Immunological conversion of triple-negative breast cancer by engineered extracellular vesicle-mediated pyroptosis

Although pyroptosis has emerged as a promising strategy for immunotherapy in triple-negative breast cancer (TNBC), its clinical translation remains hindered by systemic off-target toxicity and pronounced immune exclusion. To address these limitations, we developed a high-throughput double nanosecond pulsed microfluidic electroporation system to generate engineered extracellular vesicle (GDEV). These extracellular vesicles co-encapsulate gasdermin E N-terminal (GSDME-N) mRNA to induce pyroptosis and discoidal domain receptor 1 (DDR1) shRNA to suppress immune exclusion, respectively. Proteomics analysis of clinical TNBC specimens identified CD156 as a TNBC-specific surface biomarker. To achieve tumor-specific delivery and minimize off-target effects, GDEV was further functionalized with anti-CD156 antibody and the GALA pH-sensitive peptide, yielding pTGDEV. In the acidic tumor microenvironment, pTGDEV facilitates the precise release of its therapeutic cargo, simultaneously activating pyroptotic signaling and mitigating immune exclusion. In both orthotopic murine TNBC models and patient-derived xenograft models, pTGDEV significantly inhibited tumor progression and prolonged median survival. Treatment with pTGDEV also reduced the population of myeloid-derived suppressor cells while enhancing the infiltration of dendritic cells, CD4⁺ T cells, and CD8⁺ T cells, effectively reprogramming the tumor microenvironment from immunosuppressive to immunostimulatory. These results demonstrate that pTGDEV offers a potent and targeted approach for TNBC immunotherapy, with strong potential for clinical translation.