Biometabolic Distribution of 99m Tc-3PRGD2 and its Potential Value in Monitoring Chemotherapeutic Effects

Abstract

Previous studies have reported that ^99mTc-3PRGD2 is an excellent tumor imaging agent that showed a good correlation with integrin α_vβ₃, a main factor of tumor-induced angiogenesis. In this study, we investigated the biometabolic distribution characteristics of ^99mTc-3PRGD2 with a continuous dynamic acquisition mode to explore the potential value of ^99mTc-3PRGD2 in monitoring chemotherapeutic effects in VX2 tumor models. Eighteen rabbits with 27 implanted VX2 squamous cell tumors were randomly divided into a nontreated control group (NTG, n = 8; 12 tumors) and a treatment group (TG, n = 10; 15 tumors). ^99mTc-3PRGD2 imaging was performed prior to cisplatin injection and repeated on days 0,1, 7, and 14 postinjection. Continuous dynamic scanning up to 30 minutes; static imaging at 0.5 hours, 1 hour, and 3 hours; and single-photon emission computed tomography/computed tomography (SPECT/CT)-integrated imaging at 3 hours post-^99mTc-3PRGD2 injection were performed. The peak time (time to reach peak in dynamic curve), tumor to normal (T/N) ratios, and their change rates relative to pretherapy were calculated. Autoradiography, hematoxylin-eosin (H&E) staining, and CD31 and integrin α_v immunohistochemical staining were examined. VX2 tumors were clearly visualized at 3 hours post-^99mTc-3PRGD2 injection. Tumors in the TG shrank significantly on day 7 after cisplatin administration (p < .05). The half-life (t_1/2) of the radiotracer in heart, liver, and tumor in the NTG were 3.43 ± 0.94 minutes, 13.41 ± 9.17 minutes, and 70.83 ± 33.37 minutes, respectively. The peak time was delayed in the TG immediately and continuously after cisplatin administration compared to the peak time in the NTG. The T/N values and their change rates decreased significantly in the TG compared to the NTG after therapy (p < .05). The immunostained areas were significantly decreased in the TG (p < .05) compared to the NTG. ^99mTc-3PRGD2 was an excellent imaging agent for demonstrating tumor angiogenesis. The peak time, T/N values, and their change rates were sensitive parameters to monitor early chemotherapeutic effects. Due to the specific target mechanism and the cost-effective value of ^99mTc-3PRGD2, ^99mTc-3PRGD2 SPECT imaging may have potential in detecting the therapeutic effects of anticancer therapy.

The cytotoxicity and antiangiogenic effects of most chemotherapeutic agents kill tumor cells and shrink tumors.^1,2 Cisplatin is a cell cycle-nonspecific antitumor agent that selectively inhibits synthesis of deoxyribonucleic acid (DNA) and causes DNA cross-linking while sparing ribonucleic acid (RNA) and protein synthesis.^3,4 As such, cisplatin has actions in both tumor and endothelial cells.^5,6

Integrin α_vβ₃ is highly expressed in both neoplasm and in new blood vessel endothelial cells.^7,8 Targeting tumor angiogenesis is a novel and promising approach to anticancer research. Several antitumor angiogenesis drugs have been transferred into clinic or in a clinical trial. Novel Arg-Gly-Asp (RGD) ligands have been reported that can target α_vβ₃, which can reflect angiogenesis. Several RGD peptides have been developed as in vivo imaging agents and have undergone clinical trials in a variety of cancers and other diseases.^9,10 ¹⁸F-Galacto-RGD was the first to be applied in patients and has been successfully assessed in over 100 cases.¹¹ ¹⁸F-AH111585 was used to assess changes in tumor vascularity following antitumor therapy in animals treated with low-dose paclitaxel.¹² Although most of the studies reported a significant connection between the RGD imaging and integrin α_vβ₃ expression,^13,14 some studies pointed out that only assessing a single time point of the tissue integrin expression may be less accurate than dynamic kinetic studies.¹⁴

3PRGD2 was already prepared to use in a freeze-dried kit by our collaborator, Professor Fan Wang.¹⁵ The 3PRGD2 kit was easily labeled with ^99mTcO₄⁻ with high radiochemical purity and high specific activity to provide ^99mTc-3PRGD2 for single-photon emission computed tomography (SPECT). ^99mTc-3PRGD2 SPECT can scan tumors and monitor the treatment effect in anticancer research, especially in targeting tumor angiogenesis.¹⁵

VX2 is a Shope papillomavirus-associated squamous cell carcinoma.¹⁶ It has affluent vascularity and grows quickly when implanted in muscle, liver, and other organs of large animals, such as rabbits, for many research purposes.¹⁷ Studies found heterogeneous expression of α_vβ₃ integrin predominantly in tumor vascularity and peripheral parenchyma, with virtually no vascular expression in the necrotic core of the lesion.^18–20

To evaluate ^99mTc-3PRGD2 SPECT's potential value in monitoring chemotherapeutic effects, we established a squamous cell VX2 rabbit tumor model to explore the biometabolic distribution of ^99mTc-3PRGD2 with a continuous dynamic acquisition mode before treatment, on day 0 (immediately after cisplatin), and days 1, 7, and 14 after cisplatin treatment.

Materials and Methods

Animals and VX2 Tumor Model

This study protocol was approved by the Laboratory Animal Sciences Center of Shanghai Jiao Tong University.

Twenty-two male New Zealand rabbits (2.0-2.5 kg) were used, including four VX2 tumor donor rabbits. The VX2 tumor was surgically removed from a donor rabbit under general anesthesia (20 mg/kg 1.5% pentobarbital) administered through an indwelling catheter in the auricular vein and cut into 1 mm³ pieces. Following anesthesia, each recipient rabbit was fixed in a supine position; two 10 mm deep tunnels were made bilaterally into the skin of the front extremities, and one piece of 1 mm³ VX2 tissue was implanted into each tunnel. The incisions were closed with 3-0 sutures. The rabbits were used for experimentation when the tumors had grown to approximately 10 to 30 mm in diameter.

Experimental Design

Eighteen rabbits with a total of 27 tumors were randomly divided into a nontreated control group (NTG, n = 8; 12 tumors) and a treatment group (TG, n = 10; 15 tumors). The TG received a single dose of intravenous cisplatin (4 mg/kg) injection.^21,22 ^99mTc-3PRGD2 imaging was performed before treatment and then on day 0 (immediately after cisplatin treatment) and days 1, 7, and 14 posttreatment (Figure S1, online version only); ^99mTc-3PRGD2 images were acquired at the same time points in the NTG. Three rabbits in each group were sacrificed on day 1 after imaging, and the tumors were excised for H&E staining, autoradiography, and CD31 and integrin α_v immunohistochemical staining.

Radiopharmaceutical Preparation

The 3PRGD2 kits were kindly provided by Professor Wang-Fan (Medical Isotopes Research Center, Peking University, Beijing, China) and stored at 4°C. For the ^99mTc radiolabeling, 1 to 1.5 mL of 740 to 1,110 MBq (20–30 mCi) of ^99mTcO₄⁻ saline solution was added to the vial, which was placed in a water bath at 100°C for 20 minutes.¹⁵ The solution was analyzed by instant thin-layer chromatography (ITLC) using silica-gel paper strips (Gelman Science, Ann Arbor, MI) using acetone-saline (1:1, v/v) as an eluent. The radiochemical purity was > 97%. The reaction mixture was diluted to approximately 370 MBq/mL (10 mCi/mL) with saline and was filtered with a 0.20 μm Millex-LG filter (EMD Millipore, Bedford, MA). Each rabbit was injected with 37 to 74 MBq/kg (1-2 mCi/kg) of ^99mTc-3PRGD2 in a bolus via the auricular vein for dynamic imaging.

^99mTc-3PRGD2 Imaging Protocol

Images were acquired using a Philips Precedence 6 SPECT/CT Imaging System (Philips Medical Systems, Cleveland, OH) with a dual-head γ camera, using low-energy, high-resolution collimators and a 20% energy window centered on 140 keV. The rabbits were given a combination of anesthesia consisting of 30 mg/kg intravenous pentobarbital and 1 mL/kg intramuscular Shumianxin (846 compounds; Academy of Military Medical Sciences, Chang Chun, JiLin, China) and positioned supine. The image field included the implanted tumor sites, the front extremities, head, chest, abdomen, and pelvic cavity. Both anterior and posterior images were acquired during each of the dynamic and static scanning procedures.

Ten rabbits in the TG and four rabbits in the NTG underwent the following imaging procedure (Figure S2, online version only). Immediately after intravenous injection of ^99mTc-3PRGD2, dynamic imaging was performed in two steps: the first step acquired 30 × 2 s/frames for 60 seconds, and the second step acquired 29 × 1 min/frames for 29 minutes (64 × 64 matrix; zoom 1). Three-minute static scans were performed immediately after dynamic imaging and at 1 hour, 1.5 hours, 2 hours, and 3 hours post–^99mTc-3PRGD2 injection (256 × 256 matrix; zoom 1). Integrated SPECT/computed tomography (CT) was performed at 3 hours to acquire CT images (512 × 512 matrix) and SPECT images (64 × 30 s/frame/5.625°; 64 × 64 matrix; zoom 1).

The other four rabbits in the NTG underwent six half-hour dynamic scans during the first 3 hours after injection of ^99mTc-3PRGD2, which was prior to the integrated SPECT/CT scanning (Figure S3, online version only). The first half-hour scan included two continuous imaging programs as before (30 × 2 s/frames for 60 seconds, 29 × 1 min/frames for 29 minutes). The next five half-hour scans also underwent dynamic imaging (30 × 1 min/frames for 30 minutes).

Image Interpretation and Data Analysis

All images were viewed and processed using PMOD version 3.3 software (PMOD Technologies, Zurich, Switzerland). Region of interest (ROI) analysis was used for semiquantification of the results. The ROI of tumor, head, heart, liver, kidneys, bladder, and normal background area (N) were defined. The T/N (tumor/normal contralateral front extremities) ratios in planar images and the time to reach peak (peak time)²³ in the dynamic images were calculated to compare the differences between the two groups. The change rates of T/N ratios were calculated as (T/N_dn- T/N_pre)/T/N_pre, where T/N_dn is the T/N ratio on day n and T/N_pre is the T/N ratio before treatment. The change rates of the peak time on every latter day were calculated in the same way as (PT_dn – PT_pre)/PT_pre, where PT_dn is the peak time on day n and PT_pre is the peak time before treatment.

According to the CT measurement, tumor volume (mm³) was calculated using the following formula: (W × L × H × π/6), where W was the longest width of the tumor, L was the longest length perpendicular to W, and H was the height of the tumor.

Autoradiography

Tumor tissues were resected and immediately snap-frozen in optimum-cutting-temperature compound (Sakura Finetek, Northbrook, IL). The blocks were cryosectioned into six consecutive 5 μm slices. Two slices were stained using H&E, and four slices were prepared for autoradiography. The autoradiography procedure was as follows: slices were dried in open air and exposed to BAS-SR 2025 Fuji phosphorus film for 24 hours; then the film was scanned using a TYPHOON Multifunctional Imaging System 7000 (GE Healthcare Life Sciences, Piscataway, NJ).

Immunohistochemistry

Three VX2 tumor models in the TG and NTG were sacrificed on day 1 after the imaging, and the tumors were resected for H&E and immunohistochemical staining. The tumor tissues were fixed in 10% formalin solution and then embedded in paraffin to prepare serial sections. Immunohistochemical staining was performed by the streptavidin-biotin complex (SABC) method following deparaffinization and rehydration and treated with antigen retrieval. The primary antibodies used were a mouse monoclonal CD31 antibody (ab9498, Abcam Inc., Cambridge, MA) at a 1:50 dilution and a rabbit polyclonal anti-integrin α_v antibody (ab76609, Abcam Inc.) at a 1:200 dilution and were reacted overnight at 4°C. Subsequently, the biotinylated secondary antibody and SABC complex were applied and diaminobenzidine (DAB) substrate was used to stain the sections. The sections were then treated with hematoxylin-eosin (H&E) and mounted.

The images in 2,088 × 1,550 pixels were acquired with an Olympus BX51 microscope and processed by Leica QWin version 3.4.0 software. The slides were initially screened at low power to identify the areas with the largest numbers of stained hot spots. The mean values of immunostained areas in pixels and the percentage of immunostained area per microscopic field were calculated in 200X magnification in the four most immunostained areas.

Statistical Analysis

All data were analyzed using SPSS version 19 statistical software (IBM Corporation, Armonk, NY). Differences were calculated using the Student t-test; p values < .05 were considered significant, and p values < .001 were considered highly significant.

Results

Changes in Tumor Volumes

There was no significant difference in tumor volume between the TG and the NTG pretreatment, on day 0, or on day 1 after therapy (p > .05). On day 7, the tumor volume was significantly lower in the TG compared with the NTG (p < .05). On day 14, no significant difference was observed between the two groups (p > .05) (Figure 1).

Figure 1.

Changes in tumor volumes between the treatment group (TG) and the nontreated control group (NTG) before and after cisplatin administration.

Dynamic Image Analyses

Flow Phase Comparisons

The first peak in the flow phase was observed in the tumors 20.56 ± 5.76 seconds after it was observed in the heart. There were no significant differences (p > .05) in peak time and its change rate between the TG and the NTG throughout the treatment period.

Changes and Comparisons in the Blood-Pool Phase in 1-Minute-per-Frame Scans

The tumor radioactivity of all of the animals reached a peak at 8.69 ± 1.22 minutes after injection of ^99mTc-3PRGD2 before treatment. The peak time (Table 1) and its change rate (Table 2) in the TG and NTG were significantly different on days 0, 1, and 7 (p < .05). No significant differences were found on the day of pretherapy and day 14 (p > .05).

Table 1.

Peak Time (min) of ^99mTc-3PRGD2 Uptake in Tumors over 30 Minutes (1 min/frame)

Group	Pretherapy	Day 0	Day 1	Day 7	Day 14
Treatment	8.88 ± 2.93	10.71 ± 5.25	11.09 ± 4.13	13.12 ± 8.27	9.66 ± 5.88
Nontreated control	7.25 ± 2.99	6.36 ± 3.49	6.77 ± 4.25	8.10 ± 4.80	8.89 ± 6.04
p value	.373	.032^*	.014^*	.041^*	.815

p < .05.

Table 2.

Change Rate (%) of Peak Time of ^99mTc-3PRGD2 Uptake in Tumors over 30 Minutes (1 min/frame)

Group	Day 0 (%)	Day 1 (%)	Day 7 (%)	Day 14 (%)
Treatment	49.43 ± 65.37	56.20 ± 48.94	52.16 ± 26.96	52.82 ± 53.27
Nontreated control	–15.28 ± 18.30	–6.20 ± 41.75	16.56 ± 50.31	20.07 ± 46.62
p value	.019^*	.027^*	.044^*	.535

p < .05.

Biometabolic Distribution of ^99mTc-3PRGD2

The tracer was distinctly distributed and appeared in the heart, liver, kidney, bladder, joint, intestine, and tumor. It was mostly excreted from the kidney to the bladder and partly from the liver to the intestine.

The radioactivity in the heart reached a sharp peak within 10 seconds after injection of ^99mTc-3PRGD2 (Figure 2A). The radioactivity washout in heart followed an exponential decay; the t_1/2 of the tracer in heart was 3.43 ± 0.94 minutes (Figure 2B). The radioactivity in the liver also followed an exponential decay, and its t_1/2 was 13.41 ± 9.17 minutes (Figure 2C). The radioactivity in kidneys was observed approximately 4 to 6 seconds after the heart, and its washout rate depended on the urine volume and urine drainage rate.

Figure 2.

Biometabolic distribution of ^99mTc-3PRGD2 in dynamic scanning. A, Dynamic curves of ^99mTc-3PRGD2 uptake in tumor, heart, liver, right kidney, and left kidney (2 s/frame). B–D, Dynamic uptake of ^99mTc-3PRGD2 during 3-hour scanning showing the curves of Total(SUM), their power functions (y), and correlation coefficients (R²) in heart, liver, and tumor, respectively. E, The reciprocal of the uptake counts, 1/Total(SUM), from the peak to the end, in the tumor, showing the linear function (y), correlation coefficient (R²), and trend line (in red).

The peak time in the NTG and TG was 8.88 ± 2.93 and 7.25 ± 2.99, respectively, pretherapy (Figure 2D). No significant difference was found between the NTG and the TG pretherapy (p = .373 > .05). The reciprocal of the tumor uptake counts, (1/Total (SUM)), from the peak to the end point, was a linear function (R² > .90) (Figure 2E) from which we could obtain the t_1/2 of the tracer in tumor, which was 70.83 ± 33.37 minutes.

Static Image Analyses

The tumors were clearly observed postinjection of ^99mTc- 3PRGD2, and the T/N ratios increased with the time. No significant difference in T/N values was seen between the TG and the NTG before chemotherapy (p > .05). The radiolabeled tracer uptake in the tumor and T/N values decreased in the TG after the treatment, whereas it was increased in the NTG (Figure 3). The T/N values on day 7 and the change rates of T/N values on all of the acquisition days (days 0, 1,7, and 14) were significantly different (p < .05) between the two groups at 3 hours postinjection (Table 3, 4).

Figure 3.

Representative images in the treatment group (TG) and the nontreated control group (NTG) acquired on pretreatment day and days 0, 1, 7, and 14 at 3 hours postinjection. A, Static images in the NTG. B, SPECT-integrated CT images for the same rabbit in the NTG. C, Static images in the TG. D, SPECT/CT-integrated images for the same rabbit in the TG.

Table 3.

Tumor to Normal (T/N) Values Determined from ^99mTc-3PRGD2 Scans at 3 Hours on the Different Acquisition Days

Group	Pretherapy	Day 0	Day 1	Day 7	Day 14
Treatment	2.46 ± 0.19	2.28 ± 0.20	2.31 ± 0.23	1.89 ± 0.26	2.07 ± 0.23
Nontreated control	2.29 ± 0.23	2.38 ± 0.22	2.54 ± 0.23	2.26 ± 0.27	2.67 ± 0.46
p value	.32	.48	.17	.04^*	.06

p < .05.

Table 4.

Change Rate (%) of Tumor to Normal (T/N) Values at 3 Hours Postinjection ^99mTc-3PRGD2 on Different Acquisition Days

Group	Day 0(%)	Day 1(%)	Day 7(%)	Day 14(%)
Treatment	–14.73 ± 15.93	–15.89 ± 8.83	–18.91 ± 19.17	–9.74 ± 25.06
Nontreated control	7.75 ± 16.95	11.39 ± 22.60	14.30 ± 28.07	43.71 ± 95.57
p value	.020^*	.016^*	.037^*	.038^*

p < .05.

H&E Staining and Autoradiography

H&E staining showed richer presence of deeply stained nucleus in the NTG than in the TG; however, many more necrotic cells showed in the TG than in the NTG. Corresponding autoradiography showed higher intensity in the NTG compared to the TG (Figure 4).

Figure 4.

Representative images showing hematoxylin-eosin (H&E) staining (A, B) and autoradiography (C, D) in the treatment group (TG) (A, C) and the nontreated control group (NTG) (B, D) (samples acquired on day 1 after the imaging; original magnification X200).

Immunostained Areas in Two Groups

Immunohistochemistry analysis was performed for CD31 (Figure 5, A and B) and integrin α_v (Figure 5 C and D). The immunostained areas for CD31 and integrin α_v were more highlighted around the tumor mass than within the tumor mass and the more necrotic areas. Greater immunostaining was seen on the slices of the NTG than the TG. The mean immunostained CD31 areas in the NTG and TG were 257,537 ± 149,984 and 100,224 ± 32,998 pixels, respectively (p < .05). The mean percentage of immunostained CD31 area per microscopic field in the NTG and TG was 8.54% ± 4.97% and 3.33% ± 1.09%, respectively (p < .05).

Figure 5.

Representative images in microvessel density (A, B) and immunohistochemistry staining of integrin α_v (C, D) in the nontreated control group (NTG) (A, C) and the treatment group (TG) (B, D) (samples acquired on day 1 after the imaging; original magnification X200).

Figure S1.

Diagram of the overall experimental design.

Figure S2.

Procedure of dynamic and static ^99mTc-3PRGD2 image acquisition for the experiments (10 treated rabbits and 4 control rabbits).

Figure S3.

Procedure of continuous dynamic and intermediate static ^99mTc-3PRGD2 image acquisition in four nontreated tumor models to evaluate the characteristics of biometabolic distribution of ^99mTc-3PRGD2 in the heart, liver, kidneys, and tumor.

The mean immunostained areas for integrin α_v in the NTG and TG were 303,317 ± 98,407 and 172,141 ± 55,457 pixels, respectively (p < .05). The mean percentage of immunostained integrin α_v area per microscopic field in the NTG and TG was 9.57% ± 3.21% and 5.47% ± 1.76%, respectively (p < .05).

Discussion

In this study, we investigated the use of ^99mTc-3PRGD2 peptide molecular imaging to monitor the antitumor effects of cisplatin. 3PRGD2 is available in freeze-dried kits, and ^99mTc-3PRGD2 has high radiochemical purity (> 95%). ^99mTc-3PRGD2 has rapid blood clearance, with < 1% of the initial radioactivity remaining in the blood 60 minutes postinjection. Jia and colleagues found moderate accumulation in the heart and liver at 10 minutes postinjection, but it decreased rapidly from 10 to 40 minutes postinjection.¹⁵

Our study obtained more concrete results by the continuous dynamic 3-hour acquisition and showed the tracer washout and decay of ^99mTc-3PRGD2 in heart and liver followed by exponential decay, with half-lives of 3.43 ± 0.94 minutes and 13.41 ± 9.17 minutes, respectively. The tracer washout in the tumor was 70.83 ± 33.37 minutes, and the T/N ratio increased with time. The highest uptake was at 3 hours postinjection of ^99mTc-3PRGD2. These results were consistent with the multicenter study in humans.²⁴ Our study verified that the more exact radiotracer washout and decay curves could be obtained in different organs in vivo through the continuous dynamic scanning and more parameters and results could be obtained from those curves; thus, it was very important and helpful in the new tracer study.

Microvascular density (MVD) is significantly associated with tumor metastasis and prognosis and used to monitor the response to cancer treatment.¹² Studies reported that tumors over 0.5 cm³ and the %ID/cm³ uptake of ^99mTc-3PRGD2 decreased due to necrosis.^25,26 In our study, after cisplatin chemotherapy, relatively low radioactivity was observed in the necrotic areas. H&E staining and immunohistochemistry verified the significant decrease in microvessel areas and the percentage of immunostained area per microscopic field in the TG compared to the NTG (p < .05). The results were consistent with the Schirner and colleagues study, in which all chemotherapeutic agents led to a reduction in MVD in their Caki-1 nude mouse xenograft model.⁶

We continuously studied the dynamic biometric distribution of ^99mTc-3PRGD2 acquisition and longitudinally took the images over 14 days. Our study showed that the peak time in tumor could predict the early treatment effect of cisplatin. The peak time delayed immediately after the administration of cisplatin, the T/N values, and the tumor volume were significantly different between the TG and the NTG on day 7 after the treatment. These effects may be caused by the cytotoxic and antiangiogenic properties of cisplatin. Several in vitro studies have shown that cisplatin induces the release of interleukin-1 and interleukin-6 and decreases matrix metalloproteinase 2 and urokinase-type plasminogen activator (uPA), suggesting that cisplatin may inhibit angiogenesis by inhibiting endothelial cell proliferation.² In vivo studies have investigated the use of RGD peptide molecular imaging techniques to monitor the antiangiogenic effects of cytotoxic chemotherapy drugs.^12,27 Our study results verified that ^99mTc-3PRGD2 imaging could be used to monitor the antitumor effect of cisplatin. The reduction in tumor ^99mTc-3PRGD2 uptake was followed by the reduction in tumor angiogenesis presented by immunostained CD31 and integrin α_v. Thus, ^99mTc-3PRGD2 SPECT/CT imaging may have potential in monitoring and evaluating the downstream biological effects of specific antiangiogenic target drugs, such as linifanib,²⁸ and traditional chemotherapy drugs.

The shortcoming of our study was the relatively small number of experiment animals. Further studies will be continued to carry on the research to validate the results.

Conclusion

Our study showed that the peak time obtained by dynamic ^99mTc-3PRGD2 imaging in tumors could detect the early treatment effect of cisplatin. The peak time delayed immediately after the administration of cisplatin, whereas the T/N values and tumor volume were significantly different between the TG and the NTG on day 7 after the treatment. Thus, the peak time in the dynamic phase of ^99mTc-3PRGD2 SPECT/CT imaging may be an early parameter in monitoring treatment effects.

Footnotes

Financial disclosure of authors: This work was funded by the National Natural Science Foundation of China (grant nos. 81101073, 81471708, 81471685, 81530053), the Shanghai Pujiang Program 11PJD018, 973 Project (No. 2012CB932604), and the New Drug Discovery Project (No. 2012ZX09506-001-005).

Financial disclosure of reviewers: None reported.

References

Hanahan

Bergers

Bergsland

. Less is more, regularly: metronomic dosing of cytotoxic drugs can target tumor angiogenesis in mice. J Clin Invest 2000;105:1045–7, doi:10.1172/JCI9872.

Miller

Sweeney

Sledge

Jr . Redefining the target: chemotherapeutics as antiangiogenics. J Clin Oncol 2001;19: 1195–206.

Einhorn

Williams

. The role of cis-platinum in solidtumor therapy. N Engl J Med 1979;300:289–91, doi:10.1056/NEJM197902083000605.

Williams

Whitehouse

. Cis-platinum: a new anticancer agent. Br Med J 1979;1:1689–91, doi: 10.1136/bmj.1.6179.1689.

Yoshikawa

Saura

Matsubara

Mizuno

. A mechanism of cisplatin action: antineoplastic effect through inhibition of neovascularization. Kobe J Med Sci 1997;43:109–20.

Schirner

Hoffmann

Menrad

Schneider

. Antiangiogenic chemotherapeutic agents: characterization in comparison to their tumor growth inhibition in human renal cell carcinoma models. Clin Cancer Res 1998;4:1331–6.

Barczyk

Carracedo

Gullberg

. Integrins. Cell Tissue Res 2010;339:269–80, doi:10.1007/s00441-009-0834-6.

Takada

Simon

. The integrins. Genome Biol 2007;8:215, doi:10.1186/gb-2007-8-5-215.

Schottelius

Laufer

Kessler

Wester

. Ligands for mapping α_vβ3-integrin expression in vivo. Acc Chem Res 2009; 42:969–80, doi:10.1021/ar800243b.

10.

Liu

Wang

. Development of RGD-based radiotracers for tumor imaging and therapy: translating from bench to bedside. Curr Mol Med 2013;13:1487–505, doi:10.2174/1566524013666131111115347.

11.

Schottelius

Laufer

Kessler

Wester

. Ligands for mapping α_vβ3-integrin expression in vivo. Acc Chem Res 2009; 42:969–80, doi:10.1021/ar800243b.

12.

Morrison

Ricketts

Barnett

. Use of a novel Arg-Gly-Asp radioligand, ¹⁸F-AH111585, to determine changes in tumor vascularity after antitumor therapy. J Nucl Med 2009;50:116–22, doi:10.2967/jnumed.108.056077.

13.

Haubner

Weber

Beer

. Noninvasive visualization of the activated α_vβ3 integrin in cancer patients by positron emission tomography and [18F]Galacto-RGD. PLoS Med 2005;2(3):e70, doi: 10.1371/journal.pmed.0020070.

14.

Zhang

Xiong

. Quantitative PET imaging of tumor integrin α_vβ3 expression with 18F-FRGD2. J Nucl Med 2006;47: 113–21.

15.

Jia

Liu

Zhu

. Blood clearance kinetics, biodistribution, and radiation dosimetry of a kit-formulated integrin α_vβ3-selective radiotracer ^99mTc-3PRGD2 in non-human primates. Mol Imaging Biol 2011;13:730–6, doi:10.1007/s11307-010-0385-y.

16.

Kidd

Rous

. Cancer deriving from virus papillomas of wild rabbits under natural conditions. J Exp Med 1940;71:469–94, doi: 10.1084/jem.71.4.469.

17.

Wang

Bangash

Rhee

. Liver tumors: monitoring embolization in rabbits with VX2 tumors-transcatheter intraarterial first-pass perfusion MR imaging. Radiology 2007;245:130–9, doi:10.1148/radiol.2451061689.

18.

Winter

Caruthers

Kassner

. Molecular imaging of angiogenesis in nascent Vx-2 rabbit tumors using a novel α_vβ3- targeted nanoparticle and 1.5 tesla magnetic resonance imaging. Cancer Res 2003;63:5838–43.

19.

Schmieder

Winter

Williams

. Molecular MR imaging of neovascular progression in the Vx2 tumor with α_vβ3- targeted paramagnetic nanoparticles. Radiology 2013;268:470–80, doi: 10.1148/radiol.13120789.

20.

Lijowski

Caruthers

. High sensitivity: high-resolution SPECT-CT/MR molecular imaging of angiogenesis in the Vx2 model. Invest Radiol 2009;44:15–22, doi:10.1097/RLI.0b013e31818935eb.

21.

Song

Liu

Huang

. Changes in ¹⁸F-FDG uptake within minutes after chemotherapy in a rabbit VX2 tumor model. J Nucl Med 2008;49:303–9, doi:10.2967/jnumed.107.044206.

22.

Song

Deng

Wen

. ¹⁸F-FDG PET/CT-related metabolic parameters and their value in early prediction of chemotherapy response in a VX2 tumor model. Nucl Med Biol 2010;37:327–33, doi:j.nucmedbio.2009.12.002.

23.

Kersemans

Cornelissen

Kersemans

. In vivo characterization of ^123/125I-2-iodo-L-phenylalanine in an R1M rhabdomyosarcoma athymic mouse model as a potential tumor tracer for SPECT. J Nucl Med 2005;46:532–9.

24.

Zhu

Miao

. ^99mTc-3PRGD2 for integrin receptor imaging of lung cancer: a multicenter study. J Nucl Med 2012; 53:716–22, doi: 10.2967/jnumed.111.098988.

25.

Zhou

Shao

Liu

. Monitoring breast tumor lung metastasis by U-SPECT-II/CT with an integrin α_vβ3-targeted radiotracer (99m)Tc-3P-RGD(2). Theranostics 2012;2:577–88, doi:10.7150/thno.4443.

26.

Shao

Zhou

Wang

Liu

. Monitoring glioma growth and tumor necrosis with the U-SPECT-II/CT scanner by targeting integrin αvβ3. Mol Imaging 2013;12:39–48.

27.

Jung

Lee

Paik

. Favorable biokinetic and tumor-targeting properties of ^99mTc-labeled glucosamino RGD and effect of paclitaxel therapy. J Nucl Med 2006;47:2000–7.

28.

Zhou

Voorbach

. Monitoring tumor response to linifanib therapy with SPECT/CT using the integrin αvβ3-targeted radiotracer ^99mTc-3P-RGD2. J Pharmacol Exp Ther 2013;346: 251–8, doi: 10.1124/jpet.112.202622.

Biometabolic Distribution of 99m Tc-3PRGD2 and its Potential Value in Monitoring Chemotherapeutic Effects

Abstract

Materials and Methods

Animals and VX2 Tumor Model

Experimental Design

Radiopharmaceutical Preparation

99mTc-3PRGD2 Imaging Protocol

Image Interpretation and Data Analysis

Autoradiography

Immunohistochemistry

Statistical Analysis

Results

Changes in Tumor Volumes

Dynamic Image Analyses

Flow Phase Comparisons

Changes and Comparisons in the Blood-Pool Phase in 1-Minute-per-Frame Scans

Biometabolic Distribution of 99mTc-3PRGD2

Static Image Analyses

H&E Staining and Autoradiography

Immunostained Areas in Two Groups

Discussion

Conclusion

Footnotes

References

^99mTc-3PRGD2 Imaging Protocol

Biometabolic Distribution of ^99mTc-3PRGD2