Intratumoral Administration of High-Concentration Nitric Oxide and Anti-mPD-1 Treatment Improves Tumor Regression Rates and Survival in CT26 Tumor-Bearing Mice

Hila Confino; Yogev Sela; Yana Epshtein; Lidor Malka; Matan Goldshtein; Selena Chaisson; Steve Lisi; Amir Avniel; Jedidiah Mercer Monson; Frederick M Dirbas

doi:10.3390/cells12202439

Intratumoral Administration of High-Concentration Nitric Oxide and Anti-mPD-1 Treatment Improves Tumor Regression Rates and Survival in CT26 Tumor-Bearing Mice

Cells. 2023 Oct 11;12(20):2439. doi: 10.3390/cells12202439.

Authors

Affiliations

¹ Beyond Cancer, Rehovot 7608801, Israel.
² Beyond Cancer, Atlanta, GA 30305, USA.
³ Beyond Air, Garden City, NY 11530, USA.
⁴ Beyond Air Inc., Rehovot 7608801, Israel.
⁵ California Cancer Associates for Research and Excellence, Fresno, CA 93720, USA.
⁶ Department of General Surgery, Stanford University, Stanford, CA 94304, USA.

Abstract

Background: Immune checkpoint inhibitors have transformed clinical oncology. However, their use is limited as response is observed in only ~20-50% of patients. Previously, we demonstrated that treating CT26 tumor-bearing mice with ultra-high-concentration gaseous nitric oxide (UNO) followed by tumor resection stimulated antitumor immune responses. Accordingly, UNO may improve tumor response to immune checkpoint inhibitors. Here, we investigated the ability of UNO to improve the efficacy of a programmed cell death protein-1 (PD-1) antibody in vitro and in treating CT26 tumor-bearing mice.

Methods: CT26 cells were injected into the flank of Balb/c mice (n = 15-16 per group). On day 6, CT26 cells were injected into the contralateral flank, and anti-mPD-1 injections commenced. Primary tumors were treated with intratumoral UNO on day 8. Tumor volume, response rates, toxicity, and survival were monitored.

Results: (1) Short exposure to 25,000-100,000 parts per million (ppm) UNO in vitro resulted in significant upregulation of PD-L1 expression on CT26 cells. (2) UNO treatment in vivo consistently reduced cell viability in CT26 tumors. (3) Treatment reduced regulatory T-cell (Treg) levels in the tumor and increased levels of systemic M1 macrophages. UNO responders had increased CD8+ T-cell tumor infiltration. (4) Nine days after treatment, primary tumor growth was significantly lower in the combination arm vs. anti-mPD-1 alone (p = 0.0005). (5) Complete tumor regression occurred in 8/15 (53%) of mice treated with a combination of 10 min UNO and anti-mPD-1, 100 days post-treatment, compared to 4/16 (25%) of controls treated with anti-mPD-1 alone (p = 0.1489). (6) There was no toxicity associated with UNO treatment. (7) Combination treatment showed a trend toward increased survival 100 days post-treatment compared to anti-mPD-1 alone (p = 0.0653).

Conclusion: Combining high-concentration NO and immune checkpoint inhibitors warrants further assessment especially in tumors resistant to checkpoint inhibitor therapy.

Keywords: cancer immunotherapy; gaseous nitric oxide; immune checkpoint inhibitors; metastasis; solid tumors.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CD8-Positive T-Lymphocytes
Cell Line, Tumor
Humans
Immune Checkpoint Inhibitors*
Mice
Nitric Oxide* / metabolism

Substances

Nitric Oxide
Immune Checkpoint Inhibitors

Grants and funding

This work was financed by Beyond Cancer and its parent company Beyond Air. All authors are shareholders and employees or consultants of Beyond Cancer or Beyond Air except for Dr. Frederick Dirbas.