Distinct immune responses to proton and photon radiotherapy: implications for anti-PD-L1 combination therapy in colorectal cancer

Abstract

Background: Ionizing radiation can influence the antitumor immune response, either activating or suppressing the immune system depending on the tumor type and radiotherapy modality. While photon radiation (RT) combined with immunotherapy (IT) is widely studied in clinical trials, proton radiation (PT) combined with IT has not been thoroughly investigated in clinical or preclinical studies despite its radiobiological advantages. This study aims to explore the immune effects of a hypofractionated PT scheme compared to RT and its efficacy with anti-PD-L1 immunotherapy.

Methods: Balb/c mice bearing subcutaneous CT26 colon tumors were treated with RT or PT, delivered with 3 × 8 Gy. Seven days post-treatment, transcriptomic analysis and immune response assessments to characterize lymphoid cells, myeloid cells, and PD-L1 expression were performed. Tumor growth was monitored to evaluate the efficacy of combining RT or PT with anti-PD-L1 IT.

Results: The RNA sequencing analysis demonstrated an overexpression of genes involved in the interferon type I pathway after both RT and PT. Tumor microenvironment analysis showed enhanced immune cell infiltration in tumors after both treatments. Immunoactivating cells infiltration was observed, with LT CD8 + cells infiltration after both RT and PT, more significantly after RT. NK and TAM1 cells infiltrated only after RT. Immunosuppressive cell populations were induced by PT, including MDSCs, while Tregs infiltrated both RT and PT treated tumors. PD-L1 expression was significantly induced only by RT. The combination of anti-PD-L1 with RT or PT resulted in tumor growth delay compared to RT or PT alone, with a significant survival benefit observed only after the combination of RT and IT.

Conclusions: This study demonstrates that hypofractionated RT and PT induced both similar and significantly distinct immune responses. PT triggers a stronger immunosuppressive response than RT. Optimizing the combination of PT with IT, including dose, fractionation, and sequencing is crucial for improving treatment efficacy.

Keywords: Colorectal cancer; Immune response; Photon; Preclinical model; Proton therapy; Radiotherapy.

Fig. 1

RNA-Seq profiling analysis was assessed seven days post-irradiation, comparing CT26 tumors treated with 3 × 8 Gy with either RT or PT against non-irradiated controls (Control) (A) (Created in BioRender). Heatmaps were constructed to illustrate transcriptomic immune response pathways associated with RT (B) or PT (D), with statistical significance set a P-value < 0.05 and a fold change ≥ 2 using R software. Each experimental group comprised four mice. Enrichment analysis performed with gProfiler2 yielded lists from the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and REACTOME databases, associated with immune response pathways subsequent to RT (C) or PT (E) treatments. Ratio compared to control of the induction of certain interferon pathway genes, including cxcl10, ifnar and trex1 genes was observed following RT (F) or PT (G) treatments. Statistical analyses were performed using non-parametric Mann–Whitney test. Gene-specific p values are indicated on the graph next to the corresponding gene names. *p < 0.05, n = 4 per group

Fig. 2

Tumor microenvironment modifications induced by photon or proton therapy. Comparison of immune cell infiltration in CT26 tumors 7 days after 3 × 8 Gy with either RT or PT and non-irradiated controls (A) (Created in BioRender). The proportion of lymphocyte cells and their exhausted status (B) or NK and Treg (C), or myeloid cells (D) are represented with violin plots. Proportion of all cells expressing PDL1 (E). Statistical analyses were performed using non-parametric Mann–Whitney. *p < 0.05, **p < 0.01, ***p < 0.001, n = 5–8 per group

Fig. 3

Efficacy of combination therapy involving anti-PD-L1 treatment with either RT or PT. Tumors were treated with 3 × 8 Gy and anti-PD-L1 (10 mg/kg) 3 times per week over 3 weeks, 3 ten days after the injection of colon CT26 murine cancer cells into Balb/c mice (A) (Created in BioRender). Evaluation of untreated (black) CT26 tumors or those treated with anti-PD-L1 (blue), 3 × 8 Gy delivered with RT or PT irradiation combined with IgG (control) (orange) and RT or PT combined with anti-PD-L1 (purple) on tumor volumes implanted in immunocompetent BALB/c mice (B and D). Kaplan Meier survival curves with log-rank test comparisons, **p < 0.01 (C and E). Sample size: n = 5–10 mice per group for RT and n = 10–11 mice per group for PT. CR: complete response