Treatment of tumors with ionizing radiation stimulates an antitumor immune response partly dependent on induction of IFNs. These IFNs directly enhance dendritic cell and CD8+ T cell activity. Here we show that resistance to an effective antitumor immune response is also a result of IFN signaling in a different cellular compartment of the tumor, the cancer cells themselves. We abolished type I IFN signaling in cancer cells by genetic elimination of its receptor, IFNAR1. Pronounced immune responses were provoked after ionizing radiation of tumors from 4 mouse cancer cell lines with Ifnar1 knockout. This enhanced response depended on CD8+ T cells and was mediated by enhanced susceptibility to T cell–mediated killing. Induction of Serpinb9 proved to be the mechanism underlying control of susceptibility to T cell killing after radiation. Ifnar1-deficient tumors had an augmented response to anti–PD-L1 immunotherapy with or without radiation. We conclude that type I IFN can protect cancer cells from T cell–mediated cytotoxicity through regulation of Serpinb9. This result helps explain why radiation of tumors can stimulate antitumor immunity yet also result in resistance. It further suggests potential targets for intervention to improve therapy and to predict responses.

Regulation of the programming of tumour-associated macrophages (TAMs) controls tumour growth and anti-tumour immunity. We examined the role of FGF2 in that regulation. Tumours in mice genetically deficient in low-molecular weight FGF2 (FGF2 LMW) regress dependent on T cells. Yet, TAMS not T cells express FGF receptors. Bone marrow derived-macrophages from Fgf2 LMW−/− mice co-injected with cancer cells reduce tumour growth and express more inflammatory cytokines. FGF2 is induced in the tumour microenvironment following fractionated radiation in murine tumours consistent with clinical reports. Combination treatment of in vivo tumours with fractionated radiation and a blocking antibody to FGF2 prolongs tumour growth delay, increases long-term survival and leads to a higher iNOS + /CD206 + TAM ratio compared to irradiation alone. These studies show for the first time that FGF2 affects macrophage programming and is a critical regulator of immunity in the tumour microenvironment.

The effect of radiation therapy on tumor vasculature has long been a subject of debate. Increased oxygenation and perfusion have been documented during radiation therapy. Conversely, apoptosis of endothelial cells in irradiated tumors has been proposed as a major contributor to tumor control. To examine these contradictions, we use multiphoton microscopy in two murine tumor models: MC38, a highly vascularized, and B16F10, a moderately vascularized model, grown in transgenic mice with tdTomato‐labeled endothelium before and after a single (15 Gy) or fractionated (5 × 3 Gy) dose of radiation. Unexpectedly, even these high doses lead to little structural change of the perfused vasculature. Conversely, non‐perfused vessels and blind ends are substantially impaired after radiation accompanied by apoptosis and reduced proliferation of their endothelium. RNAseq analysis of tumor endothelial cells confirms the modification of gene expression in apoptotic and cell cycle regulation pathways after irradiation. Therefore, we conclude that apoptosis of tumor endothelial cells after radiation does not impair vascular structure.

<div>AbstractPurpose:<p>To ask whether the expression of immune markers and IFN signaling in tumor biopsies changes during concurrent chemoradiotherapy (CCRT).</p>Experimental Design:<p>Tumor biopsies and peripheral mononuclear blood cells (PMBC) before and immediately after 20 Gy/10 fractions (F) of radiation treatment (RT) from 30 patients with cervical cancer receiving CCRT were evaluated by IHC and qRT-PCR for immune markers and correlated with the short-term response.</p>Results:<p>Tumor immune response to radiation before and after 10F RT as reflected by CD8⁺ T-cell infiltration had substantial heterogeneity with increases, decreases, and no change all evident. Increases in CD8⁺ T cells during CCRT correlated with the presence of nuclear IRF1 in tumor cells (<i>r</i> = 0.68, <i>P</i> < 0.0001) and the patient short-term response (<i>P</i> < 0.01). Similarly, in a subset of patients (∼40%) PD-L1 positivity in tumor cells increased, which also correlated with nuclear IRF1 staining (<i>r</i> = 0.48, <i>P</i> < 0.01). Patients with augmented PMBC IFN signature expression after 10F had a significantly higher probability of PD-L1 induction (83% vs. 7%, <i>P</i> < 0.0001). Most patients exhibited abundant expression of SERPINB9 and CD47 in tumor cells, and tumor infiltration by CD68⁺ cells. SERPINB9 expression correlated with STAT1 signaling in tumor cells.</p>Conclusions:<p>CCRT leads to differential tumor immunogenicity and IFN signaling in patients with cervical cancer, suggesting radiation induction of immunity is limited to a subset of patients and may reflect the heterogeneity of intratumoral induction of IFNs.</p><p><i>See related commentary by Mondini and Deutsch, p. 3815</i></p></div>

<div>AbstractPurpose:<p>To ask whether the expression of immune markers and IFN signaling in tumor biopsies changes during concurrent chemoradiotherapy (CCRT).</p>Experimental Design:<p>Tumor biopsies and peripheral mononuclear blood cells (PMBC) before and immediately after 20 Gy/10 fractions (F) of radiation treatment (RT) from 30 patients with cervical cancer receiving CCRT were evaluated by IHC and qRT-PCR for immune markers and correlated with the short-term response.</p>Results:<p>Tumor immune response to radiation before and after 10F RT as reflected by CD8⁺ T-cell infiltration had substantial heterogeneity with increases, decreases, and no change all evident. Increases in CD8⁺ T cells during CCRT correlated with the presence of nuclear IRF1 in tumor cells (<i>r</i> = 0.68, <i>P</i> < 0.0001) and the patient short-term response (<i>P</i> < 0.01). Similarly, in a subset of patients (∼40%) PD-L1 positivity in tumor cells increased, which also correlated with nuclear IRF1 staining (<i>r</i> = 0.48, <i>P</i> < 0.01). Patients with augmented PMBC IFN signature expression after 10F had a significantly higher probability of PD-L1 induction (83% vs. 7%, <i>P</i> < 0.0001). Most patients exhibited abundant expression of SERPINB9 and CD47 in tumor cells, and tumor infiltration by CD68⁺ cells. SERPINB9 expression correlated with STAT1 signaling in tumor cells.</p>Conclusions:<p>CCRT leads to differential tumor immunogenicity and IFN signaling in patients with cervical cancer, suggesting radiation induction of immunity is limited to a subset of patients and may reflect the heterogeneity of intratumoral induction of IFNs.</p><p><i>See related commentary by Mondini and Deutsch, p. 3815</i></p></div>

<p>Table S1-S7</p>

<p>Table S1-S7</p>

<p>Figure S1-S14</p>