Abstract 1606: Unraveling the microbiome-iNOS connection in obesity-driven triple-negative breast cancer

Abstract Introduction: A recent Phase 1/2 clinical trial evaluating the efficacy of the inducible nitric oxide synthase (iNOS, NOS2) inhibitor L-NMMA in combination with taxanes for patients with chemorefractory, locally advanced breast cancer or metastatic triple-negative breast cancer (TNBC) reported an overall response rate (ORR) of 45.8%. Notably, stratification by body mass index (BMI) revealed that obese patients had a significantly higher ORR (85.6%) compared to non-obese patients (46.7%), correlating with elevated iNOS expression in tumor biopsies from obese patients. While obesity typically worsens disease-free and overall survival in TNBC, these findings suggest a potential role for iNOS inhibition in overcoming obesity-driven tumor progression. To investigate further, we studied the impact of iNOS inhibition on tumor growth and treatment response in an obese mouse model and examined the role of the gut microbiome. Obesity was induced in C57BL/6 mice using a high-fat diet (HFD) for 10 weeks prior to orthotopic implantation of E0771 TNBC cells in the mammary fat pad of female mice. Control mice were fed a normal diet (ND). Results:- HFD-fed mice developed larger, more aggressive tumors, with detectable burden occurring ∼3 weeks earlier. Tumors in HFD mice reached humane endpoints ∼17 days sooner than ND mice, whose tumors grew to 80% of HFD tumor size in the same period. Lung metastasis was significantly higher in HFD mice. - L-NMMA combined with docetaxel delayed tumor growth in both diet groups, though the untreated/treated tumor size ratio was higher in HFD mice (6.5) than ND mice (2.1). - Gut microbiome analysis revealed diet- and tumor-associated compositional changes. FMT (fecal microbiota transplantation) from ND mice to HFD mice reduced tumor size and improved therapeutic response, while FMT from HFD mice to ND mice increased tumor size and impaired response. Immunohistochemistry showed elevated iNOS levels in tumors of HFD mice, which normalized following ND-FMT. Conversely, ND mice receiving HFD-FMT displayed increased iNOS expression. Serum cytokine profiles reflected these tumor and treatment differences. Conclusion: These findings suggest that HFD-induced gut microbiome alterations create a pro-inflammatory environment driving increased iNOS expression, promoting tumor aggressiveness and influencing therapeutic response. Targeting the gut microbiota, alongside iNOS inhibition, could provide novel strategies for managing obesity-associated TNBC. Citation Format: Ivonne Uzair, Ann C. Anselme, Wei Qian, Jianying Zhou, Adriana E. Rosato, Roberto R. Rosato, Jenny C. Chang. Unraveling the microbiome-iNOS connection in obesity-driven triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1606.