FAP-targeted ROS-responsive nanoparticles delivering siBTF3 modulate the TME to enhance immunotherapy in colorectal cancer

Abstract

The tumor immunosuppressive microenvironment is a key factor limiting the efficacy of immunotherapy in colorectal cancer (CRC). This study aims to develop a novel nanodelivery system that reverses the immunosuppressive function of tumor-associated fibroblasts (CAFs) by specifically silencing basic transcription factor 3 (BTF3), thereby enhancing the antitumor immune response. Core-shell nanoparticles (NP@siBTF3) modified with fibroblast activation protein (FAP)-targeting and ROS-responsive properties were constructed for delivering BTF3 siRNA specifically into FAP-overexpressing human colorectal CAFs. The physicochemical properties of the nanoparticles were characterized using transmission electron microscopy and dynamic light scattering. Targeting efficiency was evaluated via confocal microscopy and in vivo imaging. The gene silencing efficacy, immune microenvironment remodeling effects, and antitumor activity were systematically assessed through qPCR, Western Blot, ELISA, flow cytometry, and tumor-bearing mouse models. NP@siBTF3 exhibits uniform particle size distribution, excellent serum stability, ROS-responsive release properties, and FAP-mediated targeting capability toward CAFs. In vitro experiments demonstrate its efficient silencing of BTF3 in CAFs, significantly downregulating TGF-β1 and IL-6 secretion alongside reduced expression of CAF activation markers α‑SMA and FAP. In the MC38 CRC mouse model, NP@siBTF3 treatment was associated with modulation of the tumor immune microenvironment, including increased CD8+ T cell infiltration, reduced regulatory T cell proportion, and enhanced macrophage polarization toward the M1 phenotype, while significantly inhibiting tumor growth. This strategy showed additional antitumor benefit when combined with anti‑PD‑1 antibody, although the enhancement was modest under the present experimental conditions, with no apparent systemic toxicity observed at the optimized dose. This study developed an intelligent nanodelivery system that may modulate the tumor-suppressive immune microenvironment by targeting and silencing BTF3 in CAFs. It suggests potential additive benefit with immune checkpoint blockade therapy, offering preliminary evidence for a potential strategy to enhance immunotherapy for CRC.