Implications of Intratumor Heterogeneity on Consensus Molecular Subtype (CMS) in Colorectal Cancer.

Abstract

Simple SummaryColorectal cancer (CRC) has been divided into four consensus molecular subtypes (CMS) using the unsupervised clustering of bulk transcriptomic data. The CMS, which align with known biological differences in CRC, have helped address inter-tumor heterogeneity and have been shown to be predictive of survival. However, bulk tumors are comprised of a mix of tumor cells, non-tumor stroma, and immune cells, and the relative contributions of each are not accounted for in the current CMS classification system. Here, we build an algorithm to assign CMS classification to individual cells, which we apply to single cell RNAseq data to explore the impact of intra-tumor heterogeneity on CMS. We find that the number of stromal and immune cells present has a strong influence on the bulk CMS type and that clusters of tumor epithelial cells derived from single cell RNAseq do not align with CMS.The implications of intratumor heterogeneity on the four consensus molecular subtypes (CMS) of colorectal cancer (CRC) are not well known. Here, we use single-cell RNA sequencing (scRNASeq) to build an algorithm to assign CMS classification to individual cells, which we use to explore the distributions of CMSs in tumor and non-tumor cells. A dataset of colorectal tumors with bulk RNAseq (n = 3232) was used to identify CMS specific-marker gene sets. These gene sets were then applied to a discovery dataset of scRNASeq profiles (n = 10) to develop an algorithm for single-cell CMS (scCMS) assignment, which recapitulated the intrinsic biology of all four CMSs. The single-cell CMS assignment algorithm was used to explore the scRNASeq profiles of two prospective CRC tumors with mixed CMS via bulk sequencing. We find that every CRC tumor contains individual cells of each scCMS, as well as many individual cells that have enrichment for features of more than one scCMS (called mixed cells). scCMS4 and scCMS1 cells dominate stroma and immune cell clusters, respectively, but account for less than 3% epithelial cells. These data imply that CMS1 and CMS4 are driven by the transcriptomic contribution of immune and stromal cells, respectively, not tumor cells.