Combination CXCR4 and PD-1 Blockade Enhances Intratumoral Dendritic Cell Activation and Immune Responses Against Hepatocellular Carcinoma

Abstract

Immune checkpoint inhibitors have revolutionized the treatment of unresectable hepatocellular carcinoma (HCC), but their impressive efficacy is seen in just a fraction of patients. One key mechanism of immunotherapy resistance is the paucity of dendritic cells (DC) in liver malignancies. In this study, we tested combination blockade of PD-1 and CXCR4, a receptor for CXCL12, a pleiotropic factor that mediates immunosuppression in tumors. Using orthotopic grafted and autochthonous HCC models with underlying liver damage, we evaluated treatment feasibility and efficacy. In addition, we examined the effects of treatment using immunofluorescence, flow cytometric analysis of DCs in vivo and in vitro, and RNA sequencing. The combination anti-CXCR4 and anti-PD-1 therapy was safe and significantly inhibited tumor growth and prolonged survival in all murine preclinical models of HCC tested. The combination treatment successfully reprogrammed antigen-presenting cells, revealing the potential role of conventional type 1 DCs (cDC1) in the HCC microenvironment. Moreover, DC reprogramming enhanced anticancer immunity by facilitating CD8+ T-cell accumulation and activation in the HCC tissue. The effectiveness of anti-CXCR4/PD-1 therapy was compromised entirely in Batf3 knockout mice deficient in cDC1s. Thus, combined CXCR4/PD-1 blockade can reprogram intratumoral cDC1s and holds the potential to potentiate antitumor immune response against HCC.

Figure 1.

Combined anti-CXCR4/anti–PD-1 antibody treatment showed significant anticancer effects and survival benefits in orthotopic models of HCC with liver damage. A and B, Tumor growth delay (A) and overall survival distributions (B) after a 3-week treatment with the anti-CXCR4 antibody, anti–PD-1 antibody, their combination, or IgG (isotype control) administered intraperitoneally at doses of 10 mg/kg diluted in PBS thrice weekly in RIL-175 murine HCC orthotopic grafts in C57Bl/6 mice with CCl₄-induced liver fibrosis. C and D, Confirmation of potent anticancer effects for the combination therapy in the autochthonous HCC model (induced in Mst1^−/−Mst2^F/− mice with underlying liver fibrosis), as measured by tumor volume using ultrasound (C) and the tumor proliferation index (D) at day 14. N = 8 or 9 mice in all groups, with experiments performed at least in duplicate. Data are the mean and SD (A, C, and D); two-way ANOVA (A) or log-rank test (B) or one-way ANOVA with Tukey’s test (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Combo, combination; Ctrl, control.

Figure 2.

Transcriptomic analysis data from RIL-175 murine HCC after anti-CXCR4 therapy combined with PD-1 blockade. A, Principal component analysis of gene expression profiles of murine HCC tissues from the control and treatment groups. B, Venn diagram representing the significantly DEGs between the treatment and control groups. C, GSEA analysis showing T-cell receptor signaling pathway upregulated in the combination therapy. D, Bar plot representing the top 10 most upregulated or downregulated GO terms using GSEA. The GO terms are ranked by the NES. The color of the bar plot indicates the transformed P values. E, The heatmap represents the gene expression of DC-related genes. Combo, combination; Ctrl, control; GO, Gene Ontology; GOBP, Gene Ontology biological process; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; pDC, plasmacytoid DC.

Figure 3.

Classical cDC1 subset infiltration inside HCC increases after combined treatment with CXCR4 and anti–PD-1 blockade. A–D, Combining the anti-CXCR4 antibody with anti–PD-1 induces a significant increase in the accumulation of cDC1 (defined as XCR1, CD11c double-positive cells) orthotopic grafted (A and B) and autochthonous (C and D) HCC models. All the mice with orthotopic grafted models were sacrificed on day 8, and the mice with autochthonous tumors were sacrificed on day 13 after starting the treatment, at the time of objective immune responses. Representative IF in A and C (arrowheads point to cDC1) and quantitative analysis of IF data in B and D. N = 7–8 mice in all groups. Scale bars, 50 μm (A) and 100 μm (C).  Data are the mean and SD (B and D); one-way ANOVA with Tukey’s test (B and D).*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Combo, combination; Ctrl, control; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4.

Combined treatment with anti-CXCR4 and anti–PD-1 antibodies promoted the activation and proliferation of cytotoxic T cells via cDC1. A, The survival benefit of the combination treatment in murine HCC was compromised when tumors were grown in Batf3 knockout (cDC1-deficient) mice. B–G, Combination therapy using anti-CXCR4 and anti–PD-1 antibodies induces proliferation and accumulation of activated cytotoxic T cells in the TME of HCC: frequency (B) and absolute numbers (C) of tumor-infiltrated CD8 T cells; frequency (D), representative plots (E), and absolute counts (F) of GzmB⁺ and Ki67⁺ proliferating (G) CD8⁺ T cells in HCC tissues from mice in the control IgG, monotherapy, or combination-treatment groups. Data are the mean ± SD. (B–G). H and I, Representative fluorescent photomicrographs of orthotopic grafted HCC models of mice treated with each demonstrated condition stained with DAPI (blue), anti-CD8 antibody (green), and anti-XCR1 antibody (red; H) and the average distance between CD8⁺ T cells and DC1s in HCC tissues (I). J, The frequency of CXCR3⁺ cells in DCs in vitro. N = 7–10 mice in all groups. Scale bars, 50 μm (H). Most experiments were performed at least in duplicate. One-way ANOVA with the Tukey test (A and B). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Combo, combination; Ctrl, control; DAPI, 4′,6-diamidino-2-phenylindole; NS, not significant; GzmB, Granzyme B.