Identification of Colorectal Cancer Cell Stemness from Single-Cell RNA Sequencing

Abstract

Cancer stem cells (CSC) play a critical role in metastasis, relapse, and therapy resistance in colorectal cancer. While characterization of the normal lineage of cell development in the intestine has led to the identification of many genes involved in the induction and maintenance of pluripotency, recent studies suggest significant heterogeneity in CSC populations. Moreover, while many canonical colorectal cancer CSC marker genes have been identified, the ability to use these classical markers to annotate stemness at the single-cell level is limited. In this study, we performed single-cell RNA sequencing on a cohort of 6 primary colon, 9 liver metastatic tumors, and 11 normal (nontumor) controls to identify colorectal CSCs at the single-cell level. Finding poor alignment of the 11 genes most used to identify colorectal CSC, we instead extracted a single-cell stemness signature (SCS_sig) that robustly identified "gold-standard" colorectal CSCs that expressed all marker genes. Using this SCS_sig to quantify stemness, we found that while normal epithelial cells show a bimodal distribution, indicating distinct stem and differentiated states, in tumor epithelial cells stemness is a continuum, suggesting greater plasticity in these cells. The SCS_sig score was quite variable between different tumors, reflective of the known transcriptomic heterogeneity of CRC. Notably, patients with higher SCS_sig scores had significantly shorter disease-free survival time after curative intent surgical resection, suggesting stemness is associated with relapse.

Implications: This study reveals significant heterogeneity of expression of genes commonly used to identify colorectal CSCs, and identifies a novel stemness signature to identify these cells from scRNA-seq data.

Figure 1:. A single-cell transcriptome map of primary and liver metastatic CRC.

A single-cell transcriptome map of primary and liver-met CRC. A, UMAP plots displaying 111,224 cells from colon normal, colon primary CRC, liver normal and liver metastatic CRC tissues. Cells were colored by cluster (left), sample ID (middle) and major cell type (right), respectively. B, UMAP plot of colon and liver epithelial cells, color-coded by cluster (left), sample class (normal/tumor, middle) and epithelial subtype (right). C, Number and relative proportions of epithelial subtype in colon and liver metastatic CRC samples. D, Percentage of immune subtypes grouped by the sample type of site and class. E, Percentage of stromal subtypes grouped by the sample type of site and class, samples with total stromal cells < 20 were not presented.

Figure 2:. Identification of a single-cell stemness signature.

A, UMAP plots showing expression of canonical intestinal CSC marker genes. Color bar represents scaled expression. B, Intersection epithelial cell number that CSC gene is non-zero. LGR5, ASCL2 and EPHB2 are marked as primary CSC genes. C, UMAP showing the ‘gold-standard’ CSC expressing most of the canonical intestinal CSC marker genes (LGR5, ASCL2, EPHB2, PROM1, AXIN2, LEFTY1, RNF43, CD44 and SLC12A2). D, Gene set enrichment analysis (GSEA) results showing the hallmark gene sets related to ‘gold-standard’ CSC compared with other epithelial cells. Gene sets with FDR < 0.25 are shown. E, Feature plot showing the 10% epithelial cells (colored by dark red) with the highest SCS_sig score of 50, 25 and 100 genes. F, Violin plot of the SCS_sig score by tumor samples and merged normal colon samples. The line in each violin represents the median value.

Figure 3:. SCS_sig characterization.

A and B, SCS_sig score (left) and CytoTRACE score (right) in non-CSC/CSC (A) and in normal/tumor cells (B). C, Spearman correlation coefficient of SCS_sig score and CytoTRACE score in epithelial cells. D, SCS_sig score in the Samsung Medical Center (SMC) cohort (left) and Broad cohort (right) epithelial cells by normal/tumor. E, Histogram showing the distribution of SCS_sig score in normal and tumor cells (MD Anderson data, left; Broad cohort data, right).

Figure 4:. Associate of SCS_sig with clinical outcomes and tumor features.

A, Swimmer plot showing the patients’ treatment history and outcomes, patients were ordered by SCS_sig score. B, SCS_sig score and disease-free survival (DFS) from time of single cell collection surgery. Each patient’s SCS_sig score is represented by the median SCS_sig score of tumor cells from that patient. C, Kaplan-Meier plot showing the DFS curves for patients grouped by median of SCS_sig score.