Abstract 6066: Elucidating cancer stem cells heterogeneity in colorectal cancer by single-cell RNA sequencing

Abstract Cancer stem cells (CSCs) are a small subpopulation of cells with capabilities of self-renewal and differentiation potential and have been suggested to play a critical role in tumor-initiation, metastasis, and multidrug resistance. The marker genes used to identify CSCs in CRC and transcriptional state of these cell is not fully elucidated due to the plasticity and dynamic nature of CSCs. In this study, we perform single-cell RNA sequencing (scRNA-seq) on primary CRC and liver metastases from CRC to evaluate the CSC population. We profiled approximately 80,000 single cells from neoadjuvant chemotherapy (NAC) treated patients generated using 10X Chromium scRNA-seq and Illumina platforms. A total of 16 samples were analyzed (5 colon tumor, 3 colon matched normal, 3 liver metastasis, 5 liver matched normal), 4 samples failed quality control and were excluded. Following extensive quality control and batch effect correction, we performed dimensionality reduction and differential expression analysis with Seurat v3.0 and custom R packages. We used a set of 11 canonical colon CSC marker genes (CD44, PROM1, LGR5, LRIG1, ASCL2, EPHB2, OLFM4, AXIN2, SLC12A2, RNF43, LEFTY1) as CSCs signature to score the stemness of each cell by AddModuleScore. Established marker genes were used to identify cell type assignment including stem/TA, immature enterocytes, immature secretory, enterocytes, Best4 enterocytes, goblet, tuff, CD4, CD8, Treg, NK, B, plasma, macrophages, monocytes, dendritic cells, fibroblasts, endothelium, glia, pericytes and hepatocytes. In the 16,703 epithelial cells, the CSC signature genes were diffusely expressed in stem/TA and immature epithelium but not in a specific cell cluster. There was minimal overlap in expression of the 11 CSC marker genes at the single-cell level. By clustering the CSCs identified by high CSC signature score, we found CSCs grouped distinctly by patients, indicating significant patient to patient variability. Furthermore, CSCs did not cluster by positivity for any specific marker gene, suggesting that marker genes may just be markers, and do not define distinct transcriptional states. Gene set enrichment analysis (GSEA) showed intestinal stemness was significantly enriched (FDR < 0.01) in CSCs compared with non-CSCs. Comparing CSCs to stem cells from normal samples, suppression of the fatty acid metabolism pathway was the only common feature seen in CSCs from each tumor. The heterogeneity of CSC marker genes in CRC tumor cells suggests that defining CSCs by single or few marker genes is inadequate. GSEA verified that a CSC scoring system aggregating expression of 11 canonical colon CSC marker genes can identify CSCs from scRNA-seq. There is marked transcriptional heterogeneity in CSCs, with few common features from CSCs from different tumors. It is hoped that future study of how the diversity of CSCs states evolve under chemotherapy will shed light on overcoming chemo-resistance. Citation Format: Kangyu Lin, John Paul Shen. Elucidating cancer stem cells heterogeneity in colorectal cancer by single-cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6066.