Abstract 1336: An assay for the detection and isolation of single live circulating tumor cells using Accucyte/Rarecyte platform
Authors: Kamal Mohamed , Saremi Shahin , Klotz Remi V. , Iriondo Oihana , Yu Min 
Published: August 2019

Abstract
Introduction: Circulating tumor cells (CTCs) are critical metastatic drivers for the interface between the primary tumor and the target secondary organs. CTCs are heterogeneous, therefore, investigating their phenotypic, molecular and biological properties on a single cell level warrant a better understanding to metastasis and patient’s response to therapy. AccuCyte® - Rarecyte® system is a density based CTC isolation platform which allows for automated visual identification and retrieval of fixed rare cells from blood.
Aims: To establish an assay for the detection and isolation of live single CTCs valid for subsequent molecular and functional characterization.
Methods: Normal human whole blood samples were spiked with cells from breast cancer patient-derived CTC lines and were processed using the AccuCyte system. Buffy coats containing spiked CTCs were stained live with a cocktail of PE-594-conjugated antibodies for immune cell markers (CD14, CD16 and CD45) and either a live cell dye or a cocktail of Alexa Fluor 488-conjugated antibodies for cancer cell surface markers (EpCAM, Her2, EGFR and CDH11). Stained cells were then plated on chamber slides in the presence of serum free RPMI media and investigated using RareCyte fluorescence platform. CTCs were identified based on the lack of immune cell marker expression and the presence of the live cell dye (negative selection) or the expression of cancer cell surface markers and the lack of immune cell markers (positive selection). Single CTCs detected by both approaches were retrieved using our optimized interactive picking module. Cells detected by negative selection were tested for their ability to proliferate ex-vivo, whereas those isolated by positive selection were processed for single cell RNA sequencing analyses.
Results: Detection rates of our protocols reached up to 85% (12 out of 14 CTCs). Single CTCs isolated by negative selection showed a slower proliferation rate during the first 6 days of culture (1.1 Fold increase relative to Day 0) compared to control cells (2.5 Fold increase). However their proliferation rates were comparable to control cells at day 13 (2.7 Vs. 3.2) and day 21 (2.2 Vs. 2.5). Currently, other live cell dyes are being tested to improve the proliferation ability of CTCs isolated by negative selection. Moreover, single cells which were retrieved by positive selection are currently being validated for RNA sequencing analyses.
Conclusion: We have developed a novel assay which allows for the detection and isolation of single viable CTCs. Our assay has two modules; one uses a positive detection approach combined with a two-step cell picking protocol to provide ultrapure single CTCs valid for gene expression analyses. The other module provides an unbiased identification of CTCs using the negative selection approach and a single step of cell picking that provides viable single CTCs able to proliferate in culture.
Citation Format: Mohamed Kamal, Shahin Saremi, Remi V. Klotz, Oihana Iriondo, Min Yu. An assay for the detection and isolation of single live circulating tumor cells using Accucyte/Rarecyte platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1336.