Single-Cell Analysis Reveals BRCA1 Mutation Drives Distinct Tumor Microenvironments in Triple-Negative Breast Cancer
Triple-negative breast cancer (TNBC) represents one of the most aggressive breast cancer subtypes, with approximately 25% of patients experiencing disease recurrence compared to 15% across all breast cancer types. The 5-year survival rate for TNBC is approximately 77%, which is 8-16% lower than hormone receptor-positive breast cancers. Understanding the molecular mechanisms driving this heterogeneity has become crucial for developing effective therapeutic strategies.
BRCA1 Mutations Create Immunologically "Hot" Tumors
A comprehensive single-cell RNA sequencing analysis of TNBC samples has revealed fundamental differences in tumor microenvironments based on BRCA1 mutation status. The study analyzed 32,386 cells from eight TNBC specimens, comprising four BRCA1-mutant (BRCA1-MT) and four BRCA1 wild-type (BRCA1-WT) samples.
The research demonstrated that BRCA1-MT tumors exhibit characteristics of immunologically "hot" tumors, with significantly elevated proportions of immune and stromal cells compared to BRCA1-WT tumors. In contrast, BRCA1-WT tumors were primarily composed of tumor cells, displaying features of immunologically "cold" tumors.
Distinct Immune Cell Profiles Drive Different Therapeutic Responses
The analysis revealed striking differences in immune cell populations between the two groups. BRCA1-WT specimens exhibited more exhausted but enhanced cytotoxic T cells, alongside tolerant dendritic cells. These tumors showed robust expression of HLA-related molecules, indicating potential immunotherapeutic response probability. However, the dominant tumor cells and myofibroblastic cancer-associated fibroblasts (myCAFs) increased tumor microenvironment stiffness, suggesting poorer immune responses.
BRCA1-MT tumors demonstrated higher levels of immune infiltration with more activated tumor-associated macrophages (TAMs) and inflammatory-like CAFs (iCAFs). The study found that TAMs from BRCA1-MT tumors exhibited significantly higher M1 polarization scores, as well as elevated angiogenesis and phagocytosis scores compared to those from BRCA1-WT tumors.
ISG15 Emerges as Key Biomarker for Immunotherapy Response
Through multi-scale integrated analysis combining single-cell, spatial, and bulk RNA sequencing data, researchers identified ISG15 as a critical biomarker associated with myCAFs in BRCA1-WT patients. ISG15 expression was significantly upregulated in the BRCA1-WT group and showed strong co-localization with myCAF subclusters in spatial transcriptomics analysis.
Using this finding, the research team developed a machine learning-based predictive system for immunotherapy response. The model, trained on 13 immune checkpoint inhibitor cohorts encompassing 829 patients, achieved area under the curve (AUC) values of 0.60 in validation and 0.61 in testing sets. Patients with higher response probability demonstrated significantly better overall survival outcomes.
CXCL9-CXCR3 Axis Controls Cancer Cell Behavior
A parallel study focusing on ligand-receptor interactions in TNBC identified the CXCL9-CXCR3 signaling axis as crucial for tumor progression. Analysis of 38,007 high-quality cells from nine TNBC samples revealed 73 significantly correlated ligand-receptor pairs, with 57 showing prognostic relevance.
The research established two distinct molecular subtypes based on ligand-receptor expression profiles. Cluster 1 exhibited heightened immune infiltration with prominent activation of interferon response pathways, while Cluster 2 showed relatively lower immune activity. Patients in Cluster 1 demonstrated better overall survival in both training and validation cohorts.
Experimental Validation Confirms Therapeutic Targets
Functional studies using MDA-MB-231 cells provided experimental validation of the CXCL9-CXCR3 axis importance. siRNA-mediated knockdown of both CXCL9 and CXCR3 resulted in significant reductions in cell proliferation, colony formation ability, and migration capacity. These findings confirm the critical roles of this signaling pathway in regulating cancer cell behavior.
The study developed a six-ligand-receptor pair scoring model (LR.score) that effectively stratified patient risk and predicted immunotherapy response. The model demonstrated robust prognostic utility across multiple independent cohorts, with patients showing higher LR.score values experiencing significantly poorer survival outcomes.
Clinical Implications for Precision Medicine
These findings have important implications for TNBC treatment strategies. The research suggests that BRCA1 mutation status alone may not be sufficient for predicting immunotherapy response, highlighting the need for more sophisticated biomarker approaches. The ISG15-based predictive system and LR.score model offer potential tools for identifying patients most likely to benefit from immune checkpoint inhibitor therapy.
The study also revealed distinct cell-cell communication networks, with iCAF-mediated CXCL12-CXCR4 signaling predominant in BRCA1-MT tumors and myCAF-mediated CD99-CD99 interactions prevalent in BRCA1-WT tumors. These findings provide new therapeutic targets for both tumor types.
Future Therapeutic Directions
The research opens several avenues for therapeutic intervention. Targeting the CXCL9-CXCR3 axis could potentially enhance immune infiltration and improve checkpoint inhibitor responses. Additionally, the distinct cancer-associated fibroblast populations identified in each BRCA1 subtype suggest opportunities for stromal-targeted therapies.
The comprehensive molecular characterization provided by this study represents a significant advance in understanding TNBC heterogeneity. By integrating single-cell transcriptomics with clinical outcomes, the research provides a framework for developing more precise prognostic tools and targeted therapeutic strategies for this challenging cancer subtype.
