Metastatic colorectal cancer (mCRC) is characterized by its extensive disease heterogeneity, suggesting that individualized analysis could be vital to improving patient outcomes. As a minimally invasive approach, the liquid biopsy has the potential to longitudinally monitor heterogeneous analytes. Current platforms primarily utilize enrichment-based approaches for epithelial-derived circulating tumor cells (CTC), but this subtype is infrequent in the peripheral blood (PB) of mCRC patients, leading to the liquid biopsy's relative disuse in this cancer type. In this study, we evaluated 18 PB samples from 10 mCRC patients using the unbiased high-definition single-cell assay (HDSCA). We first employed a rare-event (Landscape) immunofluorescence (IF) protocol, which captured a heterogenous CTC and oncosome population, the likes of which was not observed across 50 normal donor (ND) samples. Subsequent analysis was conducted using a colorectal-targeted IF protocol to assess the frequency of CDX2-expressing CTCs and oncosomes. A multi-assay clustering analysis isolated morphologically distinct subtypes across the two IF stains, demonstrating the value of applying an unbiased single-cell approach to multiple assays in tandem. Rare-event enumerations at a single timepoint and the variation of these events over time correlated with progression-free survival. This study supports the clinical utility of an unbiased approach to interrogating the liquid biopsy in mCRC, representing the heterogeneity within the CTC classification and warranting the further molecular characterization of the rare-event analytes with clinical promise.

Biomedical Center Faculty of Medicine in Pilsen Charles University 32300 Pilsen Czech Republic

Catherine and Joseph Aresty Department of Urology Institute of Urology Keck School of Medicine University of Southern California Los Angeles CA 90033 USA

Department of Aerospace and Mechanical Engineering Viterbi School of Engineering University of Southern California Los Angeles CA 90089 USA

Department of Biological Sciences Dornsife College of Letters Arts and Sciences University of Southern California Los Angeles CA 90089 USA

Department of Biomedical Engineering Viterbi School of Engineering University of Southern California Los Angeles CA 90089 USA

Gilead Sciences Inc Lakeside Drive Foster City CA 94404 USA

Michelson Center for Convergent Bioscience Convergent Science Institute in Cancer Dornsife College of Letters Arts and Sciences University of Southern California Los Angeles CA 90089 USA

Norris Comprehensive Cancer Center Keck School of Medicine University of Southern California Los Angeles CA 90033 USA

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