Abstract 3991: NOS inhibition enhances docetaxel-mediated anti-tumor effect in obese mice with triple negative breast cancer
Authors: Uzair Ivonne , Sun Kai , Anselme Ann , Qian Wei , Zhou Jianying , Rosato Roberto , Chang Jenny 
Published: April 2023

Purpose of the study: To evaluate the role of obesity in tumor progression and tumor microenvironment (TME) dynamic after the inhibition of nitric oxide synthase (NOS) in syngeneic murine models engrafted with triple negative breast cancer (TNBC) tumors. For this study, we wanted to deepen the results of our recently completed phase I/II clinical trial, where treatment with NG-methyl-L-arginine (L-NMMA, a pan-NOS inhibitor) increased the response rate to chemotherapy (docetaxel) in obese metastatic TNBC (ER-/PR-/HER2-) patients to 86% vs. 60% in normal weight individuals. TME analysis from responders revealed a neutrophil phenotype shift from protumor N2 to antitumor N1. We thus hypothesize that combined treatment with L-NMMA and docetaxel enhances antitumor effect by modulating TME in obese mice with TNBC.

Experimental procedures: 3-week old female C57BL/6 mice were fed for 10 weeks either with high fat diet (HFD) or normal diet (ND). At week 13, TNBC mouse E0771 tumor cells were injected into the mice mammary fat pad. Once tumors reached 100 mm3, the animals were randomized to four groups: vehicle; docetaxel; L-NMMA; and docetaxel plus L-NMMA treatment. Tumor volume was measured throughout the experiment and growth rate was compared between groups at the end of treatment. Blood, tumor and perigonadal white adipose tissue (pgWAT) were collected. Cytokines and nitrite/nitrate levels (NO2-/NO3-, a NO indicator) were measured from blood and conditioned media (CM) from pgWAT using appropriate kits. Tumor immune cell infiltration was evaluated by immunohistochemistry. GraphPad software was used for statistical analysis and P-values &lt;0.05 were considered to describe statistically significant differences between groups.

Results: Tumor growth rate was significantly higher in HFD mice compared to ND mice. Treatment with the docetaxel/L-NMMA combination significantly slowed tumor growth in HFD mice (p=0.015), and showed a similar trend in ND mice (p=0.92) vs. the corresponding vehicle-treated groups. The reduction of tumor growth was significantly higher in HFD mice vs. ND mice (median of differences -2.0, p=0.031). HFD mice presented higher levels of pro-inflammatory cytokines, [IL-1b (p=0.011), IL-6 (p&lt;0.0001) and TNFa (p&lt;0.0001)] vs ND mice in pgWAT CM, as well as increased levels of NO2-/NO3- and expression of iNOS in tumors and in perigonadal adipose tissue.

Conclusion: HFD mice had a pro-inflammatory profile with a significantly faster tumor growth and higher expression of iNOS in tumor and adipose tissues. Treatment with the docetaxel/L-NMMA combination resulted in a more significant anti-tumor effect in HFD mice, likely through remodeling of TME. Spatial analysis, including CODEX and immunophenotyping are being conducted to determine the TME influence in obesity associated TNBC and the effect of NOS inhibition.

Citation Format: Ivonne Uzair, Kai Sun, Ann Anselme, Wei Qian, Jianying Zhou, Roberto Rosato, Jenny Chang. NOS inhibition enhances docetaxel-mediated anti-tumor effect in obese mice with triple negative breast cancer. [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 3991.