Bioinformatics analysis and experimental validation of EOMES expression and correlation with exhausted CD8+ T cell in colorectal cancer

Abstract

Colorectal cancer (CRC) ranks among the most prevalent malignancies globally, yet exhibits limited responsiveness to immunotherapy. This therapeutic challenge stems primarily from incomplete understanding of immune microenvironment regulation in CRC. Although Eomesodermin (EOMES) has been implicated in tumor immune checkpoint gene expression, its specific interaction and regulatory mechanisms within the CRC immune microenvironment require systematic investigation. RNA-sequencing expression profiles and corresponding clinical information for 33 types of tumors were downloaded from the TCGA dataset for Pan-Cancer analysis of gene EOMES. Single cell sequencing data of CRC from TISCH was used for single-gene analysis of EOMES. The primary human CRC tissue samples were obtained for tissue multiplexed immunofluorescence staining. The isolated CD8+ T cells were cocultured with MC38 cells, following by real-time PCR detection, immunoblotting assay, and flow cytometry analysis. Pan-cancer analysis demonstrated that EOMES expression was significantly elevated in tumor tissues from 33 cancer types (including COAD/READ) compared to normal tissues (BRCA, DLBC, ESCA et al.). Further immune microenvironment correlation analysis showed that EOMES expression strongly correlates with immune microenvironment modulation, particularly in COAD. Importantly, elevated EOMES levels were identified in exhausted CD8⁺ T cells within CRC tissues (COAD/READ subtypes). Moreover, EOMES expression demonstrates significant linkage to immune/inflammatory regulatory pathways in CRC. High EOMES⁺CD8⁺ T cell infiltration occurs in microsatellite-stable (MSS) CRC but not in microsatellite instability-high (MSI-H) CRC. Most importantly, EOMES expression positively correlates with exhausted CD8⁺ T cells exclusively in MSS CRC. Further in vitro experiments demonstrated the strong link between EOMES upregulation and CD8+ T cell exhaustion, thereby impairing their cytotoxic function against CRC cells. EOMES critically participates in CRC immune microenvironment regulation, with its upregulation positively associated with exhausted CD8⁺ T cell infiltration. This association is most pronounced in MSS CRC, revealing a subtype-specific immunomodulatory mechanism.