Genetically engineered extracellular vesicles with chimeric antigen receptors of human stem cell-derived neutrophils targeting fibroblast activation protein to modulate tumor microenvironment

Glioblastoma (GBM) is the most commonly occurring central nervous system tumor, accounting for over 50% of gliomas, with a poor survival rate. A primary driver of GBM progression is the fibrotic stroma, where excess extracellular matrix deposition creates a dense network that excludes immune cells and chemotherapeutic agents. While targeting tumor-associated fibroblasts (TAFs) via fibroblast activation protein (FAP)-directed chimeric antigen receptor (CAR) immune cells is a promising strategy, off-target toxicities limit the clinical utility of the treatment. To address these safety concerns, extracellular vesicles (EVs) of FAP-CAR-engineered neutrophils differentiated from human pluripotent stem cells were isolated and characterized, including proteomic profile, microRNA content, in vitro bioactivity, and in vivo biodistribution in this study. Multi-omic profiling revealed that CAR engineering enriched EVs with proinflammatory proteins, iron-transferring proteins, and proliferation regulators, and a significant upregulation of miRNAs targeting FAP and proliferation-regulating genes was observed. Functional in vitro assays demonstrated that these EVs are selectively cytotoxic to TAFs, reducing the expression of BCL-2 and RAS-MAPK-ERK signaling pathway genes. These EVs also demonstrated cytotoxicity to patient-derived GBM spheroids. Furthermore, in vivo biodistribution results confirmed that FAP-CAR EVs are capable of systemic circulation and payload delivery, showing a significant increase in accumulation within the liver and spleen compared to free Cy5.5. These findings suggest that CAR engineering enhances the ability of FAP-CAR EVs to inhibit TAF proliferation and induce apoptosis. FAP-targeting CAR EVs are positioned as a targeted bioengineered delivery platform with the potential for remodeling the tumor microenvironment and slowing the progression of desmoplasia in GBM.