Fig 2: Systemic collagen-binding domain IL-12 (CBD-IL-12) cytokine fusion therapy inhibits prostate cancer tumor growth and reprograms the tumor immune microenvironment.a Schematic of CBD-IL-12, composed of the p35 and p40 subunits fused to the CBD from von Willebrand factor domain A3. b Volumes of RM9 subcutaneous tumors in sygeneic C57Bl/6 mice over time with treatment with vehicle, anti-PD-1 (clone 29 F.1A12) 200 ?g by intraperitoneal injection every 5 days, or CBD-IL-12 25 ?g by intravenous injection every 5 days starting on day 0. n = 7 mice in vehicle and CBD-IL-12 treated groups and n = 8 mice in anti-PD1 treated group. p < 0.0001 at day 9 and 12. Bars represent mean with SEM. P-values are derived from two-way ANOVA with Dunnett’s multiple comparisons test, ns not significant. c Uniform Manifold Approximation and Projection (UMAP) plots of different immune cell subsets (top) from single-cell RNA-seq (scRNA-seq) analysis of five RM9 tumors each aggregated from mice treated with vehicle or CBD-IL-12. UMAP plots colored with gene expression of immune cell subset-specific markers for pan-monocytes/macrophages, M1 and M2 polarized marcophages, conventional type 1 dendritic cells (cDC1), natural killer (NK) cells, and T helper type 1 (Th1) cells. d Plots showing the frequency of specific immune cell populations (relative to CD45 + immune cells) identified by scRNA-seq analysis including CD4+ and CD8+ T cells, Th1 (Infg+Tbx21+) and Th2 (cMAF+Gata3+) cells, Ly6C+/- monocytes/macrophages (Ly6C+/-Adgre1+), M1 macrophages (CD80+CD86+INOS2+), M2 macrophages (CD163+Mrc1+cMAF+), cDC1 (XCR1+IRF8+), conventional type 2 dendritic cells (cDC2, CD1+IRF4+), migratory CD103+ dendritic cells (Itgae+), eosinophils (SiglecF+), neutrophils (Ly6G+), and NK cells (Klrb1c+Ncr1+) in tumors treated with vehicle or CBD-IL-12. Volcano plots showing differential gene expression in (e) tumor cells and (f) T cells from RM9 tumors of mice treated CBD-IL-12 relative to those treated with vehicle. FC fold change, FDR false discovery rate. Source data are provided in the Source Data file.

Fig 3: NSCs-OV and paquinimod enhance T cell activation and reduce immunosuppressive populations in GL-261 murine orthotopic glioma model. Flow cytometric analysis of (A) total T cells, (B) CD4+ and CD8+ T cells, (C) PD1+CD8+ cells, (D) CD4+Foxp3+ cells, (E) M1 or M2 macrophages, and (F) myeloid-derived suppressor cells (MDSCs) across different treatment groups in GL-261 orthotopic implantation models. (G–J) Quantification of CD3+CD45+ T, CD4+ cells, CD8+ cells, PD1+CD8+ cells, and CD4+Foxp3+ cells from (B–D). (K, L) Quantification of CD86+ and CD206+ macrophages. (M, N) Quantification of Ly6G+ and Ly6C+ MDSCs. *p<0.05, **p<0.01, ***p<0.001. NSCs, neural stem cells; OV, oncolytic virus.