Abstract

Immune-resistant colorectal cancer (irCRC), represented by microsatellite stable/proficient mismatch repair (MSS/pMMR) tumors, responds poorly to immune checkpoint blockade (ICB). Here, we developed an ultrasound-targeted nanodroplet destruction (UTND) strategy combined with surface anti-PD-L1-decorated nanodroplets (PPP@P@map) to enhance ICB responsiveness in MSS colorectal cancer. In CT26 and CMT93 murine subcutaneous MSS colorectal cancer models, PPP@P@map combined with UTND markedly suppressed tumor growth compared with control treatments. Single-cell RNA sequencing identified CAF_Pi16 as a candidate therapy-associated cancer-associated fibroblast subset that showed a treatment-related increase after PPP@P@map + UTND, which was further supported by flow cytometry and multiplex immunofluorescence validation. Mechanistically, UTND induced oxidative stress in tumor cells and promoted the generation of advanced glycation end products (AGEs), which activated the AGE-RAGE/PKCα/NF-κB pathway in CAF_Pi16 and increased CCL7 secretion. CAF_Pi16-derived CCL7 provided a CCR2-dependent functional cue to CD8 + T cells. In purified CD8 + T cells, CCL7 activated NF-κB-related signalling, enhanced activation and cytotoxic effector features, and promoted NKG2D expression; these effects were attenuated by CCR2 blockade or NF-κB inhibition. Functionally, CCL7 enhanced CD8 + T cell-mediated tumor-cell killing, whereas NKG2D blockade reduced this effect and weakened the antitumor efficacy of PPP@P@map + UTND in vivo. Together, these findings reveal a UTND-induced tumor cell–CAF_Pi16–CCL7–CD8+ T-cell signalling axis that enhances NKG2D-associated antitumor immunity and provides a mechanistic basis for further investigation of UTND-based strategies to improve ICB responsiveness in MSS/pMMR colorectal cancer.