A CD36-Targeting Thermosensitive Berberine Nanogel Blocks Tumor Lipid Hijacking and Potentiates Anti-PD-L1 Immunotherapy in Triple-Negative Breast Cancer

The limited efficacy of programmed death-ligand-1 (PD-L1) monoclonal antibodies (aPD-L1) in triple-negative breast cancer (TNBC) is largely attributable to the immunosuppressive tumor microenvironment (TME). Notably, the abundance of cancer-associated adipocytes (CAAs) constitutes a distinctive feature of the TNBC microenvironment, contributing significantly to its immunosuppressive nature. CAAs upregulate cluster of differentiation 36 (CD36), a fatty acid translocase on tumor cells, thereby promoting excessive fatty acids (FAs) uptake and lipid droplet (LD) accumulation, which starve immune cells and reinforce immunosuppression through this metabolic adaptation. Berberine (BBR), a bioactive alkaloid derived from <i>Rhizoma coptidis</i>, has previously been shown to ameliorate lipid metabolism disorders through downregulation of CD36 in metabolic diseases such as hepatic steatosis. We therefore hypothesize that BBR inhibits CD36-mediated FAs uptake and reduces LD accumulation in tumor cells, representing a novel mechanism that remains unexplored in the context of TNBC. In this study, we demonstrated that BBR counteracts the tumor-promoting effects of CAAs in 4T1 cells by inhibiting CD36 upregulation and its mediated FAs uptake, thereby reducing CAA-induced LD accumulation and ultimately suppressing tumor cell proliferation. Furthermore, BBR remodeled the TME by enhancing CD8⁺ T cell recruitment and activity, while reducing immunosuppressive factors. In order to improve the sustained release of BBR at the tumor site and overcome its poor aqueous solubility, we created a thermosensitive hydrogel-based nanoparticle system (BBR-NPs-GEL). This injectable hydrogel demonstrated favorable thermosensitive gelation and shear-thinning behavior, making it suitable for localized administration. It exhibited a gelation temperature of 35.3 ± 0.2 °C and a sustained release profile with 52% of BBR released within 48 h. In 4T1^Fluc tumor-bearing mice, BBR-NPs-GEL significantly suppressed tumor growth and remodeled the TME, as evidenced by increased infiltration of CD8⁺ T cells (+8.49%), activation of dendritic cells (+8.39%), and a shift toward M1-macrophages (+39.9%), accompanied by a reduction in M2-macrophages (-19.13%). Importantly, when combined with aPD-L1 therapy, the treatment elicited synergistic antitumor effects, resulting in enhanced tumor regression. This combination strategy effectively overcame metabolic immunosuppression and reversed immune resistance in TNBC.