Abstract 7459: iNOS inhibition through L-NMMA reveals immunostimulatory effects in obesity-associated triple-negative breast cancer (TNBC) tumors
Authors: Uzair Ivonne , Sun Kai , Hynes Seun , Qian Wei , Zhou Jianying , Martinez Karina Ortega , Guzman Liliana , Matre Polina , Chang Jenny 
Published: March 2024

Purpose of study: This study examines how obesity, an inflammatory condition, impacts treatment outcomes in triple-negative breast cancer (TNBC). Obesity fosters an unfavorable tumor microenvironment (TME) marked by immune suppression, metabolic shifts, and protumoral cytokine release. Elevated nitric oxide (NO) levels linked with obesity compromise vascular integrity, promoting breast cancer metastasis. This study examines whether combining standard chemotherapy (docetaxel) with NOS inhibition via L-NMMA could reshape the TME and bolster antitumor effects in TNBC within an obese mouse model.

Experimental Procedure: C57bl/6 mice, a TNBC syngeneic mouse model, were fed a high-fat diet (HFD) or normal diet (ND) for 10 weeks. Tumors were induced orthotopically via E0771 cell line injection into the mammary fat pad. Upon reaching 100 mm^3, mice were randomized into vehicle and L-NMMA treatment groups. Digital spatial transcriptomics using NanoString™ GeoMx and nCounter platforms were conducted on FFPE tumor tissue sections from ND, HFD, and HFD+LNMMA treated mice. A panel targeting 64 protein markers aided in immune cell phenotyping and TME assessment.

Results: HFD-induced tumors exhibited increased growth, elevated pro-inflammatory cytokine levels, and heightened iNOS expression. Unsupervised hierarchical clustering after nCounter analysis revealed a distinct protein expression signature in HFD tumors associated with T cell infiltration and exhaustion, as evidenced by the upregulation of CD3, CD4, PD-1/PD-L1, FOXP3, and CTLA 4. Markers for identifying immunosuppressed macrophages (F4/80 and CD163) were also elevated. Remarkably, LNMMA treatment on HFD tumors led to reduced tumor growth and lower expression levels of these markers, alongside a downregulation of survival/proliferation markers such as p53, Ki67, and key members of the Pi3k signaling pathway such as phospoAktSer473.

Conclusions: Our findings indicate a distinct immunomodulatory mechanism of iNOS inhibition in HFD tumors, which displayed expression profiles marked by heightened immunosuppressive markers when compared to ND tumors. LNMMA treatment reversed this phenotype, aligning the profile closer to ND tumors. These discoveries underscore the potential of inhibiting iNOS to reshape the immune tumor microenvironment (TME) and modulate pathways linked to cell proliferation and survival. This presents a promising avenue for enhancing the effectiveness of treatments for triple-negative breast cancer (TNBC).

Citation Format: Ivonne Uzair, Kai Sun, Seun Hynes, Wei Qian, Jianying Zhou, Karina Ortega Martinez, Liliana Guzman, Polina Matre, Jenny Chang. iNOS inhibition through L-NMMA reveals immunostimulatory effects in obesity-associated triple-negative breast cancer (TNBC) tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7459.