PlexinB1 and PlexinB2 as Regulators of the Immunosuppressive Tumor Microenvironment in Triple-Negative Breast Cancer

The tumor microenvironment (TME) is a dynamic multicellular ecosystem in which tumors, immune, and stromal cells cooperate to regulate cancer progression and therapeutic response. In triple-negative breast cancer (TNBC), immune evasion and stromal remodeling promote an immunosuppressive TME that limits the efficacy of immune checkpoint inhibitors, highlighting the need for novel therapeutic strategies targeting multiple TME components. Semaphorin/Plexin signaling has emerged as a key regulator of tumor progression, immune responses, and tissue organization. PlexinB1 inhibition in TNBC models has been shown to impair tumor progression and activate CD8⁺ T-cell1, while PlexinB2 promotes lung metastasis by acting in circulating tumor cell-monocyte clusters2. RT-qPCR analysis of 13 FFPE TNBC specimens, in collaboration with the Anatomy and Breast Unit at Istituto Nazionale Oncologico Candiolo INOC, revealed a clear upward trend in PlexinBs expression, with marked overexpression of PlexinB1 and even more pronounced upregulation of PlexinB2 in human TNBC. These findings suggest that both receptors contribute to the modulation of the immunosuppressive phenotype, consistent with prior observations in non-small cell lung cancer2. Interrogation of a public single-cell RNA-sequencing dataset (GEO: GSE305078, sample: GSM9161819) revealed cell type-specific expression patterns: PlexinB1 is preferentially expressed in T-cells, T-regulatory cells, and matrix CAFs (mCAF), while PlexinB2 is enriched in macrophages, mCAF, and tumor-associated CAFs (tCAF). Among ligands, Sema4A is predominantly expressed in macrophages, whereas Sema4D is elevated in CD8⁺ T-cells and neutrophils. These complementary expression profiles suggest that distinct PlexinB/Sema4 axes may cooperatively regulate TME composition in TNBC. In silico analyses of additional samples (GSM9161844, GSM9161845, GSM9161846) identified transcriptionally distinct CAF populations, including myCAF, mCAF, and iCAF subsets, supporting marked stromal heterogeneity in TNBC. These subpopulations are being characterized by RT-qPCR using established markers (e.g., myCAF: COL1A1, POSTN; iCAF: CFD, CD34, CXCL12) and correlated with Plexin expression profiles to investigate their contribution to TME heterogeneity and intercellular crosstalk in CAFs derived from breast cancer patients. This work aims to identify novel biomarkers and therapeutic targets to improve immunotherapy responses in TNBC.