Fig. 3: Tumor-directed radiation upregulates programs of antitumor immune surveillance to potentiate the αPD-1 ICI tumor response.

A (left) Cartoon schema of the experimental approach. WT animals injected with 4MOSC1 tumors were treated with tdRT on day 6. (right) Representative H&E and PD-L1 staining of tumor sections post-tdRT treatment. Combined Positive Score (CPS) for PD-L1 staining was 40.6. Tumor samples were fixed in zinc formalin fixative, embedded, sectioned, and stained. Immunohistochemistry for PD-L1 was performed using an anti-PD-L1 antibody, and CPS was calculated based on the ratio of PD-L1-positive cells to total viable tumor cells. Representative of n = 4 biologically independent samples; the experiment was independently repeated at least twice with similar results. B Quantification of PD-L1 expression on tumor cells post-tdRT, measured by flow cytometry, shown as normalized median fluorescence intensity (MFI) on live CD45− cells (n = 4 mice per group, p = 0.0237). Tumors were isolated, processed into single-cell suspensions, and stained with fluorochrome-conjugated anti-PD-L1 and CD45 antibodies. Flow cytometry was performed to assess PD-L1 expression levels. Data are presented as mean values ± SEM (n = 4 biologically independent samples/group); p values calculated by two-sided unpaired Student’s t test. C Pathway enrichment analysis of immune-related gene expression changes in tumors following tdRT→αPD-1 ICI, highlighting significant upregulation in pathways involved in antigen processing and presentation, phagocytosis, and T cell activation. Tumors were harvested, RNA was isolated, and RNA sequencing was performed. Gene set enrichment analysis was conducted using GSEAPreranked module on the GenePattern public server. X-axis represents gene sets ranked by normalized enrichment score (NES); Y-axis represents the −log₁₀(FDR q-value). D Cartoon schema of the experimental approach. WT animals injected with 4MOSC1 tumors were treated with tdRT on day 6 and subsequently treated with αPD-1 on day 8 or 10. E Cytokine analysis with normalized quantification of IFNγ and IFNβ levels shows significant increases in the tdRT→αPD-1 group compared to the αPD-1 only group (n = 4–5/group; p = 0.0453 for IFNγ, p = 0.0246 for IFNβ). Data are presented as mean values ± SEM (n = 5 biologically independent samples/group); p values calculated by two-sided unpaired Student’s t-test. F Tumor growth curves in 4MOSC1 and 4MOSC2-tumor bearing animals treated with αPD-1 or tdRT followed by αPD-1 (left and right, respectively). Left panels show control (0/10 responders) and αPD-1 treated groups (4/9 responders for 4MOSC1, p = 0.0156; 2/10 responders for 4MOSC2, p = 0.0156). Right panels show tdRT (0/10 responders) and tdRT followed by αPD-1 treated groups (8/10 responders for 4MOSC1, p < 0.0001; 7/10 responders for 4MOSC2, p < 0.0001). The data indicate that the combination therapy leads to a significantly higher response rate and tumor regression compared to αPD-1 alone. Best-fit lines and p values calculated by simple linear regression (two-sided). Created in BioRender. Saddawi-Konefka, R. (2025) https://BioRender.com/m675tdd. Source data are provided as a Source Data file.