Abstract 5192: Detection of disseminated prostate cancer cells in bone marrow using tyramide signal amplification

Abstract

Abstract Approximately 28,000 men die of prostate cancer (PCa) each year in the US, and 90-100% of them are due to bone metastasis. It has been demonstrated that PCa cells detach from the primary tumor, “home” to bone, and develop into these incurable metastases. Existing methods of detecting cells early that can lead to bone metastasis have limited sensitivity and current efforts involve liquid biopsy assays to capture and detect disseminated and circulating tumor cells (DTCs and CTCs respectively). A significant challenge during this detection lies in the optimization of the signal-to-noise ratio during immunofluorescent staining. Noise during immunostaining can originate from endogenous immune cells and other bone marrow matrix components and lead to false staining which reduces sensitivity. We have developed an optimized protocol to reduce background noise from these factors while also enhancing signal using tyramide signal amplification (TSA). Following immuno-staining with PCa cell-specific markers, TSA amplifies beyond primary and secondary antibody binding using horseradish peroxidase (HRP) and fluorophore binding. To further apply this novel signal amplification protocol to the visualization of disseminated tumor cells in bone marrow, we will detect, quantify, and image parental and modified PCa cells injected into murine blood. We have previously shown that C-X-C chemokine receptor 4 causes PCa cells to home to bone. We have stably overexpressed CXCR4 in several PCa cell lines and will be able to determine the role of CXCR4 expression on the development of bone metastases. This robust protocol will be quickly modifiable for automated staining to yield higher throughput analysis. Citation Format: Sounak Roy, Kenneth C. Valkenburg, Kenneth J. Pienta. Detection of disseminated prostate cancer cells in bone marrow using tyramide signal amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5192.