [MRM 43:226–36 (2000)]. However, for cells having appropriate loading levels (0.1 pg of iron per cell), R2* values exceeded R2 by a factor of 70 and were sensitive to iron concentration alone and agreed with SD regime theory predictions. Our results represent an important advance toward quantifying cellular iron content and ultimately mapping a targeted cell population density.
Abstract ID: 309
David S. Lester
Clinical Technologies, Pharmacia
Other
Biomedical imaging has had more of an impact on clinical sciences than any other technology in the 20th century. However, its integration into the drug discovery and development processes has been limited. This is due to trepidations and concerns expressed both by the pharmaceutical industries and the Food and Drug Administration. In addition, there have been relatively no efforts from the radiologic community to transfer the technology from a diagnostic tool to a methodology for monitoring therapeutic efficacy in clinical drug trials. Imaging has the potential to have considerable influence on all aspects of drug discovery and development. The initial task is to identify areas of therapeutic interest where there is a necessity for new clinical/biological endpoints and whether imaging is capable of fulfilling this need. Many pharmaceutical companies have “experiences” with imaging but the successes are relatively limited. This is in large part due to a lack of internal expertise for appropriate clinical trial design with imaging methodologies and, in addition, an understanding of what is the specific goal of the study in question. In terms of the drug development process, there are three levels of information that contribute to the drug development process that can be obtained using imaging. They are: (1) drug hits the target, (2) drug affects mechanism, (3) drug affects disease progress. During this presentation, some potential applications of biomedical imaging to these three areas of drug development and the risk/benefit issues that are associated will be discussed.
Abstract ID: 311
Alan Jasanoff Phillip Sun David Cory Andrew Dennis Peter Fam
Whitehead Institute
Other
We are using molecular imaging approaches to study neural activity at high resolution in intact animals. We have adapted a 14-T MRI system for near cellular resolution imaging (20–40 μm)3 of unanesthetized flies during visual stimulation. Because of its physiological stability, relatively large neurons, and absence of hemodynamic effects, the fly preparation is an ideal platform for the validation of new functional imaging agents. We have exploited cross-linked hemoglobin as an oxygen sensor for high-resolution studies, and demonstrate its efficacy in vivo; this approach could be used for neuro-imaging through a “metabolic filter,” analogously to conventional functional MRI. We are also developing ion-sensitive peptide-based imaging agents using combinatorial synthesis and screening methods. Hits will be tested by cell-specific injection into fly neurons, and may allow direct MRI-based recording of neuronal firing rate correlates.
Abstract ID: 312
A. S. Arbab B. R. Miller E. K. Jordan B. K. Lewis, et al.
Laboratory of Diagnostic Radiology and Research, National Institute of Health
Targets
Magnetic labeling of stem cells (SC) and other cells provides the ability to in vivo monitor the migration and trafficking of these cells following transplantation or infusion using MRI. Cellular labeling using dextran-coated superparamagnetic iron oxide nanoparticles (SPIO) has been limited because the native form is not efficiently incorporated into cells. Modification of the SPIO by conjugation with either monoclonal antibodies or exogenous proteins has been used to facilitate cellular uptake. Alternatively, coating SPIO with transfection agent (TA) such as dendrimers results in a nanoparticle that can be used for nonspecific labeling of mammalian cells. Commercially available TAs, incubated with Feridex or MION-46L, effectively coated the agent through electrostatic interaction. Stem cells and other mammalian cells were then incubated for 2–48 hr with TA-SPIO mixtures. Iron uptake and cell viability were evaluated. Disappearance of iron incorporated into the cells was also studied. Prussian blue staining showed intracytoplasmic iron particles when TA-SPIOs were incubated with human mesenchymal stem cells, hemopoetic stem cells (CD34+ cells), human cervical and small cell lung carcinoma cells, and marmoset and mouse lymphocytes. MR images and relaxometry revealed a 50–90% decrease in signal intensity and prolongation of 1/T2 of the magnetically labeled cell suspensions compared to unlabeled cells. Long-term cell viability and growth rate were unaffected by the incorporation of nanoparticles as compared to unlabeled cells. In nondividing cells, incorporated iron could be found into the cells even after 7 weeks. These results may facilitate the introduction of MR monitoring of magnetically labeled cells following transplantation in a clinical setting.
Abstract ID: 313
A. S. Arbab B. R. Miller E. K. Jordan B. K. Lewis, et al.
Experimental Neuroimaging Section, National Institutes of Health
Targets
Magnetic labeling of stem cells (SC) and other cells provides the ability to in vivo monitor the migration and trafficking of these cells following transplantation or infusion using MRI. Cellular labeling using dextran-coated superparamagnetic iron oxide nanoparticles (SPIO) has been limited because the native form is not efficiently incorporated into cells. Modification of the SPIO by conjugation with either monoclonal antibodies or exogenous proteins has been used to facilitate cellular uptake. Alternatively, coating SPIO with transfection agent (TA) such as dendrimers results in a nanoparticle that can be used for nonspecific labeling of mammalian cells. Commercially available TAs, incubated with Feridex (Berlex Laboratories), effectively coated the agent through electrostatic interaction. Stem cells and other mammalian cells were then incubated for 2–48 hr with TA-SPIO mixtures. Iron uptake and cell viability were evaluated. Disappearance of iron incorporated into the cells was also studied. Prussian blue staining showed intracytoplasmic iron particles when TA-SPIOs were incubated with human mesenchymal stem cells, hemopoetic stem cells (CD34+ cells), human cervical and small cell lung carcinoma cells, and marmoset and mouse lymphocytes. MR images and relaxometry revealed a 50–90% decrease in signal intensity and prolongation of 1/T2 of the magnetically labeled cell suspensions compared to unlabeled cells. Long-term cell viability and growth rate were unaffected by the incorporation of nanoparticles as compared to unlabeled cells. In nondividing cells, incorporated iron could be found into the cells >7 weeks. These results may facilitate the introduction of MR monitoring of magnetically labeled cells following transplantation in a clinical setting.
Abstract ID: 314
H. Li M. Zhang G. Zheng D. Blessington, et al.
Radiology, University of Pennsylvania
Targets
The lack of selectivity and specificity is still the key problem in cancer diagnosis and treatment. Low-density lipoprotein (LDL) has been used for the delivery of cytotoxic drugs to tumor cells on the basis of a higher uptake of LDL by overexpressed LDL receptors (LDLRs) in these tumor tissues. We are utilizing LDL or apolipoprotein E labeled lipid vesicles to specifically deliver near-infrared (NIR) contrast agents to these tumors for noninvasive detection. Chlorophyll analog-pyropheo-phorbide was synthesized and attached to cholesterol ester, then the conjugate was reconstituted into the lipid core of LDL as an NIR fluorescence probe (NIRF). Laser scanning confocal microscopy was employed to monitor receptor internalization of NIRF in cultured human hepatoblastoma G2 (HepG2) tumor cells. Intense fluorescence signal was observed throughout the cell except for the nucleus, but no fluorescence could be detected in the presence of an excess of unlabeled LDL or the photosensitizer alone without the LDL, which clearly indicates the specific internalization of this NIRF by LDLRs. Intravenous delivery of NIRF to in vivo subcutaneous HepG2 tumors in nude mice was also demonstrated by ex vivo 3-D redox ratio-scanning after rapid freezing. The fluorescence was distributed uniformly throughout the tumor at different depths. Both in vitro and ex vivo experiments indicated that the NIR dye labeled LDL could be used for the noninvasive early detection and specific antitumor drug delivery to tumors that overexpress LDLRs.
Abstract ID: 315
A. S. K. Dzik-Jurasz D. J. Collins
NMR, Institute of Cancer Research
Targets
Ligand–substrate interactions form the basis several important biological processes, including enzyme action, cell membrane signaling, and as generic therapeutic targets. Therapeutically, competitive binding is recognized as a cause of altered drug pharmacodynamics and toxicity. Despite the detailed characterization of competitive binding in vitro, the process remains to be demonstrated by in vivo magnetic resonance. We demonstrate, using an animal model, the in vivo competitive binding of the antibiotic flucloxacillin with tetrafluorosuccinic acid (TFSA) using 19F-MRS (magnetic resonance spectroscopy) via changes in the longitudinal relaxation time of TFSA. All experiments were performed on a clinical 1.5-T scanner using a dual-resonant 1H/19 F transmit/receive coil. Following administration of TFSA, incremental doses of flucloxacillin were delivered up to a molar ratio of 1.5 relative to TFSA. In all animals (n = 5), the T1 of TSFA exhibited a typical competitive profile, rising incrementally to, but not beyond, a plateau phase at a molar ratio of one with flucloxacillin. The mean maximum percentage increase in the T1 of TFSA following administration of flucloxacillin was 47 ± 9% (SD). There was a significant (70%) enhancement in the TFSA (19F-relaxivity: 1 × 45 sec−1 mM−1), but not the flucloxacillin (19F-relaxivity: 3 × 12 sec−1 mM−1) resonance following intravenous administration of Gd-DTPA. In summary, behavior strongly indicative of competitive binding has been demonstrated in vivo via changes in the T1 of TFSA. The absence of enhancement in the 19F signal of flucloxacillin indicates that flucloxacillin is predominantly intracellular and the detected competitive binding is therefore occurring intracellularly. The substrate and site for this event remains to be determined.
Abstract ID: 316
L. Josephson M. Kircher U. Mahmood Y. Tang, et al.
Radiology, Center for Molecular Imaging Research
Targets
The design of smart or environmentally sensitive probes is now a major effort of the molecular imaging community. These include DNA binding, fluorescent molecular beacons, enzyme-activated NIRF probes, and paramagnetic enzyme substrates that change their relaxivities due to the action of enzymes. Here we describe magnetic nanoparticle-based MR contrast agents that have a near-infrared fluorescence (NIRF) that is activated by certain enzymes. The probes are prepared by conjugation of arginyl peptides to cross-linked iron oxide amine (amino-CLIO), either by a disulfide linkage or a thioether linker, followed by the attachment of the indocyanine dye Cy5.5. The NIRF of disulfide-linked conjugate was activated by DTT, while the NIRF of thioether-linked conjugate was activated by trypsin. Fluorescent quenching of the attached fluorochrome occurs in part due to the interaction with iron oxide, as evident by the activation of fluorescence with DTT when nanoparticles have less than one dye attached per particle. With a subcutaneous injection of the probe, axillary and brachial lymph nodes were darkened on MR images and were easily delineated by NIRF imaging. The probes may provide the basis for a new class smart nanoparticles, capable of pinpointing their position through their magnetic properties, while providing information on their environment by optical imaging techniques.
Abstract ID: 317
H. Handl J. Vagner V. Hruby R. J. Gillies
Biochemistry and Cancer Center, University of Arizona
Targets
Many of the current targeting methods for cancer imaging and therapy rely on the overexpression of cell surface epitopes on the target cell. Targeting combinations of cell surface receptors with multimeric ligand complexes can theoretically improve the binding specificity and affinity of imaging and therapeutic agents for defined cell targets. In this study, we have utilized a competitive binding assay with 125-I radiolabeled ligands to investigate the specific binding of melanotropin (MSH) multimers to cells displaying the corresponding melanocortin (MC1R and MC4R) receptors. Initial studies have shown that a dimeric MSH ligand has up to a 16-fold increase in binding affinity compared with the monomeric MSH ligands. In order to empirically determine the optimal linker rigidity and length for a dimeric ligand, a succession of different spacers for the multimeric ligand complexes has been designed which allows for a range of flexibilities and lengths. Assessment of flexible PEG linkers of lengths up to 38 atoms has shown that the optimal linker length for a dimeric truncated MSH ligand is 19 atoms. Data are currently being obtained for a set of more rigid linkers consisting of repeating chains of b-alanine and galactose. Based on results from these studies, dimeric and multimeric ligands can be tested in vivo to determine the binding affinity and specificity of the multimeric ligands compared to monomeric ligands.
Abstract ID: 318
G. Shi S. K. Hobbs R. J. Homer G. R. Harsh S. W. Atlas M. D. Bednarski
Department of Radiology, Stanford University
Targets
Gliomas are the most common type of brain cancer and glioblastoma multiforme (GBM) is the most malignant form. Classic MRI appearance of GBM reflects the tumor's heterogeneous pattern of hypercellularity, necrosis, neovascularity, and invasion. Image-guided proteomics may help elucidate mechanisms responsible for the imaging patterns and pathologic variability seen in GBM and offers a novel technique for discerning new molecular targets. In this study, image-guided protein expression profiling was performed on human GBM specimens. Samples were taken from MRI Gd-enhanced (CE) and nonenhanced (NE) regions of each tumor during surgical resection. Proteins were isolated from the fresh–frozen tissue samples followed by proteomic mass spectral analysis. Protein expression profiles of enhanced regions differ from nonenhanced regions. The NE regions across the four tumors and from different NE regions within the same tumor are markedly homogeneous. In contrast, no common protein profile can be determined from the CE regions. This first use of MRI-guided proteomics in human tumors identified a protein fingerprint that correlates with spatial contrast enhancement patterns in solid tumors. Specific proteins identified by this method to account for the different patterns may prove useful as diagnostic markers and/or therapeutic targets.
Abstract ID: 319
W. Hundt E. L. Yuh R. Seip M. Bednarski, et al.
Radiology, Stanford University
Targets
Abstract ID: 320
Martin Rausch Diana Baumann Christoph Wiessner Markus Rudin
Novartis Central Technologies, Novartis Pharma
Targets
Labeling of phagocytotic cells can be achieved by systemic application of ultra small particles of iron oxide (USPIO). While macrophages of the primary lymphatic organs can get in direct contact with USPIO during the plasma phase, the mechanism of USPIO accumulation in brain tissue is still unclear. Under nonpathological conditions, brain parenchyma is efficiently isolated from the luminar side of blood vessels by the blood-brain barrier (BBB). In the present study, patterns of USPIO enhancement detected by T1- and T2-weighted MRI in rats with unilateral pMCAO were compared to dynamic contrast-enhanced (DCE-), CBV-, and immunohistological data. USPIO (AMI-227) was administered 5 hr after onset of ischemia and images were acquired 19 hr later. Signal enhancement was found in T1-weighted images in the affected hemisphere within the basal ganglia and the cortex. T2-weighted images showed signal reduction in almost the same areas. However, edema formation strongly interfered with USPIO-associated signal reduction. CBV was reduced in the same areas to a variable extent. DCE-imaging revealed integrity of the BBB throughout the cortex despite some small spots of increased vascular permeability. By combined analysis of DCE- and CBV-maps, it could be excluded that vascular permeability was masked by reduced blood flow. Therefore, USPIO particles did not accumulate in brain tissue by passive diffusion followed by endocytosis by activated microglia. The data suggest that signal alterations were due to blood-borne monocytes, which had incorporated the tracer prior to transcytosis. Prussian blue staining showed clusters of labeled monocytes within the lesion, which were spatially matched with ED-1 positive cells. In conclusion, systemic administration of USPIO allows effective labeling of blood-borne macrophages in neuroinflammatory diseases.
Abstract ID: 321
Samira Guccione Carina Mari Yi-Shan Yang Mark Bednarski
Radiology, Stanford University
Targets
The recent explosion of information in the fields of genomic and proteomic has provided a rich ground for the discovery of molecular targets for therapeutic and diagnostic purposes. However, due to the heterogeneity within most tumors, tissue sampling for genomic and proteomic analysis is spatially dependent. In addition, molecular targets vary with temporal changes during tumor growth. We believe that there is a need to resolve tissue analysis both spatially and temporally so that the most relevant molecular targets can be identified. Clinically, regions of high contrast enhancement in magnetic resonance imaging (MRI) are believed to be related to areas of increased vascularization, angiogenesis, and tumor aggressiveness. We hypothesized that image-guided tissue sampling from the contrast-enhanced (CE) and nonenhanced (NE) regions within the same tumor will facilitate the identification of molecular targets. To test this hypothesis, a murine squamous cell carcinoma model was used to obtain tissues from CE and NE areas of the tumor after MR imaging. While histologic staining (H&E) showed no significant differences between the CE and NE regions, the MR images clearly showed differences in the diffusion and perfusion of the small molecule contrast agent and no significant necrosis. Oligonucleotide microarray analysis of these spatially distinct areas within the same tumor revealed that nine genes were up-regulated in the CE areas of the tumor relative to the NE regions. Only one gene was down-regulated. Six out of these nine up-regulated genes in the CE regions are associated with the extracellular matrix. The only major growth factor gene up-regulated in the CE areas was the platelet-derived growth factor receptor (PDGF-R). Both PDGF and its receptors have been shown to have an angiogenic effect. Furthermore, it has been proposed that the overproduction of PDGF may be involved in autocrine and paracrine growth stimulation of human tumors.
Abstract ID: 322
Samira Guccione Dan Lee Mark Bednarski
Radiology, Stanford University
Targets
We have developed a molecular imaging agent that targets a class of cell surface receptors associated with tumor angiogenesis, the integrins, in an effort to spatially resolve and map their expression in vivo. The integrins avb3 and avb5 have been identified as important cell surface markers of angiogenesis found to be overexpressed in the majority of solid malignant tumors. We have used a nanoscale lipid-polymer-based agent (NP, described elsewhere) with a multivalent array of a novel synthetic peptidomimetic (PM) to specifically target this class of receptors known to be expressed on vascular endothelial cells and certain tumor cells. By labeling the PM-NP with 111-Indium, a gamma-emitting radioisotope, we were able to obtain whole body scintigraphic images in a murine model of malignant melanoma. Tumors were implanted into test animals, grown to significant mass, and then scanned following the administration of a single bolus intravenous injection of 111-Indium-labeled PM-NP. Time course scanning demonstrated a persistent blood-pooling effect. Tumor accumulation was dependent on the presence of the PM moiety since nontargeted contrast agent did not show significant tumor localization. Furthermore, integrin specificity of the PM was examined both in vitro using a cell-adhesion blocking assay and in vivo by immunohistochemistry and autoradiography. We have found that the PM-NP was functionally superior to the monomeric PM in our in vitro assay, demonstrating the increased binding avidity conferred by a multivalency effect. We have also observed this increased binding avidity in vivo as a function of percent PM moiety on the NP. This approach to visualizing receptors associated with clinically relevant pathological processes has a general application. We propose to use this type of molecular imaging agent to confirm and follow the progression of tumor angiogenesis during cancer therapy using clinical medical imaging techniques.
Abstract ID: 323
D. Schellingerhout L. Hsu K. Hynynen R. Weissleder F. A. Jolesz
Radiology, Massachusetts General Hospital
Targets
We investigated the use of MR image-guided focused ultrasound (FUS) to achieve focal transient disruption of the blood–brain barrier (BBB) and, by this means, achieve targeted delivery of viral gene therapy to the brain. We hypothesized that anatomically targeted delivery of virus could be achieved, and that the amount of vector delivered could be controlled by the FUS energy deposited. White New Zealand rabbits (n = 3) were anesthetized and MR studies were obtained to guide the FUS beam. FUS lesions were generated by a custom-built magnet-integrated system. Lesion localization was confirmed using SPGR and MR thermometry sequences; BBB disruption was confirmed using gadolinium-enhanced T1-weighted images. Genetically engineered Herpes Simplex Virus (HSV) was radiolabeled using 111-Indium-oxine, and was injected intravenously. Brains were harvested 24 hr later, and subjected to serial sectioning, autoradiography, and histology. Foci of viral deposition were shown at the sites of the FUS lesions, indicating focal viral delivery. ROI analysis showed target-to-background ratios of 3.53 for a 1.1-W location, 3.56 for a 0.3-W location, and 1.74 for a 0.06-W location. Histology showed neuronal necrosis at high-energy sites, but none at low-energy sites. We conclude that FUS does disrupt the BBB in a directed focal manner, allowing the targeted delivery of viral vectors. A dose–response relationship exists between FUS energy and vector delivered. BBB disruption occurs at relatively low FUS energy levels, with an apparent saturation effect above 0.3 W. Higher energies show no benefit in delivery, but do contribute to neuronal necrosis.
Abstract ID: 324
D. Blessington L. Zhou H. Li G. Zheng, et al.
Biochemistry/Biophysics, University of Pennsylvania
Targets
NIR imaging allows for deep tissue penetration and localization of tumors within tissue when labeled with a molecular beacon delivered to the tumor. Images we have conceived were produced by the low-temperature redox scanner in response to the intensities of fluorescent signals. Two light sources (100 W mercury arc lamp and 660 nm laser diode), specific-wavelength optical filters, and a PMT were used to produce and collect the fluorescent signals. Tumors typically express an increase in the reduced nicotinamide adenine dinucleotide (NADH) and a decrease in flavin adenine nucleotide (FAD). These two fluorescent intensity signals were recorded and applied to the ratio NADH/(FAD + NADH) to demonstrate the oxidation–reduction rate of the tumor. A large ratio value would indicate a high metabolic (less oxidized) state of the tumor. The blood volume content and metabolic state are maintained by freeze-trapping the tissue in liquid nitrogen. In this study, the imager is used to monitor the delivery of the molecular beacon, reconstituted pyropheophorbide chlorophyll labeled LDL (pyro-CE), administered via the tail vein to a mouse model bearing the human hepatoma (HepG2) tumor. We also observed the effects of photodynamic therapy (PDT) to the HepG2 tumor, by monitoring changes in the NADH and FAD signals, after light treatment with a laser at 660 nm and pyro-CE, the photosensitizing agent. Signals were collected at 366 nm excitation/420 nm emission for NADH, 450 nm excitation/525 nm emission for FAD (flavoprotein), and 670 nm excitation/720 nm emission for pyroCE, the molecular beacon specifically targeted to LDL receptors, overexpressed in the HepG2 tumor.
Abstract ID: 325
Y.-S. Yang S. Guccione G. Shi M. Bednarski
Targets
Gene and protein expression profiles in tumors can vary with temporal progression and spatial distribution. We hypothesize that using MRI, one can determine temporal changes in tumor progression and reveal spatial heterogeneity within the tumor at different stages of growth for genomic analysis, and combination of MR imaging and microarray analysis can be used to validate potential molecular targets for new molecular imaging agents useful for cancer diagnosis and therapy. C3H/Km mice were transplanted with murine squamous cell carcinoma VII (SCCVII) cells. Tumors were imaged sequentially starting at a diameter of ~6 mm using a 3-T MR Scanner with standard spin-echo (T1-wt) and fast spin-echo (T2-wt) sequences. Postcontrast T1-wt image was obtained after an injection of gadolinium-DTPA. MRI contrast-enhancing and nonenhancing regions were spatially marked at different stages of tumor growth, and the tumor tissue was surgically harvested for genomic analysis. Four distinct stages of tumor progression in the SCCVII murine tumor model were defined based on the contrast enhancement patterns of their T1- and T2-weighted MR images. Microarray analysis was performed on the periphery and center of these tumors to obtain differences in gene expression profiles between each of these four stages of tumor growth. Sets of interesting gene products for each stage have been identified as potential candidates of molecular targets for cancer therapeutics. We conclude that tissue sampling of tumors for genomic analysis using MRI-guided biopsy is sensitive to spatial and temporal changes in tumors. Clinical imaging can therefore serve as a powerful tool for discerning regions of distinct patterns of gene expression.
Abstract ID: 326
Y.-S. Yang S. Guccione G. Shi M. Bednarski
Radiology, Stanford University
Targets
Gene and protein expression profiles in tumors can vary with temporal progression and spatial distribution. We hypothesize that using magnetic resonance imaging (MRI) one can determine temporal changes in tumor progression and reveal spatial heterogeneity within the tumor at different stages of growth for genomic analysis, and combination of MRI and microarray analysis can be used to validate potential molecular targets for new molecular imaging agents useful for cancer diagnosis and therapy. C3H/Km mice were transplanted with murine squamous cell carcinoma VII (SCCVII) cells. Tumors were imaged sequentially starting at a diameter of ~6 mm using a 3-T MR Scanner with standard spin-echo (T1-wt) and fast spin-echo (T2-wt) sequences. Postcontrast T1-wt image was obtained after an injection of gadolinium-DTPA. MRI contrast-enhancing and nonenhancing regions were spatially marked at different stages of tumor growth, and the tumor tissue was surgically harvested for genomic analysis. Four distinct stages of tumor progression in the SCCVII murine tumor model were defined based on the contrast enhancement patters of their T1- and T2-weighted MR images. Microarray analysis was performed on the periphery and center of these tumors to obtain differences in gene expression profiles between each of these four stages of tumor growth. Sets of interesting gene products for each stage have been identified as potential candidates of molecular targets for cancer therapeutics. We conclude that tissue sampling of tumors for genomic analysis using MRI-guided biopsy is sensitive to spatial and temporal changes in tumors. Clinical imaging can therefore serve as a powerful tool for discerning regions of distinct patterns of gene expression.
Abstract ID: 327
B. L. Franc H. M. Song M. T. Longaker C. H. Contag
Stanford University, Nuclear Medicine
Apoptosis
Elucidation of the underlying mechanisms leading to craniosynostosis, the premature fusion of cranial sutures, could facilitate development of less invasive treatments for this debilitating craniofacial anomaly. An increase in apoptosis in the posterior-frontal suture (PF) of the Sprague-Dawley rat during physiologic fusion (postnatal (PN) days 12–22) has recently been demonstrated in situ using Tc-99m- and fluorescent-labeled annexin. The present work focused on an in vivo imaging method to detect apoptotic events within the fusing PF suture and the sagittal suture (SAG), a suture that remains patent. Fluorescent-labeled annexin (Allexa-Fluor 647, Molecular Probes) was injected (100mg/kg) into 5, 15, and 30 day PN animals (n = 5). Animals were anesthetized and imaged one hour later using a CCD-based imaging system equipped with an excitation source and appropriate filters (IVIS system, 550–645 nm excitation filter, 650–710 emission filter). The cranium and the intersection of the PF, SAG, and coronal sutures were imaged following sacrifice. Ratios of the activity within each suture to the background activity (S/B) were produced using ROI analysis. A linear relationship was found between the average S/B measured from in vivo images and that measured from in situ images (R2 = .90) demonstrating the feasibility of imaging apoptotic events within superficial structures in vivo. A significant increase (p < .05) in apoptotic activity within the PF at 15 days PN was measured in both in vivo and in situ images, supporting previously published findings. Future work will investigate the spatial progression of apoptosis in the fusing cranial suture using this method in a cohort of animals.