Deciphering Functional Heterogeneity of Cancer-Associated Fibroblasts Across Molecular Subtypes of Breast Cancer

Breast cancer heterogeneity has been well understood based solely on tumor components. How-ever, the tumor microenvironment, which plays a crucial role in cancer by regulating tumor cells' differentiation, maturation, and malignant potential, remains under-explored. This study con-ducted high-throughput RNA-Seq analysis on fresh breast cancer tissues from each molecular subtype and a pure population of cancer-associated fibroblasts (CAFs) isolated from cancer tissue and adjacent normal parts. Based on their functions, we identified three populations of CAFs: a) immune response-related, b) ECM remodeling, and c) calcium/protein binding. Validation of differentially expressed genes among these CAF populations revealed subtype-specific correla-tions. For instance, CAFs expressing immune response-related genes were significantly enriched in Luminal A/B and TNBC (log +1-fold; p =0.00047), compared to Her-2 positive (log -2.3-fold; p =0.0026). ECM remodeling genes exhibited greater expression in Her-2 positive and TNBC (log +4 to +11-fold; p =7.87E-08) when compared to Luminal subtypes (log +1.5-fold to +6-fold; p =8.66E-05). Calcium binding genes displayed overall similar expression across breast cancer subtypes. Thus, this study identified and underscored the functional heterogeneity of CAFs within the tumor microenvironment across the molecular subtypes of breast cancer.