Ultrasound-targeted nanobubble destruction induces Macro Ccl8 to suppress treg function and enhance immunotherapy efficacy in CRC

Microsatellite stable (MSS) colorectal cancer (CRC) is largely resistant to immune checkpoint blockade due to its immunosuppressive tumor microenvironment (TME). Ultrasound-targeted nanobubble destruction (UTND) offers a localized physical delivery method, but its active immunomodulatory mechanisms remain underexplored. Ultrasound-responsive NBs loaded with CTLA-4 antibody (PF4) were fabricated and characterized. A CT26 mouse CRC model was used to evaluate anti-tumor effects across four treatment groups. Single-cell RNA sequencing (scRNA-seq) analyzed cellular changes in the TME. The activation of the NF-κB pathway was verified through western blot analysis, qPCR, and immunofluorescence staining. Intracellular ROS production was assessed using flow cytometry, confocal microscopy, and an enzyme immunoassay. Furthermore, the effects of Ccl8 on regulatory T (Treg) cells were examined by flow cytometry, qPCR and western blot. Combination treatment with UTND and anti-CTLA-4 suppressed tumor growth relative to single-agent treatments. ScRNA-seq identified a CCL8-expressing macrophage subpopulation (Macro Ccl8) that was more abundant in the combination group. UTND promoted ROS production, which activated NF-κB signaling and increased Ccl8 transcription. CCL8 secreted by Macro Ccl8 modulated Treg function through the Ccl8-Ccr2 axis. Together with direct CTLA-4 checkpoint inhibition, this cascade contributed to elevated CD8 + T cell activity. In the CT26 mouse model, UTND combined with CTLA-4 antibody modulates the immunosuppressive TME partially through inducing Macro Ccl8 via the ROS–NF-κB–Ccl8 axis, in addition to direct CTLA-4 blockade. This dual mechanism attenuates Treg function and enhances antitumor immunity, providing a preclinical rationale for combining ultrasound-based physical intervention with immunotherapy in MSS CRC.