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.

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

Background: Nitric oxide (NO) is a gaseous free radical produced from L-arginine by the nitric oxide synthase (NOS) enzymes. NO exerts a dose-dependent biphasic effect on lung cancer development, angiogenesis, and dissemination. The widespread contribution of nitric oxide signaling to lung cancer biology has cast a spotlight on the identification of NO-based therapeutic approaches as well as the use of fractional exhaled NO (FeNO) as a prognostic biomarker of clinical control. However, the significance of lung cancer treatment and prognosis has not been fully elucidated. Objective: This narrative review gives an overview of NO in lung cancer, focusing on its therapeutic and prognostic implications. Results: FeNO may help to assess the complications associated with non-pharmacological treatments, including postoperative pneumonia and radiation pneumonitis. By contrast, the role of FeNO dynamics during pharmacological treatment is still largely unexplored due to the suppressive effect of chemotherapy on FeNO levels. The rise of immunotherapy may pave the way to a better evaluation of FeNO as a prognostic biomarker of treatment response. The dichotomous involvement of NO in lung cancer events has led to the adoption of several NO-centered treatments that are focused on both inhibiting and enhancing NO signaling. However, NO chemical and biological characteristics have hindered its implementation in clinical practice. Conclusions: In the coming years, the advancements in drug delivery systems may lead to more effective anti-cancer applications of NO by improving tumor targeting and minimizing the systemic side effects. Together, our findings emphasize the promising role of NO in lung cancer treatment, underscoring the challenges and avenues for future research. 

From Prognostic Marker to Therapeutic Agent: The Role of Nitric Oxide in Lung Cancer


 Background: We have previously shown that treating mouse colon carcinoma (CT26) tumor-bearing mice with ultra-high concentrations of nitric oxide (UNO) upregulates innate and adaptive immune cells both locally and systemically. As immune-checkpoint molecules, such as the PD-L1 receptor PD-1, are expressed on immune cells, our group assessed the potential synergy of UNO and an anti-PD-1 antibody for treating tumors in vivo. Indeed, primary tumors regressed in 53% of the CT26 tumor-bearing mice treated with UNO and anti-PD-1. Moreover, these mice were primary and distant tumor-free and survived 100 days post-treatment. In the current study, we assessed PD-L1 expression levels on cancer cells in vitro following short-term exposure to UNO.
 Methods: CT26 cells were exposed to 25,000 - 100,000 ppm NO for 10 - 60 seconds. The percentage of PD-L1-expressing cells was assessed by flow cytometry analysis.
 Results: 1. Exposing CT26 cells to 100,000 ppm NO for 10 seconds upregulated PD-L1 expression. 85.1% of cells, which are still viable or at early apoptosis, expressed PD-L1 compared to 71% of untreated cells (p<0.0001). Exposure of CT26 cells to 25,000 ppm or 50,000 ppm NO for 10 seconds did not change the level of PD-L1 expression significantly. 2. Exposure to 100,000 ppm NO for 30 seconds further increased the percentage of cells expressing PD-L1 to 96.7% (p<0.0001 compared to untreated cells). For this duration, lower concentrations of NO also increased PD-L1-expression in viable early apoptotic CT26 cells to 82.8% - 92.3% when treated with 25,000 ppm or 50,000 ppm NO, respectively. 3. Finally, a 60 second exposure of CT26 cells to 25,000 ppm, 50,000 ppm, or 100,000 ppm NO resulted in the highest percentage of PD-L1 expressing cells. Approximately 95% of all viable or early apoptotic CT26 cells express PD-L1 under all NO conditions. This finding is significant compared to 71% of cells in the control arm that express PD-L1 (p<0.0001).
 Conclusion: Short-term exposure (10 - 60 seconds) to UNO upregulates PD-L1 expression on cancer cells in an NO concentration-dependent and exposure-dependent process. A 10-second exposure to 100,000 ppm NO is sufficient to significantly induce PDL-1 expression. Therefore, NO therapy can potentially be combined with anti-PD-L1 antibody treatment to improve therapeutic outcomes.
 Citation Format: Yana Epshtein, Matan Goldshtein, Selena Chaisson, Jedidiah Monson, Matt Johnson, Amir Avniel, Steve Lisi, Hila Confino. Short-term exposure of cancer cells to ultra-high concentrations nitric oxide induces PDL-1 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6363.

Abstract 6363: Short-term exposure of cancer cells to ultra-high concentrations nitric oxide induces PDL-1 upregulation


 Background: For the majority of metastatic triple-negative breast cancer (mTNBC) patients, chemotherapy is the standard first-line treatment, however, outcomes in this population are poor. A monoclonal antibody against the immune checkpoint inhibitor anti-programmed death 1 (PD-1) also has antitumor activity in patients with mTNBC. Previously, we reported that delivering ultra-high concentrations of nitric oxide (UNO) to mouse colon tumors stimulated innate and adaptive immune responses leading to the rejection of secondarily-induced tumors. Moreover, adding an anti-PD-1 antibody to UNO treatment resulted in primary tumor regression in 53% of the mice, the rejection of a distant tumors in all mice, and prolonged survival compared to control and anti-PD-1 arms. In this in vivo study, we investigated the ability of UNO to improve the efficacy of anti-PD-1 antibody treatment in the aggressive mouse breast cancer model, 4T1.
 Methods: We tested two treatment protocols: 4T1 mice tumors were treated with either a single dose of UNO and anti-PD-1 or a repeated dose of UNO and anti-PD-1. In the single dose design, 4T1 tumors were treated with a single administration of UNO combined with Anti-PD1 administration starting one day after UNO treatment (n=9-10 mice per group). In the repeated dosing design, 4T1 tumors were treated with UNO twice combined with Anti-PD-1 administration starting on the first UNO treatment day (n=8 mice per group). • UNO treatment regimen: 50,000 ppm, 10 minutes, flow rate ~0.2 liters per minute • Anti-PD1 treatment regimen: 10 mg/kg antibody was administered every 3 days for a total of 5 treatments.
 Results (UNO Single Dose): The primary tumors of 4T1 tumor-bearing mice were 13.2% smaller in the combined single-dose UNO group compared to anti-PD-1 alone 15 days post-UNO treatment.
 Results (UNO Repeated Dosing): The primary tumors of 4T1 tumor-bearing mice were 25.2% smaller in the combined repeated dose UNO group compared to the anti-PD-1 alone group 16 days after the first UNO treatment.
 Conclusion: Treating 4T1 tumors with single and repeated doses of UNO improved outcomes compared to anti-PD-1 alone. This suggests that local short-term treatment with UNO can serve as a treatment option for cancer patients with tumors that are not amenable to immune checkpoint inhibitor treatment.
 Citation Format: Yana Epshtein, Matan Goldshtein, Selena Chaisson, Jedidiah Monson, Matt Johnson, Amir Avniel, Steve Lisi, Hila Confino. Intratumoral administration of ultra high-concentration nitric oxide (UNO) is more efficacious than anti-PD1 therapy in 4T1 tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6327.

Abstract 6327: Intratumoral administration of ultra high-concentration nitric oxide (UNO) is more efficacious than anti-PD1 therapy in 4T1 tumor-bearing mice

In-situ tumor ablation provides the immune system with the appropriate antigens to induce anti-tumor immunity. Here, we present an innovative technique for generating anti-tumor immunity by delivering exogenous ultra-high concentration (> 10,000 ppm) gaseous nitric oxide (UHCgNO) intratumorally.The capability of UHCgNO to induce apoptosis was tested in vitro in mouse colon (CT26), breast (4T1) and Lewis lung carcinoma (LLC-1) cancer cell lines. In vivo, UHCgNO was studied by treating CT26 tumor-bearing mice in-situ and assessing the immune response using a Challenge assay.Exposing CT26, 4T1 and LLC-1 cell lines to UHCgNO for 10 s-2.5 min induced cellular apoptosis 24 h after exposure. Treating CT26 tumors in-situ with UHCgNO followed by surgical resection 14 days later resulted in a significant secondary anti-tumor effect in vivo. 100% of tumor-bearing mice treated with 50,000 ppm UHCgNO and 64% of mice treated with 20,000 ppm UHCgNO rejected a second tumor inoculation, compared to 0% in the naive control for 70 days. Additionally, more dendrocytes infiltrated the tumor 14 days post UHCgNO treatment versus the nitrogen control. Moreover, T-cell penetration into the primary tumor was observed in a dose-dependent manner. Systemic increases in T- and B-cells were seen in UHCgNO-treated mice compared to nitrogen control. Furthermore, polymorphonuclear-myeloid-derived suppressor cells were downregulated in the spleen in the UHCgNO-treated groups.Taken together, our data demonstrate that UHCgNO followed by the surgical removal of the primary tumor 14 days later induces a strong and potent anti-tumor response. 

https://cancerci.biomedcentral.com/counter/pdf/10.1186/s12935-022-02828-z

Gaseous nitric oxide tumor ablation induces an anti-tumor abscopal effect

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Abstract 6363: Short-term exposure of cancer cells to ultra-high concentrations nitric oxide induces PDL-1 upregulation

Abstract 6327: Intratumoral administration of ultra high-concentration nitric oxide (UNO) is more efficacious than anti-PD1 therapy in 4T1 tumor-bearing mice

Gaseous nitric oxide tumor ablation induces an anti-tumor abscopal effect