Background: Metastases are responsible for a significant portion of the morbidity and mortality in cancer patients. In situ destruction of tumor mass has been reported to provide the immune system with an antigen source for the induction of antitumor immunity, which can then attack distant metastases. Nitric oxide (NO) acts as a signaling molecule in multiple disease states, including cancer. NO has been shown to activate innate and adaptive responses of the immune system against tumors. In a previous Beyond Air study, 89% of CT26 tumor bearing mice treated with 50,000 parts per million (ppm) gaseous NO (gNO), rejected a secondary tumor induction. Here, we assessed the effects of a single intra-tumoral injection of gNO at concentrations of 20,000-50,000 ppm on immune cell recruitment in the tumor, spleen, and blood.

Methods: Mouse colon carcinoma (CT26) tumor bearing Balb/c mice underwent a single intra-tumoral treatment with 20,000 (n=3) or 50,000 (n=4) ppm gNO for 5 minutes. Mice treated with N2 (n=3), under the same conditions, served as a control. Tumors were removed 14 days post treatment and were histologically analyzed to test for immune cell penetration, inflammation, and necrosis. Immune cells from the spleen and blood were harvested 21 days post treatment and assessed via flow cytometry.

Results: 1. Treatment with 50,000 ppm gNO resulted in higher levels of inflammation, lymphocytes and T-cell infiltration, and necrosis within the tumor compared to both N2 and 20,000 ppm NO, as viewed histologically. 2. Flow cytometry analysis of immune cells from the spleen revealed a 1.3-fold increase of both B cells and IFN-γ expressing T cells with 50,000 NO treatment, and a 1.3-fold increase of B cells and 1.1-fold increase of IFN-γ expressing T cells with 20,000 NO treatment, compared to N2. 3. A 29% reduction of myeloid-derived suppressor cells (MDSCs) was detected in spleens of mice treated with 50,000 ppm gNO, and 40% for 20,000 ppm, compared to mice treated with N2.

Conclusion: Intra-tumoral injection of ultra-high doses of NO at 20,000 and 50,000 ppm gNO led to an increased recruitment and concentration of T and B cells. Additionally, 50,000 ppm NO led to an increase of lymphocyte infiltration to the tumor. Taken together, the data suggest that the increase in lymphocyte infiltration, B and T cell population, and the reduction of MDSCs, are indicative of an adaptive immune response that results in rejection of secondary tumors in gNO-treated mice.

Citation Format: Hila Confino, Matan Goldshtein, Yakir Levi, Lidor Malka, Amir Avniel, Steve Lisi, Ido Wolf. Single intra-tumoral injection of gaseous nitric oxide induces an adaptive immune response in a mouse CT-26 solid tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1283.