THSD4 is a novel mediator of T cell exclusion and anti-PD-1 resistance in hormone receptor-positive breast cancer

Breast cancer remains the most prevalent cancer among women, with hormone receptor-positive (HR +) tumors accounting for approximately 70% of breast cancer cases. While the immune checkpoint inhibitor (ICI) anti-programmed cell death 1 (PD-1) pembrolizumab has demonstrated efficacy in triple-negative breast cancers (TNBCs), its benefit in HR + subtypes is limited. ICI resistance in breast cancer is largely due to a "cold" tumor immune microenvironment characterized by low tumor-infiltrating lymphocytes (TILs). To identify novel genetic determinants of immune exclusion and pembrolizumab resistance, we analyzed multi-omics and clinical datasets from the I-SPY2 clinical trial and The Cancer Genome Atlas (TCGA), focusing on genes associated with low T cell infiltration and poor response to pembrolizumab. We identified thrombospondin type-1 domain containing 4 (THSD4) as a top candidate. THSD4 expression was significantly elevated in breast tumors with low T cells and in breast cancer patients exhibiting resistance to pembrolizumab, particularly within the HR + subtype. THSD4 expression is enriched in HR + breast cancers. Validation in local patient cohorts using RNA sequencing and multiplex immunofluorescence confirmed that both high THSD4 expression and anti-THSD4 antibody staining correlated with reduced T cell infiltration in the tumor epithelium and associations with poorer clinical outcomes. Functional studies in a syngeneic mouse HR + tumor model demonstrated that THSD4 promotes an immunosuppressive tumor microenvironment, with reduced T cells, resistance to anti-PD-1, and altered collagen fiber abundance. Collectively, these findings establish THSD4 as a prognostic biomarker of pembrolizumab resistance and a potential therapeutic target to enhance immunotherapy efficacy in breast cancer.