Dual Targeting of FAP-Directed Nanoparticles and FRα-Specific CAR-T Cells Induces Additive Anti-Tumor Effects in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive malignancy with limited treatment options. It lacks hormone receptors and human epidermal growth factor receptor 2. The immunosuppressive tumor microenvironment (TME), particularly cancer-associated fibroblasts (CAFs), significantly hinders chimeric antigen receptor (CAR)-T cell therapy success. Novel strategies to overcome TME-mediated immunosuppression are urgently needed. We evaluated whether targeting CAFs with fibroblast activation protein alpha (FAP)-coated, 8--methylfusarubin-loaded nanoparticles called anti-FAP@OMF-NPs could enhance the anti-tumor efficacy of folate receptor alpha (FRα)-specific CAR-T cells against TNBC in a 3D cancer cells-CAFs co-culture heterospheriod (HS) model. FRα and FAP expression in TNBC cells and primary breast CAFs were assessed using immunofluorescence and flow cytometry. Anti-FRα-CAR-T cells were generated via lentiviral transduction and characterised for activation markers. Cytotoxic activity of CAR-T cells, anti-FAP@OMF-NPs, and their combination was evaluated in 3D-HS comprising FRα-high TNBC cells and FAP-high CAFs. A fluorescent transfection assay measured cell viability. Cytokine bead arrays quantified IFN-γ, granzyme A, and granzyme B levels to assess anti-tumor immune activation. PC-B-130CAFs and PC-B-132CAFs demonstrated high FAP expression compared with PC-B-004CAFs and normal human dermal fibroblast cells (HDFa). Anti-FRα-CAR-T cells selectively targeted FRα-positive TNBC cells whilst showing minimal cytotoxicity towards normal MCF-10A cells. Anti-FAP@OMF-NPs induced potent cytotoxic effects specifically in FAP-expressing CAFs. Combined treatment significantly enhanced the destruction of MDA-MB-231/130CAF and MDA-MB-231/132CAF HSs compared with monotherapies. This combination increased secretion of IFN-γ, granzyme A, and granzyme B from anti-FRα-CAR-T cells. Targeting CAFs using anti-FAP@OMF-NPs enhances the cytotoxic efficacy of FRα-specific CAR-T cells in TNBC. This combinatorial approach offers a promising strategy to overcome TME-mediated immunosuppression. These findings support further development of dual-targeting approaches to improve therapeutic outcomes in TNBC.