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

Abstract

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.

Charting the Heterogeneity of Colorectal Cancer Consensus Molecular Subtypes using Spatial Transcriptomics

“…Recently, several studies applied scRNA-seq on CRC samples to further reveal the diversity of and the dynamic relationships between cellular components of CRC tumors and their TME 7,9,[12][13][14] . These analyses disclosed CMS features at the individual tumor cell level and stressed the high prevalence of multiple CMS phenotypes in the same patient 7,9,12 .…”

Genetic and microenvironmental intra-tumor heterogeneity impacts colorectal cancer evolution and metastatic development

“…Besides the differential content in stromal and immune cells, CRC tumors also showed ITH at the level of epithelial cells. Indeed, a variety of CMS-like signatures were detected inside each tumor sample, as previously described 30 , 42 . As expected, the mesenchymal subtype CMS4 showed the lowest epithelial ITH, while CMS3 tumors harbored the highest levels of each CMS-like cell.…”

“…This is in agreement with highly mutated tumors harboring more immunogenic mutations and consequently attracting more immune-cell infiltrates 31 . In fact, a recent study suggests that the CMS1 and CMS4 signal is mostly derived from immune and stromal cells, respectively 42 , with little contribution from tumor epithelial cells. Besides the differential content in stromal and immune cells, CRC tumors also showed ITH at the level of epithelial cells.…”

“…A favorable outcome can be explained by the high levels of immune cells found in highly heterogeneous tumors. Accordingly, immune-cells infiltration in primary tumors has been associated with reduced CRC tumor dissemination 42 , 47 . However, the protective capacity of the immune system seems to be overrun by cases of excessive clonal diversity.…”

Artificial intelligence, molecular subtyping, biomarkers, and precision oncology

“…Although the promise of using molecular subtyping to achieve precision oncology is currently more achievable today than at any time in the past, significant obstacles still remain. While the idea of molecular subtyping was created to address the problem of inter-tumor heterogeneity, advances in single cell sequencing have revealed that intra-tumor heterogeneity is another major issue to be addressed [83][84][85]. Intra-tumor heterogeneity is particularly challenging with transcriptomic measurements, where contribution from non-tumor stroma and immune cells can confound analysis of tumor intrinsic transcription and also increase risk of spatial sampling bias [86].…”