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

Abstract

Introduction

Colorectal 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.

Methods

This 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.

Results

Combination 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.

Conclusion

The 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.

Introduction

Colorectal cancer (CRC) is among the most common malignancies worldwide. In 2020, it accounted for an estimated 1.93 million new cases and 930 000 deaths, representing almost one-tenth of all cancer incidence and mortality and ranking third for incidence and second for mortality. ¹ This upward trend is particularly marked in China, where CRC now ranks behind only lung and gastric cancers in incidence. ² Approximately 20% of patients present with distant metastases at diagnosis, and the 5-year survival rate for those with locally advanced or metastatic disease remains unsatisfactory despite surgery, chemotherapy, and radiotherapy. ³

Radiotherapy remains a cornerstone of CRC management. IR generates reactive oxygen species that cause sustained DNA double-strand breaks, leading to tumor cell apoptosis, senescence, or a permanent loss of clonogenicity. ⁴ Neoadjuvant IR can shrink tumors, facilitate tumor resection, and reduce local recurrence. IR can also induce immunogenic cell death, exposing tumor-associated antigens and recruiting and activating dendritic cells (DCs), thereby enhancing T cell-mediated immunity.^5,6 In addition, IR up-regulates major histocompatibility complex molecules (MHC) and death receptors on tumor cells, broadening tumor visibility to the immune system and converting ‘cold’ tumors into ‘hot’ lesions for immune infiltration. ⁷ Conversely, IR may increase immunosuppressive mediators such as PD-L1 and TGF-β or attract regulatory T cells and myeloid-derived suppressor cells, restricting durable systemic responses. At our center, the pathological complete-response rate after preoperative chemoradiotherapy is only approximately 9%, underscoring the need for radiosensitizing strategies.

Immune checkpoint inhibitors (ICIs) block suppressive pathways such as PD-1/PD-L1 and CTLA-4, reinvigorating cytotoxic T cells.^8,9 In patients with mismatch-repair-deficient (dMMR)/microsatellite-instability-high (MSI-H) CRC, the anti-PD-1 antibody pembrolizumab confers markedly superior progression-free survival compared to chemotherapy.^10–12 However, 95% of advanced CRCs are proficient in mismatch repair and microsatellite stable (pMMR/MSS) and exhibit minimal response to ICI monotherapy.^8,13 As such, enhancing the sensitivity of MSS tumors to immunotherapy is a key clinical challenge; therefore, combination regimens that pair ICIs with chemotherapy, anti-angiogenic agents, targeted drugs, or IR are being actively explored. ⁵

Among these approaches, IR plus ICIs is emerging as a particularly promising strategy. ¹⁴ Preliminary clinical studies and extensive pre-clinical data have shown that ICIs relieve tumor-induced T-cell exhaustion, while IR generates neo-antigens and an inflammatory milieu that facilitates T-cell infiltration and tumor recognition. ¹⁵ In murine CRC models, this combination increases tumor-infiltrating lymphocytes, diminishes suppressor populations, and controls both primary and metastatic lesions.^5,7 Simultaneous blockade of multiple checkpoints (eg, PD-1 and CTLA-4) together with local IR can further potentiate antitumor immunity. ⁷ Nevertheless, the precise mechanisms underlying this synergy and the collective impact of IR and ICIs on the tumor microenvironment (TME) have not yet been fully elucidated. ¹⁶ The TME, comprising cancer cells, immune cells, vasculature, and stroma, dictates tumor evolution and the therapeutic response. ¹⁷ The TME is closely linked to multiple signalling pathways in tumor cells. The JAK/STAT pathway, particularly STAT1 activated downstream of interferon-γ (IFN-γ), governs tumor cell apoptosis. ¹⁸ We therefore hypothesized that hypofractionated IR combined with anti-PD-L1 remodels the TME, elevates IFN-γ, and triggers STAT1-dependent cell death in pMMR/MSS CRC.

In the present study, we integrated publicly available transcriptomic data from both in vivo and in vitro experiments. Bioinformatic analyses of paired CRC biopsies ¹⁹ (GSE179351) suggested that IR plus dual ICIs augments CD8⁺ T-cell infiltration, sustains IFN-γ-mediated JAK/STAT signalling and promotes apoptosis of cancer cells. We then established CT26.WT syngeneic mouse models to compare the antitumor efficacy and safety of IR, anti-PD-L1, and their combination, and we characterized changes in the TME and peripheral immunity by immunohistochemistry, flow cytometry, and ELISA. Finally, using CT26.WT and MC38 cell lines, we examined the downstream effects of IFN-γ on JAK-STAT activation and apoptosis in vitro. Together, these investigations delineated how IR synergizes with anti-PD-L1 therapy through the IFN-γ–STAT1 axis and provided a mechanistic rationale for clinical translation of radio-immunotherapy in pMMR/MSS CRC.

Materials and Methods

Mice. Six-week-old male BALB/c mice (Sibeifu Biotechnology, Beijing, China) were housed in the Specific Pathogen-Free (SPF) facility of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences. Conditions were maintained at 22 ± 2 °C, 50 ± 10% relative humidity, with a 12 h light–dark cycle. Mice had free access to autoclaved chow and water (five mice per cage, individually ventilated). All experimental protocols were approved with ethics number: 2024-SYDWLL-000269. The reporting of this study conforms to ARRIVE 2.0 guidelines. ²⁰

Cell lines and culture. Murine colon-carcinoma cell lines CT26.WT and MC38 (Boster Biotech, Wuhan, China) were maintained in RPMI-1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin/100 µg/mL streptomycin at 37 °C in 5% CO₂. For resuscitation, cryovials were thawed in a 37 °C water bath, centrifuged at 1000 rpm for 3 min, and the pellet was resuspended in 1 mL of fresh medium before being transferred to a 25 cm² flask with 3–4 mL of complete medium. All consumables were UV-irradiated for 30 min before use.

Bioinformatic Analysis. Transcriptomic data for paired CRC biopsies before and after IR (24 Gy/3 fractions) combined with dual immune checkpoint inhibition (dataset GSE179351, MSS cohort) were downloaded from the Gene Expression Omnibus (GEO). Differential expression was calculated with limma or DESeq2 (|log₂FC| > 1, Benjamini–Hochberg adjusted P < 0.05). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed, pathway scores were generated using GSVA, and significant pathways were identified using Wilcoxon rank-sum testing (P < 0.05). Immune cell fractions were estimated using CIBERSORT (22-cell signature) and stromal/immune scores were estimated using ESTIMATE. Associations among interferon-γ (IFN-γ), JAK–STAT pathway genes, and apoptosis signatures were assessed using Spearman correlation.

Key Reagents and Antibodies. Therapeutic agents: anti-mouse PD-L1 antibody (clone 10F.9G2), recombinant mouse IFN-γ and Itacitinib (both MedChemExpress). Primary antibodies for immunohistochemistry (IHC) and western blotting (WB) included CD3 and CD8 (Starter), p-STAT1(Ser727), STAT1, p-JAK1(Tyr1022), JAK1 (all Affinity Biosciences), cleaved caspase-3, and β-actin (Cell Signalling Technology). Detailed catalogue numbers and dilutions are listed in the Supplementary Methods. For immune checkpoint inhibition, we employed a single-agent anti–PD-L1 antibody (clone 10F.9G2, 10 mg/kg, intraperitoneal) because robust murine safety and efficacy data are available for this agent, and single-agent blockade allowed us to isolate the contribution of PD-L1 inhibition to the observed synergy with irradiation.

In Vivo Tumor Study. Tumor establishment: CT26.WT cells (1 × 10⁶ cells in 100 µL PBS) were injected subcutaneously into the right flank of mice. When the tumors reached approximately 6 mm in diameter (seven days), the mice were randomized (n = 4 per group) to: 1. Control (no treatment), 2. IR: local 6 MV x-ray, 6 Gy on days 1, 3, and 5 (field: 2 cm; dose rate: 2 Gy/min); 3. ICI: anti-PD-L1 10 mg/kg intraperitoneally (days 2, 4, and 6); and 4. IR + ICI: combined schedules as described above. Anaesthesia for irradiation used 2.5% tribromoethanol (0.10 Ml/10 g); mice were positioned left-lateral with 1.5 cm bolus for build-up. Tumor length (L) and width (W), and body weight were recorded every other day as follows: volume = L × W² / 2. End-points were day 7 after the first treatment or humane criteria (≥ 20% weight loss, diameter > 20 mm, or impaired mobility). The mice were euthanized using 3% isoflurane, followed by cervical dislocation. Tumors were harvested and processed in two different ways: two tumors were paraffin-embedded for histology and immunohistochemistry, while the remaining two were snap-frozen in liquid nitrogen for subsequent cytokine and protein analysis. We have made efforts to minimize the number of animals utilized and to decrease their suffering. ²¹ The radiation regimen (6 Gy × 3 fractions, total 18 Gy) and immune checkpoint inhibition (a single-agent anti–PD-L1 antibody) were chosen based on published preclinical studies.^22,23 Each treatment arm comprised four mice (n = 4), a number consistent with prior preclinical studies employing syngeneic CT26.WT models to evaluate combined radiotherapy and immune checkpoint blockade. This cohort size was selected as an exploratory proof-of-concept study to minimize animal use while ensuring preliminary detection of treatment-related differences in tumor growth and immune activation. Given the pilot nature of the work, no prospective power calculation was performed; instead, we relied on effect sizes reported in the literature for similar designs.

In Vitro Functional Assays. CT26.WT and MC38 cells were assigned to: Control; IR (single 6 Gy, source-to-skin distance 1 m, 3 Gy/min); IFN-γ (100 ng/mL added 2 h before IR); or IR + IFN-γ. The cells were harvested 6 h after treatment for Cell Counting Kit-8 (CCK-8) viability assays and WB. For apoptosis assays, cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. STAT1 inhibition assay: CT26.WT cells were divided into five groups: Control, STAT1 inhibition (Itacitinib, 0.5 μM), IR + Itacitinib, IFN-γ+Itacitinib, and IR + IFN-γ+Itacitinib (same dose of IR and IFN-γ. For WB, lysates were probed for phospho-STAT1, cleaved caspase-3, and β-actin. The dose of recombinant murine IFN-γ was selected based on prior studies demonstrating that nanogram-level IFN-γ effectively activates JAK–STAT signaling and induces pro-apoptotic gene expression in murine tumor cell lines.^24–26 Our chosen concentration which was sufficient to robustly induce STAT1 phosphorylation without causing nonspecific cytotoxicity, remains within the biologically relevant range for receptor occupancy and signaling activation in vitro.

Immunohistochemistry. Tumors were fixed in 10% neutral buffered formalin, paraffin-embedded, and sectioned at 4 µm. After dewaxing with xylene and rehydration through graded ethanol, heat-induced epitope retrieval was carried out in EDTA buffer (pH 8.0). Endogenous peroxidase activity was quenched with 3% H₂O₂ for 15 min, followed by blocking with 5% goat serum for 20 min. The sections were incubated overnight at 4 °C with primary antibodies (negative control, PBS). Detection was performed using an SABC-HRP polymer and DAB substrate, slides were counterstained with haematoxylin, dehydrated, cleared, and mounted. Immunohistochemistry quantification: staining intensity was quantified as integrated optical density (IOD) using ImageJ v1.51. The measurement principle of the Image J software is to determine the expression level of the target protein based on the color intensity and distribution area of the target protein. For each biological sample, four randomly selected fields were analyzed, and the mean IOD value was calculated. The results are presented as bar graphs showing mean ± SD, with individual data points overlaid to indicate biological variability. Representative high-resolution images (100×) are provided in the Supplemental Materials.

Protein Extraction and WB. Tumor or cell lysates were prepared in RIPA buffer containing 1 mM PMSF, sonicated on ice and cleared at 12000 rpm, 4 °C, 10 min. Protein concentrations were determined through BCA assay; 30 µg protein per lane were separated on 10% SDS-PAGE (80 V stacking, 120 V resolving) and transferred to PVDF membranes (methanol-activated) at 200 mA (duration dependent on molecular weight). Membranes were blocked with 5% BSA for 1 h, incubated with primary antibodies (1:1000) overnight at 4 °C, and then with HRP-conjugated secondary antibodies (1:5000, 1 h). Bands were visualized with an ECL kit (Vazyme) and densitometry was normalized to β-actin. Primary antibodies used: p-STAT1 (Ser727) (#AF3299, Affinity); STAT1 (#AF6300, Affinity); p-JAK1 (Tyr1022) (#AF2012, Affinity); JAK1 (#AF5012, Affinity); Cleaved Caspase-3 (Asp175) (#9661, Cell Signaling Technology); β-actin (#3700, Cell Signaling Technology). Dilute primary antibodies 1:1000 in WB antibody dilution buffer (Beyotime Biotechnology, Shanghai, China).

Cytokine Quantification (ELISA). Tumor and spleen homogenates were prepared with magnetic-bead disruption; supernatants were analyzed for IFN-γ using a commercial ELISA kit (Elabscience, cat. E-EL-M0048) in duplicate wells. After incubation steps (37 °C, 90 min for capture; 1 h biotin-antibody; 30 min HRP conjugate) and TMB development (max 15 min), absorbance was read at 450 nm. The cytokine concentrations were determined from a four-parameter logistic standard curve.

Flow Cytometry. The spleens were gently pressed through a 200-mesh sieve into PBS, red blood cells were lysed (1 × buffer, 3 min), and the cells were washed and filtered. The suspension was adjusted to 1 × 10⁷ cells/mL and stained for 30 min at 4 °C (antibodies: CD45-ER780, CD3-EV450, CD4-FITC, CD8-APC, and CD49b-APC; Elabscience). Data were acquired on a BD FACSymphony A1 (4-laser, 16-colour) with voltage and compensation set by single-stain controls and analyzed using FlowJo v10.8.1. Flow cytometry and gating strategy：Debris was excluded by FSC-A versus SSC-A, and doublets were removed by FSC-H versus FSC-W and SSC-H versus SSC-W. CD45⁺ leukocytes were then selected, followed by CD3⁺ T cells, and finally CD4⁺ and CD8⁺ subsets were resolved on bivariate plots. Gates were set using single-stain controls for compensation and unstained controls and population distributions were used to confirm gate placement. Annexin V-FITC/PI apoptosis detection: after staining, the cell suspension is incubated at room temperature (20-25 °C) in the dark for 20 min. Apoptosis is then detected using flow cytometry, and the apoptosis rate is calculated. Annexin V-FITC exhibits green fluorescence, while propidium iodide (PI) emits red fluorescence. Apoptosis rate = early apoptotic cells (Annexin V+/PI-) + late apoptotic cells (Annexin V+/PI+).

Statistical Analysis. Data are presented as mean ± standard deviation (SD). Tumor growth curves were compared using two-way repeated-measures ANOVA (factors: Treatment, Time; Geisser-Greenhouse correction) with Sidak-adjusted pairwise contrasts where interactions were significant. Multi-group endpoints were analyzed by one-way ANOVA, and non-normal data were analyzed by Wilcoxon rank-sum tests. Family-wise error was controlled using the Holm–Bonferroni adjustment. Analyses were performed using GraphPad Prism 7 and IBM SPSS Statistics 28, and an adjusted P < 0.05 was considered statistically significant.

Detailed standard operating procedures (SOPs) and full reagent lists are provided in the Supplementary Methods.

Results

Differential expression and pathway enrichment after radio-immunotherapy. To profile treatment-induced transcriptional changes, we analyzed paired biopsies obtained before and after IR plus immune checkpoint blockade (24 Gy/3 fractions + dual ICIs; GSE179351). With cut-offs of |log_2FC| > 1 and adjusted P < 0.05, 701 genes were differentially expressed; 568 were upregulated and 133 were downregulated (Figure 1A). Downregulated genes were enriched for GO terms linked to cell cycle regulation, differentiation, and metabolism (Figure 1B), whereas upregulated genes were mapped chiefly to apoptosis-related GO categories (Figure 1C, supplementary Tables S1 and S2 represent upregulation group and downregulation group, respectively) and, in the KEGG (supplementary Table S3) analysis, to apoptosis and the JAK–STAT cascade (Figure 1D). These data indicated that JAK–STAT activation and programmed cell death were the central consequences of the combined regimen.

Figure 1.

Differential transcriptomic analysis. The number of patients: Pre (N = 26) and Post (N = 6). (A) Volcano plot showing 701 differentially expressed genes (DEGs): 568 upregulated and 133 downregulated. (B) Top Gene Ontology (GO) terms enriched among downregulated DEGs (the top 30 pathways with P < 0.05). (C) Top GO terms enriched among upregulated DEGs (the top 30 pathways with P < 0.05). (D) KEGG enrichment pathways in the upregulated gene group (all pathways with P < 0.05).

Immune cell composition shifts towards a cytotoxic phenotype. CIBERSORT deconvolution revealed marked reshaping of the immune landscape (Figure 2A). After therapy, the numbers of CD8⁺ T cells and resting dendritic cells increased notably, whereas those of follicular helper T cells and M1 macrophages decreased (Figure 2B). We conducted a supplementary analysis of the transcriptional expression levels of the classic T cell exhaustion markers (including PDCD1, HAVCR2, LAG3, ENTPD1, CTLA4, and TIGIT) in the tumor microenvironment. The results are shown in the Figure 2C. Although the infiltration of CD8⁺ T cells was significantly increased in the treatment group, the expression of these exhaustion markers did not show statistically significant differences between the pre-treatment and post-treatment groups (P > 0.05). Gene-set variation analysis (GSVA, supplementary Table S4) supported these findings: both the “Apoptosis” and “JAK–STAT signaling” KEGG signatures trended upward after treatment (Figure 3A–C). Collectively, the transcriptomic data suggested that radio-immunotherapy drives an immunologically “hot” and pro-apoptotic milieu.

Figure 2.

Immune-cell landscape inferred by CIBERSORT. (A) Stacked bar chart of 22 immune-cell subsets in each sample. (B) Box-plots comparing key immune-cell fractions before and after treatment. (C) Box-plots comparing the transcriptional expression levels of the classic T cell exhaustion markers (including PDCD1, HAVCR2, LAG3, ENTPD1, CTLA4, and TIGIT) in the tumor microenvironment.

Figure 3.

GSVA of KEGG pathways. (A, B) Heat maps of pathway scores across 32 paired samples. (C) Boxplot illustrating expression patterns of JAK-STAT signaling pathway and apoptosis-related pathways before and after treatment.

Combined therapy enhances tumor control in a CT26.WT model. Therapeutic efficacy was examined in BALB/c mice bearing syngeneic CT26.WT tumors. Animals (n = 4 per arm) received control, anti-PD-L1 alone, IR alone (6 Gy × 3), or IR + anti-PD-L1 (Figure 4A). The tumors were inoculated dorsally, and the mice were irradiated in the left lateral decubitus position to confine the field (Figure 4B). As the fractionated IR caused appreciable weight loss, all animals were euthanized on day 7 for tissue collection (Figure 4C). Tumor volume change normalized the baseline size differences. Anti-PD-L1 monotherapy produced negligible inhibition, whereas IR alone curtailed growth by 59% versus controls (ΔV 486 mm³ vs 1195 mm³; P-value 0.0016). The combination further reduced growth to 174 mm³, but high inter-individual variability precluded statistical separation from IR alone (Figure 4D). Body-weight curves mirrored treatment stress; IR induced a notable decline that was not exacerbated by the combination of the antibodies (combination 20.28 g vs IR 18.95 g; P-value 0.132; Figure 4E). Histological examination of the liver and colon revealed no checkpoint-related hepatitis or colitis, indicating an acceptable safety profile.

Figure 4.

In vivo treatment schedule and therapeutic outcomes. (A) Experimental timeline for control, radiotherapy (IR), anti-PD-L1 (ICI), and combination groups. (B) Positioning of tumor-bearing mice for 6 MV x-ray irradiation. (C) Representative image of tumor harvesting on day 7. (D) Tumor-growth curves (mean ± SD; n = 4 per group); statistical methods: one-way ANOVA. ∗, ∗∗, ∗∗∗, or ∗∗∗∗ represent p-values <0.05, < 0.01, < 0.001, and <0.0001, respectively. X-axis: Number of days, corresponding to the treatment timeline, with the first day being the first day of treatment. Y-axis: Tumor volume growth, measured as the difference between the tumor volume on the day of measurement and the tumor volume before treatment (first day). (E) Body-weight changes over the study period (mean ± SD; n = 4 per group); statistical methods: one-way ANOVA. X-axis: Number of days. Y-axis: Mouse weight, measured as mouse weight on the day of measurement.

IR + anti-PD-L1 creates a pro-inflammatory TME. Immunohistochemistry revealed a stepwise increase in the number of intratumoral CD3⁺/CD8⁺ lymphocytes across the treatment gradient. Integrated optical density (IOD) values were: control 6.42 × 10⁶, IR 10.18 × 10⁶, and combination 13.20 × 10⁶ (P-value: CTR vs IR + ICI 0.0249; IR vs IR + ICI 0.0217; Figure 5A). To enhance transparency, IOD values are now displayed as bar graphs with individual data points. High-resolution representative images are included in Supplemental Materials. Flow cytometry of splenocytes confirmed systemic activation, with combination therapy yielding the highest proportions of CD4⁺ and CD8⁺ T cells (eg CD4⁺: 25.5% vs 19.9% [IR] vs 13.6% [control]; Figure 5B). Consistent with heightened T-cell activity, ELISA detected significantly greater interferon-γ (IFN-γ) levels in both tumor and spleen after combined therapy (P-value: CTR vs IR + ICI 0.0406; Figure 5C).

Figure 5.

Immune activation in tumor and spleen. (A) Immunohistochemical staining for CD3 and CD8 in tumor sections (100 × and 400×) with integrated optical-density quantification (CD3 IOD + CD8 IOD); n = 4, mean ± SD; Representative fields were randomly selected from biological samples, with multiple technical replicates per sample. Statistical analysis: one-way ANOVA. (B) Flow cytometric analysis of splenic CD4⁺ and CD8⁺ T-cell subsets. First row of images: Heat map of CD45⁺ cells in each group of spleen cells. Values among the figure: Percentage of CD45⁺ cells in spleen cells. Second row image: Heat map of T cell subsets. The quadrants represent: Q2 CD3⁺ CD4⁺, ie, CD4⁺ T cells; Q3 CD3⁺ CD4⁺, ie, CD8⁺ T cells. The values among the image represent the percentage of the cell subset in CD45⁺ cells. (C) IFN-γ concentrations in tumor and spleen measured using ELISA (n = 4); Tumors were divided for different downstream assays: two frozen for cytokine analysis. ELISA results therefore reflect technical duplicates from available biological samples (n = 4, mean ± SD, technical replicates). Statistical analysis: one-way ANOVA.

IFN-γ–driven JAK–STAT signalling underlies enhanced apoptosis. Given the central role of IFN-γ in STAT1 activation, we examined pathway status in tumor tissues. The combination-treated tumors displayed the strongest p-STAT1 staining (P-value: CTR vs IR + ICI 0.0314; Figure 6A). To determine causality, CT26.WT and MC38 cells were exposed to IR (6 Gy), recombinant IFN-γ (100 ng/mL), or both. WB confirmed that IFN-γ, alone or with IR, markedly up-regulated phospho-JAK1, phospho-STAT1 and cleaved caspase-3, whereas IR alone did not (Figure 6B). Apoptosis was quantified by Annexin V-FITC/PI staining in CT26.WT cells. We found compared with control (5.3 ± 0.1%), both IR (20.0 ± 0.3%) and IFN-γ (20.0 ± 0.4%) significantly increased apoptosis (P < 0.0001). The IR + IFN-γ combination further elevated apoptosis to 34.7 ± 0.3%, which was significantly higher than either IR or IFN-γ alone (P < 0.0001) (Figure 6C). To directly test the requirement of STAT1 signaling, CT26.WT cells were treated with the JAK1 inhibitor Itacitinib. Western blot analysis showed that IFN-γ or IR + IFN-γ markedly increased phospho-STAT1 and cleaved caspase-3, whereas these effects were abolished by Itacitinib pretreatment (Figure 7A). Consistently, Annexin V-FITC/PI staining showed that although IR and IFN-γ each significantly increased apoptosis in CT26.WT cells, pretreatment with Itacitinib abolished these effects. In the presence of Itacitinib, apoptosis rates across all treatment arms (IR, IFN-γ, IR + IFN-γ) dropped to near-control levels (2.6-3.7%). One-way ANOVA showed apoptosis was significantly reduced in Itacitinib-treated cells compared with control (4.6% vs 2.6%, P = 0.0082), confirming that IFN-γ engages the JAK–STAT1 pathway to promote apoptosis (Figure 7B). These findings indicate that therapeutic synergy relies on IFN-γ-mediated activation of the JAK1/STAT1 axis, culminating in tumor-cell apoptosis.

Figure 6.

Function of the IFN-γ–JAK–STAT axis. (A) Immunohistochemistry for phospho-STAT1 in tumor tissues (100 × and 400×); n = 4, mean ± SD. Four representative fields were randomly selected from biological samples (technical replicates). Statistical analysis: one-way ANOVA. (B) Western blots showing expression of p-JAK1, p-STAT1, and cleaved caspase-3 in CT26.WT and MC38 cells under control, IR, IFN-γ, and IR + IFN-γ conditions. Grayscale densitometry for all Western blots has been performed using ImageJ, normalized to β-actin (n = 3 independent experiments). Bars represent mean ± SD. Statistical comparison was performed using an unpaired two-tailed t-test. (C) Apoptosis was quantified by Annexin V-FITC/PI staining in CT26.WT cells (n = 3 independent experiments per group). One-way ANOVA revealed a significant overall effect of treatment (P < 0.0001).

Figure 7.

STAT1 inhibition assay. (A-B) Western blots and Annexin V/PI staining demonstrated that pharmacologic inhibition of STAT1 by Itacitinib abolished IFN-γ induced caspase-3 cleavage, and apoptosis in CT26.WT cells. Grayscale densitometry for all Western blots has been performed using ImageJ, normalized to β-actin (n = 3 independent experiments). Bars represent mean ± SD. Statistical comparison was performed using an unpaired two-tailed t-test.

Discussion

In the present study, we first investigated a publicly available GEO dataset comprising MSS CRC samples from patients who had received IR (24 Gy in 3 fractions) plus dual immune checkpoint inhibition. Differential expression and enrichment analyses highlighted pronounced rewiring of tumor-immune pathways, which we validated in vivo and in vitro. Although our mouse protocol (18 Gy/3 fractions) was not identical to the clinical schedule, published biologically effective dose calculations indicate that the two regimens are broadly comparable, especially after allowing for species-specific dose conversion. ²³ Similarly, we selected anti-PD-L1 instead of the anti-PD-1/anti-CTLA-4 combination used clinically because (i) prior murine safety data are robust for single-agent PD-L1 blockade and (ii) PD-1/PD-L1 signalling converges mechanistically on the same axis. ²² We will explicitly state that while clinical protocols may pair IR with dual ICIs, our single-agent choice prioritized internal validity for mechanistic readouts and animal welfare; evaluation of alternative ICIs (eg, PD-1) or combinations will be pursued in a prospectively powered follow-up study.

Using a CT26.WT syngeneic model, which mimics the pMMR/MSS phenotype and is largely refractory to single-agent immunotherapy, we demonstrated that IR combined with anti-PD-L1 achieved the greatest tumor-growth delay. ²⁷ Body-weight monitoring revealed noteworthy weight loss after irradiation, but the addition of anti-PD-L1 did not exacerbate the toxicity. Histological assessments of the liver and colon confirmed the absence of checkpoint-related hepatitis or colitis, thereby supporting a favorable safety profile. ²⁸ Notably, the superiority of combination therapy over IR alone did not reach statistical significance, most likely because of (i) substantial inter-mouse variability and (ii) the small cohort size (n = 4 per arm), which limited the power of the two-factor repeated-measures ANOVA. Future experiments will increase the sample size and apply prospective power calculations to detect the interaction effects between treatment and time more sensitively.

Immunohistochemistry showed that the combination regimen elicited the highest intratumoral CD3⁺/CD8⁺ T-cell infiltration, whereas flow cytometric profiling of splenocytes revealed parallel increases in peripheral CD4⁺ and CD8⁺ T-cell subsets. The recovery of viable immune cells was low and inconsistent across samples, thereby limiting flow cytometric profiling. We relied on spleen-derived lymphocytes as a surrogate for systemic immunity. The spleen is the largest secondary lymphoid organ, reflects systemic T-cell activation, and thus complements tumor-infiltrating lymphocyte data obtained by immunohistochemistry. These immunological changes were accompanied by significantly elevated IFN-γ concentrations in both tumor and spleen, implicating IFN-γ as a pivotal mediator linking local radiation effect to systemic immune activation. ²⁶ Consistent with this notion, KEGG and GSVA analyses flagged JAK-STAT signalling as a dominant pathway up-regulated after therapy, with nuclear phospho-STAT1 being most abundant in the combination group.

To determine causality, we exposed CT26.WT and MC38 cells to IR, recombinant IFN-γ, or both. IR alone failed to trigger downstream signalling, whereas IFN-γ robustly phosphorylated JAK1 and STAT1 and increased cleaved caspase-3, either alone or in conjunction with IR, confirming that IFN-γ is sufficient to activate the pro-apoptotic JAK1/STAT1 axis in CRC cells. ²⁹ Functional validation confirmed that pharmacologic inhibition of STAT1 by Itacitinib abolished IFN-γ– and IR + IFN-γ–induced caspase-3 cleavage, and apoptosis in CT26.WT cells. These results provide direct mechanistic evidence that STAT1 activation is not merely correlative but essential for the pro-apoptotic effects of IFN-γ in the context of radio-immunotherapy. This strengthens the translational relevance of our findings and supports future efforts to modulate STAT1 signaling in clinical strategies. These findings aligned with the emerging view that IR “primes the engine” by releasing neo-antigens and other signals, while checkpoint blockade prevents IFN-γ-driven adaptive resistance, thereby transforming an immunologically “cold” MSS tumor into a “hot” lesion. ³⁰ The mechanistic link we detailed here provides a biological explanation for the TORCH clinical trial conducted at our institution. ³¹ This research primarily validates the clinical efficacy of ‘total neoadjuvant immuno-radiotherapy’ in pMMR/MSS CRC, ¹⁵ while suggesting future strategies to expand therapeutic benefits for pMMR/MSS populations through optimized IR fractionation or targeting the IFN-γ/JAK-STAT pathway. ³²

However, this study has some limitations. Notably, the superiority of combination therapy over IR alone did not reach statistical significance, most likely because of substantial inter-mouse variability and the small cohort size (n = 4 per arm), which inevitably limited the statistical power of the repeated-measures ANOVA. Furthermore, some downstream assays were constrained by the allocation of tumor tissues: in the combination group, two tumors were paraffin-embedded for immunohistochemistry, while the other two were frozen for cytokine analysis, leading to lower biological replicates for individual assays. In future experiments, we will increase the cohort size, perform prospective power calculations to ensure adequate sensitivity, and employ multiplexed immunophenotyping methods to validate and extend the findings. Another limitation of our study is the low yield of viable immune cells from dissociated tumor tissues, which made direct flow cytometric profiling of the tumor microenvironment technically challenging. We attempted enzymatic digestion, but cell recovery was inconsistent and often insufficient for robust multiparametric analysis. To mitigate this, we used immunohistochemistry for mapping intratumoral T-cell infiltration and employed splenocytes as a surrogate for systemic immune responses. Nevertheless, we recognize that this approach provides only an indirect measure of systemic immunity. In future experiments, we plan to adopt optimized digestion protocols, high-parameter flow cytometry, and single-cell RNA sequencing or multiplex immunofluorescence, which will enable simultaneous spatial quantification of multiple immune subsets and a more refined characterization of the tumor–immune interface.^33,34 Although we titrated a single IFN-γ concentration (100 ng/mL) that effectively activated STAT1 in vitro, we acknowledge that this level likely exceeds the average concentrations detected in tumor homogenates. ²⁹ Nonetheless, such nanogram-per-milliliter doses are commonly employed in vitro to ensure receptor engagement and downstream signaling. To better approximate physiological gradients, future studies will incorporate autologous T-cell co-culture systems or conditioned medium assays, which can generate IFN-γ concentrations more closely reflecting the tumor microenvironment. Numerous studies have shown that STAT1 acts as a key transcription factor regulating the expression of downstream pro-apoptotic genes.^24,25 Finally, although we focused on the IFN-γ/JAK-STAT axis, IR also activates cGAS-STING DNA-sensing pathways that could cooperate with, or parallel, the responses described here. ¹⁷

Despite these caveats, our integrated bioinformatic and experimental data demonstrated that hypofractionated IR synergizes with PD-L1 blockade to remodel the TME, heighten IFN-γ production, activate STAT1-dependent apoptosis, and ultimately suppress MSS CRC growth. The combination trended toward improved tumor control versus IR alone but did not reach statistical separation in this small-n pilot; accordingly, we frame these results as mechanistic and hypothesis-generating, warranting adequately powered confirmation and clinical translation. These insights provide a mechanistic foundation for rationally designed radio-immunotherapy trials aimed at large patient populations with pMMR/MSS CRC.^35–37

Conclusion

In summary, combining IR with PD-L1 blockade markedly increases CD8⁺ T-cell infiltration in the CRC TME, elevates local and systemic IFN-γ levels, activates the JAK–STAT signalling cascade in tumor cells, and promotes apoptosis. These coordinated effects were associated with improved tumor growth control in pMMR/MSS models compared to IR alone. However, the small cohort size and inter-individual variability limit the statistical power of our findings, and the superiority of combination therapy over IR alone should therefore be interpreted with caution. Our findings support radio-immunotherapy as a promising strategy for a large population of patients with pMMR/MSS CRC and provide mechanistic insights to guide future clinical trials, while underscoring the need for larger, adequately powered studies to validate these findings and inform future clinical trial design.

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