Abstract C109: Single-nucleus sequencing reveals a functionally distinct breast tumor microenvironment by ancestral group with implications for disease progression and therapy

Abstract Breast cancer biology is influenced by both genetic ancestry and the environment with implications for disease outcomes. However, we lack the knowledge how non-cancerous cells within tumors are functionally altered by these factors at single-cell resolution. Here, we created a single-nucleus (sn) dataset from 33 African American, 25 Kenyan, and 24 European American women with breast cancer using combined RNA and ATAC sequencing. We successfully isolated intact, high-quality single nuclei from archival frozen breast tumor tissue using an optimized combination of enzymatic digestion and automated tissue homogenization. SnMultiome sequencing of 82 tumors was performed using the 10x Genomics platform. Following filtering, normalization, peak calling, and integration, our dataset includes a total of 292,458 nuclei. Cancerous (163,419 nuclei) and non-cancerous (129,039 nuclei) cells were distinguished based on DNA copy number. Within the microenvironment, 11 major immune, epithelial, and stromal cell types were identified. CD45+, EPCAM- cells were further analytically extracted, clustered, and manually annotated based on known marker genes, detecting 26 distinct immune subpopulations across our samples. After adjustment for potential confounders (age, body mass index, and tumor molecular subtype) in all analyses, we found that women of African descent possessed distinct, activated mesenchymal and endothelial cell subpopulations within the tumor vasculature that promote inflammation, angiogenesis, and cancer cell invasion. Women of African descent exhibited a more immune-suppressive tumor microenvironment, with elevated expression of immune checkpoint markers (PD-1, TIGIT, CTLA-4, and LAG-3), functional impairment of dendritic cells, and enrichment of regulatory and tolerogenic lymphocytic subpopulations. Further, malignant cells from African descent patients expressed a discrete set of CAR-T targets with potential implications for precision immunotherapy. Lastly, we developed three gene signatures within the microenvironment that are predictive of survival with external validation in both tumor-adjacent normal and cancerous tissues from the TCGA. Together, we uncovered molecular breast tumor characteristics of clinical significance in women of African ancestry. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany D. Jenkins, Sanna Madan, Tiangen Chang, Tiffany H. Dorsey, Eytan Ruppin, Shahin Sayed, Francis Makokha, Gretchen L. Gierach, Stefan Ambs. Single-nucleus sequencing reveals a functionally distinct breast tumor microenvironment by ancestral group with implications for disease progression and therapy abstract. In: Proceedings of the 18th AACR Conference on the Science of Cancer Health Disparities; 2025 Sep 18-21; Baltimore, MD. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2025;34(9 Suppl):Abstract nr C109.