Abstract 2454: Identification of colorectal cancer stem cells from single cell RNA sequencing
Authors: Lin Kangyu , Zeineddine Mohammad , Chowdhury Saikat , Hornstein Nicholas , Mani Sendurai , Kopetz Scott , Shen John Paul 
Published: April 2023

Abstract
Cancer stem cells (CSCs) play a critical role in metastasis, relapse and therapy resistance in colorectal cancer (CRC). Although the normal lineage of cell development in the intestine has identified many of the genes involved in the induction and maintenance of pluripotency, recent studies of CSCs suggest significant heterogeneity and lack of common surface markers for identification. In this study, we perform single cell RNA sequencing (scRNA-seq) to extract a single cell stemness signature (SCS_sig) for colorectal CSCs identification and as a potential predictor of relapse after surgical resection. A total of 26 fresh surgery specimens (6 colon tumor, 3 colon matched normal, 9 liver metastasis, 8 liver matched normal) from neoadjuvant chemotherapy (NAC) treated patients with single-cell transcriptome generated using 10X Chromium scRNA-seq and Illumina platforms were analyzed. Following extensive quality control and batch effect correction, we identified 20k epithelial cells using canonical epithelial cell marker genes (EPCAM, KRT8 and KRT18) from a total of 111,200 cells, with mean 1113 epithelial cell per sample (excluding liver normal samples).To obtain the CSCs profiling signature, we first annotated a ‘gold-standard’ set of CSC that expressed all canonical colon CSC marker genes (LGR5, ASCL2, EPHB2, PROM1, AXIN2, LEFTY1, RNF43, CD44 and SLC12A2), identifying 346 CSCs (1.71% of epithelial cells). Published stemness signatures were significantly enriched in the gold standard stem cells (false discovery rate (FDR) &lt; 0.05). Then we selected the 50 most significantly up-regulated genes in this gold standard set (relative to other epithelial cells) as a single-cell stemness signature (named SCS_sig hereafter). Interesting, we saw that in every tumor there was a continuum of stemness, rather than distinct stem and differentiated populations. The stemness state by SCS_sig was positively correlated with less differentiated state calculated by CytoTRACE score (r = 0.76, p&lt; 0.001), a computational method that predicts the differentiation state of cells from scRNA-seq data. The SCS_sig was significantly higher in tumor cells compared to normal cells in our cohort as well as the previously published SMC cohort (fold change = 2.52 and p &lt; 0.001, fold change = 1.44 and p &lt; 0.001, respectively). There was notable heterogeneity in the fraction of stem-like tumor cells with in 15 tumor samples, with the tumor with greatest CSC percentage having the shortest time to relapse. The SCS_sig generated from stem-like cells can identify the stemness state of epithelial cells in scRNAseq data. A signature score is more simple and robust than using few or panel of CSCs marker genes to identify single CSC because of the reads dropout in scRNA-seq. Further investigation of the SCS_sig as a predictive and prognostic biomarker in CRC is ongoing in larger patient cohorts.
Citation Format: Kangyu Lin, Mohammad Zeineddine, Saikat Chowdhury, Nicholas Hornstein, Sendurai Mani, Scott Kopetz, John Paul Shen. Identification of colorectal cancer stem cells from single cell RNA sequencing [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 2454.