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Immune checkpoint blockade therapy (ICB) has achieved remarkable successes in a subset of cancer patients across different malignancies, but responses are limited in breast cancer (BC), prompting investigations into combination therapies that can synergize with ICB. In pre-clinical models focal radiotherapy has been shown to induce systemic responses when combined with ICB in otherwise unresponsive tumors. Mechanistically, the DNA damage response induced by radiation is coupled with the accumulation of DNA in the cytosol of the cancer cells and leads to the production of interferon type I (IFN-I) in the tumor microenvironment (TME). IFN-I and IFN-stimulated genes (ISGs) orchestrate the recruitment and activation of immune cells.  Hypoxia reduces the effects of radiotherapy and promotes an immunosuppressive TME, in part mediated by high levels of adenosine. Here we investigated the effect of hypoxia on the radiation-induced IFN-I pathway activation. Furthermore, we assessed the regulation of immuno-suppressive adenosine generating enzymes CD73 and CD38 as well as PD-L1.

BC spheroids were generated from TSA (murine) and MDA-MB-231 (human) BC cells. 1000 cells (1T) and 40.000 cells (40T) were seeded, respectively, on day 0 and cultured for 3 days before treatment to establish non-hypoxic (1T) and hypoxic (40T) spheroids. Radiation was delivered as single doses (0 Gy, 8 Gy) or with 3 fraction of 8 Gy on 3 consecutive days. Cytosolic dsDNA was quantified fluorescence-based after subcellular fractionation (SpectraMax® Quant™ dsDNA Assay Kit). Expression of IFN-I, ISGs, PD-L1, and the adenosine generating ectonucleotidases CD73 and CD38 were assessed using RNAseq, qRT-PCR and flow cytometry. Spatial distribution and co-localization of hypoxia and proteins of interest were visualized in paraffin-embedded spheroids with immunofluorescence. Hypoxia was verified with pimonidazole-staining, yH2AX was assessed as a marker for DNA damage and Ki67 for proliferation.

Both breast cancer models yielded comparable results. Radiation-mediated accumulation of cytosolic dsDNA induced a robust IFN-I response in 2D standard cell cultures and small, non-hypoxic spheroids. Hypoxia markedly reduced cytosolic dsDNA accumulation (p<0.001) and abolished the IFN-I response. In contrast, the radiation-induced expression of adenosine-generating enzymes CD73 and CD38 was higher in hypoxic spheroids compared to 2D and non-hypoxic spheroids. Likewise, radiation increased PD-L1 expression strongly in hypoxic spheroids.

Our findings suggest that hypoxia is a major regulator of the ability of radiation to induce anti-tumor immune responses. We identified a twofold mechanism: hypoxia abolished the radiation-induced activation of IFN-I, and enhanced the expression of adenosine generating enzymes and PD-L1. Thus, strategies to counter hypoxia may be needed to improve the success of combinations of focal radiotherapy with ICB.