Sub Topic | Secondary Topic: Biotherapeutics and Biotechnology - Bioassay | Activity Based Assays (Mainly for Product Characterization)
Authors: Yu-Ping Yang, University of Miami (Main Author, Presenting Author); Ram Datar, University of Miami; Richard Cote, University of Miami; Sylvia Daunert, University of Miami
Presenting Author: Yu-Ping Yang
Purpose: Enumeration of circulating tumor cells (CTCs) have shown a great value as a surrogate marker for patient prognosis and therapy response. Characterization of CTCs also provides novel insight into personalized medicine for guiding treatment decisions. However, widespread utilization of CTC assays in a clinical setting is still challenged due to multiple procedural steps of current techniques. In most cases, identifying CTCs remains on the use of a gold standard method, immunocytochemistry (ICC) that is a labor-intensive and time-consuming procedure. Moreover, ICC requires a fixation step, resulting in the limitation of post-detection analysis of CTCs, such as single-cell analysis and ex vivo culture expansion. In this study, we develop the dual bioluminescence/fluorescence assay for rapid detection of circulating tumor cells without a fixation step, and subsequently characterization of those viable CTCs in single-cell level.
Methods: Gaussia luciferases (GLuc) were genetically fused with three different antibody mimetics respectively, which serves as targeting molecules that bind specifically to tumor surface antigens (EpCAM, HER2, and EGFR). The antibody-mimetic-GLuc fusion proteins were expressed in the E. coli expression system, and purified using affinity chromatography. A panel of the breast cancer cell lines was employed for determining the targeting specificity of fusion proteins. The cell viability assay was used to determine the toxic effect of the assay on tumor cells. The fusion proteins were conjugated with fluorescent dye for identifying individual tumor cells for single-cell analysis. Spiking experiments and metastatic mouse models were used to investigate the feasibility of the dual functional assay.
Results: The fusion proteins were able to specifically bind to the target tumor cells but not leukocytes. Additionally, multi-marker combination strategy for CTC detection was used to address the issue of the heterogeneous nature of CTCs and consequently, improved the detection sensitivity. With a combination of density gradient CTC enrichment technique, our assay showed high sensitivity in detecting spiked tumor cells with no observed cell toxicity. Subsequently, those spiked tumor cells were able to grow in culture. Moreover, incorporation of fluorescent dyes with the fusion proteins was able to identify individual tumor cells spiked in blood after bioluminescent detection, and those cells can be precisely selected by fluorescence-based single-cell isolation devices for single-cell analysis. In the metastatic animal model, we demonstrated the dual functional assay was able to detect CTCs in the blood of tumor-bearing mice. Following the detection step, expansion of those CTCs in culture can be achieved and individual CTCs can be identified for downstream characterization.
Conclusion: Our study shows that the dual functional assay is capable of rapidly detecting viable CTCs and permits further downstream analysis of these cells such as ex vivo culture and single-cell analysis. Our assay will have the potential point-of-care application in clinical settings as a diagnostic and prognostic tool for CTC detection as well as allow for characterization of CTCs for guiding treatment decision.
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