Single-Cell Analysis Reveals Immune Profile of T Cell Exhaustion- Associated Microenvironment in Colorectal Cancer

Abstract Background: Distinguishing the unique immune microenvironment of left and right colorectal cancer (CRC) is essential for better the development of tailored therapeutic strategies and improving patient outcomes. However, few data was available on single-cell RNA-seq (scRNA-seq)-based analysis of the immune profile in these regions. Therefore, we aimed to depict and compare the immune landscape among left and right colorectal cancer and the paired normal tissue. Targeted genes and pathways was analyzed for constructing transgenic Tumor Infiltrating Lymphocyte (TIL) to improve its efficacy in solid tumors. Methods: Bioinformatics’ analysis was performed on GSE200997 obtained from GEO (Gene Expression Omnibus) database. By analyzing the scRNA-seq data, immune cell clusters were identified and their proportions and differentially expressed genes(DEGs)on the meaningful immune cell clusters were compared among left and right colorectal cancer tissues and the paired normal tissue. Monocle3 was used to analyze the developmental sequence of cell clusters and genes involved in the progression of CRC. CellChat package was conducted to characterize the underlying mechanism of immune cell recruiting. Results: In this study, we found that more CD8 T cells were in exhausted state in right tumor tissue (RT) than in normal tissue (N) and left tumor tissue (LT) microenvironments, where CXCL13 and MTRNR2L8 were not only mainly involved in the differentiation of CD8 from naive T cells to exhausted T cells but also a factor in the poor prognosis of CRC patients. CD8 T cell exhaustion was also associated with the presence of BATF, FOXP3, TIGIT, and TNFRSF4 on Treg that inhibit CD8-mediated immune responses. In addition to that, we found that in tumor tissue, especially RT and MSI-H state the proportion of epithelial cells was higher, wherein invasive cancer cells developed from epithelial cells and shared the same input and output signal patterns as epithelial cells. Further crosstalk analysis of epithelial cells on cytotoxic T lymphocytes (CTLs) revealed that comparing to epithelial cells, the invasive cancer cells expressed TFG-β and CXCL signaling axis in LT, and TGF-β was highly expressed by epithelial and endothelial cells with CTL cells in RT, which may be a key mechanism to promote tumor invasion, progression, and treatment resistance. Conclusions: Most CD8 in RT are effector memory T cells and are more likely to differentiate into exhausted T cells with high expression of CXCL13 and MTRNR2L8 and less expression of chemokines. Cells responsible for this phenomenon are proliferative cancer cell, Treg, invasive cancer cell, and epithelial cell in LT, and proliferative cancer cell, endothelial cell, Treg, and cancer stem cell in RT. The pathway involved in the interactions between above cells and cytotoxic T cell is mainly TGFB-TGFBR. Keywords: Colorectal cancer; Tumor microenvironment; Single-cell RNA sequencing; Cell crosstalk