Abstract 4155: Simultaneous quantitation of multiple signaling molecules in individual circulating tumor cells (CTCs) by multi-color laser scanning cytometry

Introduction: Recent advances in circulating tumor cell (CTC) technology provide a new alternative approach to examine proteomic and molecular characteristic of tumor cells from cancer patients. The current trend in anti-cancer drug development focuses on new compounds that modulate multiple signaling pathways. With CTC detection used as a potential liquid biopsy, the simultaneous examination of signaling molecules for multiple pathways on the same CTC could provide insight into the drug activity in tumor cells. We used proprietary laser scanner cytometry (LSC) technology and developed a multi-color immunofluorescence (IF) procedure to detect 7 biomarkers within the same CTC. We validated this new staining technology using the (1) receptor tyrosine kinase pathways based molecules included total and phospho-IGF-1R, in combination with total and phospho-EGFR, or with total and phospho-ERK1/2, or with total and phospho-AKT (S473) in HT-29 colorectal tumor cells, and (2) same marker expression in CTCs enriched from cancer patients. Purpose: To validate the new multiple marker IF staining procedure and laser scanning cytometry (LSC) platform using ligand treated or untreated tumor cells, and in CTCs enriched from cancer patients. Method: A target biomarker staining panel was combined with standard CTC staining (DAPI, CK+ and CD45-) to create a 7-color IF staining panel to examine marker expression in tumor cell lines and in CTCs from cancer patients. Cultured tumor cells and CTCs enriched from Cellsearch Profile were cytospun onto slides, stained, scanned (by LSC) and analyzed. Results: Using a simultaneous co-staining procedure and LSC-based analysis, we demonstrated that treatment of HT29 cells with IGF1 for 15-30 minutes induced significant increases in both p-IGF1R expression and p-ERK1/2 and p-AKT expression on HT29 cells. In addition, anti-IGF1R and/or anti-EGFR, or with multi-kinase inhibitor treatment leads to differential reduction of p-ERK1/2 and p-AKT. Using CTCs enriched from cancer patient, simultaneous detection of multiple signaling molecules was also observed. Summary A multi-color, LSC-based methodology was developed to quantify expression of up to 4 biomarkers in CTCs. Application of this technology in clinical studies to examine the activation status of CTCs from patients could provide important information on pathway-specific drug activity and guide dosing selection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4155. doi:10.1158/1538-7445.AM2011-4155