Cancer stem cells orchestrate immune evasion through extracellular vesicle-mediated non-canonical signaling pathways

Cancer stem cells (CSCs) within tumors exhibit a remarkable capacity to evade immune surveillance, thereby escaping elimination by the host immune system, yet the molecular mechanisms underlying this immune-evasive plasticity remain incompletely defined. Compared to other breast cancer subtypes, triple-negative breast cancer (TNBC) is particularly enriched with CSCs exhibiting basal-like molecular features characterized by elevated stemness indices, positioning it as an exemplary model for investigating bidirectional CSCs-TME crosstalk. Here, we mapped the spatial immunometabolic landscape of TNBC using high-dimensional flow cytometry to simultaneously interrogate 50 TME-associated proteins at single-cell resolution, including CSC markers tetraspanin-8 (TSPAN8) and CD44. A total of 161 treatment-naïve TNBC specimens were analyzed, stratified into CSC-high (CSCHi) and CSC-low (CSCLo) cohorts based on TSPAN8 and CD44 protein expression thresholds. TNBCs with elevated stemness indices drive effector-to-regulatory T cell (Treg) phenotypic conversion, establishing immunosuppressive niches. Blocking EV-associated TSPAN8 using an anti-TSPAN8 monoclonal antibody synergized with PD-1 checkpoint inhibition, providing preclinical validation for dual targeting of CSC plasticity and immune checkpoint pathways. The raw and processed data from high-dimensional single-cell proteomics analysis performed on the BD FACSymphony Flow Cytometry system, as shown in Figures 1B, 2B, and 2G, have been deposited.