To investigate the effect of exosomes secreted by microsatellite stability(MSS) colorectal cancer cells after radiotherapy on the function of CD8⁺T cells and to explore the underlying mechanisms.

CT-26 cells were treated with 0 Gy, 10 Gy, and 20 Gy radiation doses, and cell viability was assessed using the CCK-8 assay, while apoptosis was measured by flow cytometry to determine the optimal radiation dose. Exosomes were isolated by ultracentrifugation and characterized by transmission electron microscopy and Western blot. CD8⁺T cells were cultured alone (control group) or co-cultured with exosomes (experimental group), followed by functional analysis via flow cytometry. Exosomal transcriptomic and metabolomic approaches were employed to investigate the impact of radiotherapy on the composition of CT-26 derived exosomes.

A 10 Gy radiation dose did not significantly affect the proliferation of CT-26 cells. Radiation did not significantly alter the morphology or secretion quantity of exosomes released by CT-26 cells, but it modified the proportions of some non-coding RNAs and the composition of metabolites within the exosomes. Differential metabolite enrichment analysis revealed that the altered metabolites in exosomes from irradiated CT-26 cells were primarily enriched in immune-related metabolic pathways such as neuroactive ligand-receptor interaction, serotonergic synapse, retinoic acid metabolism, and sphingolipid metabolism. Compared to the blank group, CT-26 exosomes (control group) suppressed CD8⁺T cell function (blank group vs. control group: CD44⁺CD8⁺T cells: 60.720±3.529 vs. 42.640±2.378, t=9.501, P<0.001; IFN-γ⁺CD8⁺T cells: 2.362±0.418 vs. 1.632±0.198, t=3.532, P=0.008). However, exosomes from irradiated CT-26 cells reversed this suppression, restoring CD8⁺T cell function to a level close to that of the blank group (radiation group vs. control group: CD44⁺CD8⁺T cells: 61.720±3.891 vs. 42.640±2.378, t=9.357, P<0.001; IFN-γ⁺CD8⁺T cells: 2.512±0.469 vs. 1.632±0.198, t=3.858, P=0.010).

Radiotherapy can reprogram the metabolism of exosomes derived from MSS colorectal cancer cells, thereby activating the anti-tumor immune function of CD8⁺T cells.