Abstract
Diagnostic Technology
Cancer Cell Profiling by Barcoding Allows Multiplexed Protein Analysis in Fine-Needle Aspirates
The use of biopsies to obtain tissue samples for patient diagnostics remains a widely used method. Conventional approaches include the subsequent use of immunohistochemistry that can typically study a limited range of proteins that can serve as biomarkers for cellular response during the course of treatment. Genomic analysis is also conducted to monitor driver oncogenes. These biopsies are typically invasive and conducted several times during the course of treatment, which can challenge the practicality of the approach.
Conventional fine-needle aspirates obtain cells from bodily fluids that are more readily obtainable. However, the limited number of cells available may limit the proteins that can be assayed. An innovative recent study harnesses low cell count fluids for multiplexed protein analysis using a photocleavable DNA barcode system that is able to uniquely tag antibodies for the subsequent analysis of clinical lung cancer samples. This work demonstrates the translational potential of this technology and its ability to interrogate a broad range of disease models. (Ullal, A.V., et al., Sci. Transl. Med.
Dielectrophoretically-Assisted Electroporation Using Light-Activated Virtual Microelectrodes for Multiple DNA Transfection
Electroporation is a widely used method to introduce genetic material into cells for the purposes of modulating gene expression or inducing RNA interference, for example. Current electroporation methods and their voltage requirements can sometimes lead to cell membrane damage.
A recent study uses virtual microelectrodes that are photoactivated using dielectrophoresis, which is a well-tolerated approach toward porating cell membranes. Using this technology, triangular microneedles are projected onto cells using a photoconductive layer that effectively creates electric fields that mediate DNA transfection with demonstrated efficacy. An important part of this approach is based on its ability to induce electroporation in a massively parallel and less invasive nature to preserve cell health while optimizing transfection efficacy. (Wang, C. H., et al., Lab Chip
Electrokinetic Stringency Control in Self-Assembled Monolayer-Based Biosensors for Multiplex Urinary Tract Infection Diagnosis
Urinary tract infections (UTIs) are serious issues that often accompany other medical disorders. UTIs themselves have been known to cause serious complications, such as premature birth, and have also been associated with mortality in critically ill patients following catheter use. With regard to UTI therapy, the administration of antibiotics is a common course. However, an increase in drug-resistant strains of bacteria can decrease the efficacy of treatment. Given the prevalence of UTIs, the development of increased sensitivity and specificity of treatment is becoming increasingly important.
A recent study uses a monolayer electrochemical electrode sensor approach to harness electrokinetic stringency control to concentrate bacterial nucleic acid samples using electrothermal fluid motion. The ability to focus the bacterial 16S ribosomal RNA (rRNA) over the electrode decreases the background noise in clinical urine samples by 60%. Furthermore, this device impressively detects similar 16S rRNA samples from three clinical samples. This work demonstrates the clinical relevance of this approach due to the ability to process clinical urine with high specificity and sensitivity. (Liu, T., et al., Nanomed. Nanotech. Biol. Med.
Quantitative Diagnosis of Malignant Pleural Effusions by Single-Cell Mechanophenotyping
Changes in cellular mechanical properties can serve as indicators of cell health and diagnostic markers associated with disorders such as cancer. Cellular phenotyping based on these characteristics enables accurate diagnosis that is label free and can identify cancerous cells that are hard to track using conventional cytology.
An exciting methodology that harnesses single-cell mechanophenotyping, or deformability cytometry (DC), demonstrates that hard-to-identify malignant cells from pleural effusions can be rapidly sorted at a rate of 1000 cells per second, a rate that is comparable to conventional flow cytometry. Currently, certain samples present a challenge to cytopathologists in terms of accurate diagnosis, and these samples require further analysis. The DC approach enables high confidence classification in more than half of the cases that are analyzed. This strategy may direct cytopathologists to focus on specific/smaller populations of cells during the course of diagnosis to improve accuracy and efficiency. As label-free methods for cellular phenotyping continue to evolve toward clinical application, DC serves as a promising, translationally relevant approach that can be applied to a broad range of cell types. (Tse, H. T. K., et al., Sci. Transl. Med.
Microfluidic CD4+ and CD8+ T Lymphocyte Counters for Point-of-Care HIV Diagnostics Using Whole Blood
The ability to rapidly diagnose human immunodeficiency virus (HIV) in resource-limited settings remains a challenge, as comprehensive cellular processing is often laborious and expensive. In addition, the operation of equipment that is typically needed requires specialized training and personnel. Therefore, a fully integrated point-of-care diagnostic could potentially transform the way that patient HIV diagnostics are conducted and markedly enhance the reach of this technology in resource-limited environments.
A technology with integrated modules that are capable of erythrocyte lysis, leukocyte preservation, isolates target CD4+ and CD8+ T lymphocytes, and cellular enumeration recently was unveiled. This exciting platform is capable of achieving CD4+ and CD8+ T-cell counts via antibody-functionalized microstructures that are comparable to conventional flow cytometry with a range of 40 to 1000 cells per microliter. Conventional flow cytometry is known to be a costly process that is not portable. The newly reported microfluidic platform is able to complete its diagnosis within 20 min with handheld portability, signifying a promising step toward widespread implementation of rapid and specific HIV diagnosis from a single drop of blood. (Watkins, N. N., et al., Sci. Transl. Med.
Highly Efficient Enrichment of Low-Abundant Cells under Continuous Flow by Standing Surface Acoustic Waves
The ability to sort and isolate cells from highly diluted biological samples is an important technique for a wide variety of biomedical applications, particularly in oncology, immunology, and regenerative medicine-based research and therapy. The advancement of noncontact methods of cell trapping and enrichment is important as this minimizes the mechanical stress and surface interactions that may adversely affect cellular properties. In this report, the authors demonstrate the use of standing surface acoustic waves (SSAW) to trap and enrich cells under continuous flow. This is particularly important as this allows for the use of SSAW in cell isolation and enrichment techniques, such as flow cytometry, where continuous flow is required to isolate cells from a large volume of liquid. SSAW-based cell enrichment is able to successfully enrich human blood cells from highly diluted samples with greater than 90% efficiency and concentrate the cells by a factor of 100 to 1000. With this report, SSAW should be considered a viable approach to noncontact cell enrichment in a wide variety of bioanalytical systems. (Chen, Y., et al., Lab Chip
Drug Development
Gel–Liposome-Mediated Co-Delivery of Anticancer Membrane-Associated Proteins and Small-Molecule Drugs for Enhanced Therapeutic Efficacy
The use of novel materials to improve cancer therapy efficacy and safety has made significant strides over recent years because of their ability to markedly decrease toxicity while improving efficacy. Several compounds that currently serve as clinical standards have been improved via delivery with nanoparticles, particularly against cancers that are drug resistant, where compounds are effluxed or ejected before they have a chance to function. While several important advances have been realized both preclinically and in the clinic with single-drug administration via nanotechnology, recent efforts have sought to deliver multiple compounds to further improve efficacy.
A new study uses a liposome/hyaluronic acid network–based hybrid to co-deliver tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and doxorubicin (Dox), a widely used small-molecule therapeutic in the clinic, which results in a significant enhancement in cytotoxicity of the MDA-MB-231 triple-negative breast cancer line. More specifically, the Dox molecules are packaged within the liposomal core while the TRAIL is delivered in the hyaluronic acid network. Degradation of the hyaluronic acid mediated by the tumor environment results in TRAIL delivery, liposomal internalization, and Dox delivery. The dual drug delivery approach improves the efficacy of preclinical tumor growth inhibition, representing a promising pathway toward multidrug delivery using a single nanomaterial platform. (Jiang, T., et al., Adv. Func. Mat.
Enhancing Tumor Cell Response to Chemotherapy through Nanoparticle-Mediated Codelivery of siRNA and Cisplatin Prodrug
Drug resistance represents a major challenge in chemotherapy that can preclude several classes of compounds from inducing apoptosis in cancer cells. Several strategies pertaining to modifying conventional drugs with nanoparticles have resulted in significant improvements to treatment efficacy and safety by enhancing drug retention. However, synergistic effects can be harnessed when multiple compounds are delivered together.
A recent study realizes an innovative poly(lactide-co-glycolide)-b-poly(ethylene glycol) diblock copolymer platform that simultaneously carries small interfering RNA (siRNA) for the REV1 and REV3L complexes, which had previously been shown to play a role in mediating resistance in relapsing cancers, as well as cisplatin, a platinum-containing chemotherapeutic that crosslinks DNA to induce subsequent apoptosis. Silencing of REV1 and REV3L has been shown to sensitize resistant cancer cells to chemotherapeutic treatment. In vitro and preclinical studies demonstrate that the administration of siRNA/cisplatin-functionalized nanoparticles is markedly more effective in treating a lymph node carcinoma model compared with the administration of unmodified cisplatin. This work provides a pathway for combining siRNA and small-molecule delivery to improve therapeutic outcomes for hard-to-treat cancers. (Xu, X., et al., Proc. Nat. Acad. Sci. U. S. A.
Identifying Novel Regulatory Proteins through High-Throughput High-Content Analysis of siRNA Screens
Screening with siRNA libraries is a powerful tool for identifying novel functions of proteins. Particularly in drug development, siRNA screens can identify novel therapeutic targets as well as identify molecular mechanisms of actions for drugs as well as drug resistance. Traditional siRNA screens rely on a single end-point readout that provides limited information about the effects of knockdown of specific proteins. In this report, the researchers report developing a high-throughput siRNA screening procedure that uses high-content analysis (HCA) to identify lead regulatory protein candidates for estrogen receptor alpha (ERα) protein levels and activity through multiple end points. HCA allows for the analysis of ERα nuclear protein levels, chromatin remodeling, and transcriptional activation. Using this approach, known ERα regulatory proteins are confirmed as well as novel proteins such as ubiquitin protein ligase E3 component n-recognin 5 (UBR5). The application of a high-throughput HCA approach to siRNA screening is an efficient technique that will be useful in a wide range of biomedical and drug development research projects where multipoint analysis will yield greater information and a more intelligent identification of lead candidate proteins. (Bolt, M. J., et al., Oncogene