Nanocrystalline berberine is associated with improved antitumor activity of PD-1 blockade in ovarian cancer.

Ovarian cancer remains a highly lethal gynecologic malignancy with limited responsiveness to immune checkpoint blockade and few well-tolerated strategies for improving antitumor immunity. Here, we developed a nanocrystalline berberine formulation (nanoBBR) using high-pressure homogenization and evaluated whether nanocrystal reformulation is associated with enhanced antitumor activity and improved responsiveness to programmed cell death protein 1 (PD-1) blockade. NanoBBR retained the crystalline structure of berberine and exhibited stable dispersion under the tested conditions. Across two-dimensional ovarian cancer cell models, three-dimensional human ovarian cancer organoids, and an immunocompetent intraperitoneal ID8-luc mouse model, nanoBBR showed greater inhibitory activity than conventional berberine (BBR). Transcriptomic analyses revealed immune-related pathway enrichment associated with nanoBBR treatment, including cytokine signaling and T cell receptor-related programs. In combination studies, nanoBBR plus anti-PD-1 antibody produced greater tumor suppression than either monotherapy, accompanied by increased intratumoral CD8⁺ T cell abundance and altered inflammatory and immune-related marker profiles. CXCL10 emerged as a prominently upregulated candidate chemokine in the combination group and showed spatial association with CD8⁺ T cell enrichment. These findings identify nanocrystalline berberine as a formulation strategy associated with enhanced antitumor activity and support further investigation of nanoBBR as a potential immunomodulatory adjunct to PD-1 blockade in ovarian cancer.