Adjuvant COX inhibition augments STING signaling and cytolytic T cell infiltration in irradiated murine 4T1 TNBC tumors

Immune therapy has emerged as the new frontier of cancer treatment. Therapeutic radiation is a known inducer of an immune response that can be limited by immunosuppressive mediators including cyclooxygenase-2 (COX2), which is highly expressed in triple negative breast tumors. A clinical cohort of triple negative breast cancer (TNBC) tumors revealed that elevated COX2 tumor expression predicted poor radiation therapeutic efficacy. Using the aggressive murine 4T1 TNBC model, we show that treatment with ionizing radiation and adjuvant NSAID (indomethacin) therapy to inhibit COX2 provides a powerful combination to reduce both primary tumor growth and lung metastasis through immune modulation. Using whole tumor RNAseq and multiplex fluorescence imaging, this study reveals spatial immunological changes in the treated primary tumor. Indomethacin alone increased the infiltration of lymphoid populations into the primary tumor. Importantly, the combined treatment improved lymphoid infiltration into the tumor epithelium, augmented cGAS/STING1 and type I IFN gene expression and altered key immune checkpoints including PD1, IDO, and CTLA4. Markedly increased B cell and T cell populations with increased CD8+ T cell activation and reduced T cell exhaustion were observed. Thus, adjuvant NSAID treatment combined with radiation therapy shifts “immune desert phenotypes” toward anti-tumor immune mediators favoring M1/Th1 signatures in the immunologically challenging 4T1 tumor model. Importantly, radiation/indomethacin combination improved local control of the primary lesion, reduced metastatic burden, and increased median survival when compared to mice treated with single agent radiation. These results show that clinically available NSAID's augment radiation therapeutic efficacy through improved antitumor immune response and augmented local generation of cGAS/STING1 and type I IFNs Overall design: In this study, we explored the effects of pan-NOS and -COX inhibitors on radiation therapeutic efficacy in the murine 4T1 TNBC model. After implanted 200k 4T1 TNBC cells at the age of 8 weeks old, mice were divided into 4 groups: (1) control, (2) 6 Gy radiation, (3) indomethacin, and (4) 6 Gy radiation plus indomethacin.