Tumor NOS2 and COX2 Spatial Juxtaposition with CD8⁺ T Cells Promote Metastatic and Cancer Stem Cell Niches that Lead to Poor Outcome in ER^- Breast Cancer

Estrogen receptor–negative breast cancer is an aggressive subtype with limited therapeutic options. Elevated nitric oxide synthase 2 (NOS2) and COX2 mediate immunosuppression and poor survival in these tumors. Therefore, the influence of tumor NOS2/COX2 on immune architecture was examined in 16 African American and five Caucasian estrogen receptor–negative tumors. Elevated tumor NOS2/COX2 limited CD8⁺ T-cell infiltration at 5-year survival. Distinct CD8^+/-NOS2^+/-COX2^+/phenotypes defining metastatic and cancer stem cell niches and immune desert regions were identified. These results were supported by an unbiased, unsupervised nonlinear dimensionality reduction technique, Uniform Manifold Approximation and Projection for Dimension Reduction, incorporating spatial relations between cells and validated in a separate gene expression cohort using NOS2/CD8 and COX2/CD8 ratios. Additionally, elongated tumor cells were specifically in CD8^-NOS2⁺COX2⁺ regions, suggesting metastatic hot spots. This work demonstrates predictive power of spatial analyses of CD8/NOS2/COX2 architecture and supports the use of clinically available NOS2/COX2 inhibitors for improved survival in patients with these aggressive tumors. Significance: This work identifies CD8^-NOS2⁺COX2⁺ and CD8^-NOS2^-COX2⁺ unique cellular neighborhoods that drive the tumor immune spatial architecture of CD8⁺ T cells predictive of clinical outcome and can be targeted with clinically available NOS inhibitors and NSAIDs.