Modulating the tumor immune phenotypes by radiotherapy: formulating and validating the combination therapy of radiation, PD-L1, and TIM-3 blockade in colorectal cancer

Background Most colorectal cancers (CRCs) are mismatch repair-proficient (pMMR) and microsatellite stable (MSS), and they respond poorly to immune checkpoint inhibitors (ICIs). Radiotherapy (RT) can promote antitumor immunity but may also trigger adaptive immune suppression through checkpoint upregulation, providing a rationale for combination therapies. Methods We integrated transcriptomic analyses from The Cancer Genome Atlas and Gene Expression Omnibus cohorts—annotated for microsatellite instability (MSI)/MMR status—with single-cell RNA-sequencing data from the Human Colorectal Cancer Atlas. Mechanistic and translational experiments were conducted using CT26 (MSS/pMMR) and MC38 (microsatellite instability (MSI)-high/mismatch repair-deficient (dMMR)) mouse models, patient-derived organoid (PDO)-immune co-cultures, and clinical CRC specimens. Assessments included multicolor flow cytometry, immunohistochemistry/immunofluorescence, bulk RNA-sequencing, and immune profiling. Results HAVCR2 (T cell immunoglobulin and mucin-domain containing-3 (TIM-3)) and LGALS9 (galectin-9) were broadly expressed in CRC, remaining relatively high in MSS/pMMR tumors compared with most other checkpoints. In CT26 tumors, RT preferentially increased programmed cell death protein 1 (PD-1) and TIM-3 co-expression on intratumoral CD8 + T cells and natural killer (NK) cells, whereas these changes were weaker and less consistent in MC38 tumors. The addition of TIM-3 blockade to RT plus programmed death-ligand 1 (PD-L1) blockade produced the most durable antitumor activity in CT26 tumors, improving primary tumor control, abscopal effects, and protection against tumor rechallenge. In PDO-immune co-cultures, pMMR PDOs showed a consistent incremental benefit when TIM-3 blockade was added to RT+PD-L1 blockade, whereas this added benefit was less consistent in dMMR PDOs. In clinical datasets and specimens, RT-containing treatment was associated with increased T-cell infiltration and higher TIM-3/PD-1 signals; stereotactic body radiotherapy was accompanied by systemic immune alterations, including TIM-3 induction on circulating immune subsets. Across pan-cancer cohorts treated with ICIs, concomitantly high expression of HAVCR2 and PDCD1 correlated with an immune-activated transcriptional profile and improved clinical outcomes. Conclusions These findings identify TIM-3/PD-L1 as a context-dependent adaptive resistance axis following RT. The greatest incremental value of TIM-3 blockade was observed in MSS/pMMR settings, where RT more consistently increased PD-1 and TIM-3 co-expression on intratumoral CD8 + T cells and NK cells. Combining RT with dual TIM-3/PD-L1 blockade warrants further clinical evaluation for immunotherapy-refractory CRC.