Combining Radiation and anti-PD-L1 Enhances the Antitumor Activity in Colorectal Cancer via IFN-γ-Dependent Activation of STAT1

IntroductionColorectal Cancer (CRC) remains a leading cause of cancer-related mortality, and proficient mismatch-repair/microsatellite-stable (pMMR/MSS) tumors respond poorly to immune checkpoint inhibitors (ICIs). Ionizing radiation (IR) can convert 'cold' tumors into 'hot' lesions but rarely elicits durable immunity alone.MethodsThis study investigated the synergistic antitumor effects of hypofractionated ionizing radiation (IR) combined with anti-PD-L1 therapy in CRC. Transcriptomic analysis of paired CRC biopsies (GSE179351) was performed to identify gene expression changes following combination therapy. In vivo efficacy was assessed in syngeneic BALB/c mice bearing CT26.WT tumors treated with 18 Gy IR (in 3 fractions) and anti-PD-L1 (10 mg/kg). Immunohistochemistry, flow cytometry, cytokine quantification, and Western blotting were used to evaluate immune cell infiltration, IFN-γ expression, and activation of JAK1/STAT1 signaling and Apoptosis. To directly test the requirement of STAT1 signaling, CT26.WT cells were treated with the JAK1 Inhibitor Itacitinib.ResultsCombination therapy induced 701 differentially expressed genes enriched in JAK-STAT signaling and Apoptosis pathways. In vivo, IR + anti-PD-L1 significantly delayed tumor growth versus monotherapy without added systemic toxicity. Enhanced CD8⁺ T-cell infiltration and increased IFN-γ levels were observed in both tumor and spleen. Mechanistically, IR alone did not activate STAT1 signaling, while exogenous IFN-γ or IR + IFN-γ induced JAK1/STAT1 phosphorylation and Caspase-3 cleavage in CRC cells, promoting STAT1-dependent Apoptosis. These findings highlight IR's role in priming an IFN-γ-rich tumor microenvironment that enhances the efficacy of PD-L1 blockade.ConclusionThe study supports radio-immunotherapy as a promising approach for patients with pMMR/MSS CRC and provides a mechanistic rationale for clinical trials optimizing fractionation or targeting the IFN-γ/JAK-STAT pathway.

COLORECTAL CANCER; RADIOTHERAPY; immune checkpoint inhibitors; interferon-γ; tumour micro-environment.