Fig. 3: USP2 inhibition enhances macrophage phagocytosis and reshapes an inflamed tumor microenvironment.

a–d In vitro phagocytosis assay. Representative flow cytometry plots displaying THP-1 macrophage phagocytosis of PC9 cells (a) and H1975 cells (c). Quantitative results for phagocytosis to show the percentage of CFSE⁺ cells (PC9 cells for b and H1975 cells for d) in CD11b⁺ cells (THP-1 macrophages). PC9 or H1975 cells were pre-treated with DMSO or 2 µM ML364 for 16 h and pre-stained with carboxyfluorescein diacetate succinimidyl ester (CFSE). Subsequently, PC9 or H1975 cells were co-cultured with THP-1 cells, which have been stimulated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h to induce THP-1 monocytes into macrophages. After 6 h of co-culture at 37 °C, cells were harvested for the flow cytometry analysis. n = 3 per group for (b, d). e A schematic treatment plan for immunocompetent C57BL/6J mice bearing LLC tumors. Mice were subcutaneously implanted with 2 × 10⁶ LLC cells and treated with a control vehicle or USP2 inhibitor (ML364, 5 mg/kg), respectively. i.p., intraperitoneal; s.c., subcutaneous. f–h The growth of subcutaneous LLC tumors was assessed. Images of LLC tumors at the endpoint (f). Tumor volume for different treatment groups was measured with a caliper, and the tumor growth curve was plotted (g). The weight of LLC tumors was measured at the endpoint (h). i Uniform Manifold Approximation and Projection (UMAP) of single-cell RNA sequencing (scRNA-seq) data showing the myeloid lineage subsets from LLC tumors treated with ML364 (5 mg/kg) or control vehicle for 12 days. Color coding indicates the different cell types. M1, M1 macrophages; M2, M2 macrophages; M0, M0 macrophages; DCs, dendritic cells; MDSCs, myeloid-derived suppressor cells. j Dot-plot showing the selected representative gene markers enriched in myeloid lineage subsets. k Comparative abundance of the myeloid lineage subsets in LLC tumors treated with control vehicle (blue) versus ML364 (green) from the scRNA-seq data. l, m Quantification of MHCII⁺ (M1 macrophage) cells (l) or CD206⁺ (M2 macrophage) cells (m) represented as a percentage of F4/80⁺ macrophages in subcutaneous LLC tumors derived from mice with indicated treatments. n Quantification of Gr1⁺Ly6C⁺cells (myeloid-derived suppressor cells, MDSCs) represented as a percentage of CD11b⁺ monocytes in subcutaneous LLC tumors derived from mice with indicated treatments. o Quantification of CD8⁺ T cells represented as a percentage of CD3⁺ lymphocytes in subcutaneous LLC tumors derived from mice-indicated treatments. p–r Representative IF staining images of CD86 (orange) and CD206 (green) in subcutaneous LLC tumors with indicated treatments (p). Each point represents the average counts of positive cells within three high-power fields (q, r). Scale bars, 100 μm (left); zoom scale bars, 50 μm (right). For (b, d, h, l–o, q, and r), unpaired two-tailed Student’s t-test. Two-way ANOVA for (g). n = 5 mice per group for (g, h, l–o, q, and r), Data are shown as the mean ± SD. P < 0.05 was considered statistically significant. Source data are provided as the Source Data file.