Abstract 2296: Engineering and evaluation of a novel circulating tumor cell capture assay for urothelial carcinoma
Authors: Zhou Qinhan , Poellmann Michael , Sultanpuram Nikhila Reddy , Gunenc Damla , English Krista , Chen Luke , Zhao Runze , Hong Seungpyo , Wang Andrew , Zhang Tian 
Published: June 2023

Abstract
Background: Circulating tumor cells (CTCs) are promising real-time, noninvasive biomarkers to assess therapy response and to monitor disease. However clinical applications of CTCs in urothelial carcinomas (UC) remain limited. There is a need for sensitive and disease-specific CTC assay for urothelial cancer. As CTCs are very rare in blood compared to blood cells and may vary due to tumor heterogeneity, detection and isolation of them solely based on one biomarker (e.g., EpCAM) would be insufficient. To achieve better results and increase clinical relevance, we designed a CTC capture device that was functionalized with multiple antibodies specific to UC.
Methods: We engineered a novel UC-CTC capture device using nanotechnology and biomimetic cell rolling. To capture UC cells, we examined 4 biomarkers: EpCAM, Nectin-4, Trop-2, and FGFR3, that are highly expressed in UC. We engineered CTC assays using single and multi-biomarker capture surfaces. The capture sensitivity and selectivity of the device were investigated using four bladder cancer cell lines as well as a leukocyte model, HL-60, as negative control. CTCs are defined as CK+/DAPI+/CD45- cells. The capture sensitivity was compared between the different surfaces with each of the different antibodies and the final UC-CTC chip with optimized antibody mix was chosen to detect CTCs from UC patients.
Results: We examined the CTC capture using 4 UC cell lines: UM-UC-1, T-24, HT1376, RT4. The devices that utilized single antibodies were much less sensitive (capture efficiencies less than 82%). In comparison, combination antibody capture improved sensitivity to &gt;90%. Negative control using the HL-60 cells showed minimal non-specific binding, &lt;0.3%. We were also able to validate the CTC assay using patient samples and demonstrated high sensitivity. For example, we captured ~302 CTCs/mL in a patient who had early-stage T2 muscle invasive UC.
Conclusion: We developed a novel CTC capture device using four UC-specific capture antibodies for the detection and isolation of CTCs in UC. Compared to the configuration of EpCAM alone, the mixture of multiple antibodies improved the capture efficiency up to 1.4-fold for in vitro bladder cancer cell spiking assays. High specificity was also achieved, with low levels of non-specific leukocyte detection. High sensitivity and specificity suggest that this platform has potential impact to detect CTCs from patients with UC.
Citation Format: Qinhan Zhou, Michael Poellmann, Nikhila Reddy Sultanpuram, Damla Gunenc, Krista English, Luke Chen, Runze Zhao, Seungpyo Hong, Andrew Wang, Tian Zhang. Engineering and evaluation of a novel circulating tumor cell capture assay for urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2296.