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Single-Cell Analysis of Colorectal Cancer Microenvironment Uncovers Somatic Copy Number Alterations

NEW YORK – Stromal cells found in the tumor microenvironment of colorectal cancer patients harbor a number of genomic alterations, some of which can indicate poor prognosis, a new single-cell multi-omics study has found.

Cells within the tumor microenvironment may contribute to disease progression through their interactions with cancer cells, but as researchers from Peking University noted, little is known about the degree to which these non-malignant cells are transformed by cancer cells. 

Using a parallel single-cell genome- and transcriptome-sequencing approach, they analyzed tumor and normal tissue samples from 21 colorectal cancer patients and samples from six cancer-free elderly controls. As they reported on Thursday in the journal Cancer Cell, the researchers found that somatic copy number alterations (SCNAs) are common in immune cells, fibroblasts, and endothelial cells from both the tumor microenvironment and normal tissues, though they were more prevalent among colorectal cancer patients. In particular, they noted an enrichment of chromosome 7 gains within fibroblasts of the tumor microenvironment.

"Overall, using single-cell multi-omics sequencing of FACS-sorted cells isolated from CRC patients as well as elderly cancer-free individuals, we detected SCNAs at single-cell resolution and provided convincing evidence for pervasive permanent genetic alterations in fibroblasts, endothelial cells, and immune cells in every individual we analyzed," senior author Fuchou Tang from Peking University and his colleagues wrote in their paper.

The researchers isolated those cell types from normal, tumor, blood, and lymph node samples from patients and isolated immune cells from blood samples from elderly controls without cancer. Overall, they generated RNA profiles of more than 15,000 cells, of which nearly 13,500 also had DNA profiles. The profiled cells included almost 1,500 epithelial cells, 2,000 fibroblasts, 9,000 immune cells, and slightly more than 1,000 endothelial cells.

From this data, they found that SCNAs were prevalent among cells from the tumor microenvironment and immune cells from both normal and tumor tissue. 

The researchers additionally noted that SCNAs were found in every cell type they analyzed from every colorectal cancer patient and elderly normal individual, suggesting that the alterations are common. For instance, between 2.9 percent and 10.2 percent of immune cells from the cancer-free elderly controls harbored SCNAs.

The portion of SCNA-affected cells, though, was higher among individuals with cancer. In particular, the proportion of fibroblasts with SCNAs from tumor tissue ranged between 11.1 percent and 47.7 percent, while adjacent normal tissue had between 1.1 percent and 11.5 percent fibroblasts with SCNAs.

Chromosome 7 gains were especially prominent in the tumor microenvironment, the researchers noted. Fibroblasts from tumor tissue were enriched for a whole-chromosome gain of chromosome 7, affecting about a quarter of such fibroblasts and suggesting that there is a clonal expansion process specific to fibroblasts. Intriguingly, the average expression level of genes on chromosome 7 among cells with gains in that chromosome only increased by 26 percent, not the expected 50 percent.

The researchers additionally identified a set of genes that are differentially expressed between fibroblasts from tumor samples and those from adjacent normal tissue. Five of the upregulated genes — BGN, RCN3, TAGLN, MYL9, and TPM2 — were previously linked to poorer prognosis in The Cancer Genome Atlas colorectal cancer cohort, and their fibroblast expression could serve as markers of poor prognosis.

"Our study provides evidence and functional relevance of pervasive genomic alterations in the stromal cells of [the tumor microenvironment] in CRC," Tang and his colleagues wrote.