A Natural CCR2 Antagonist Relieves Tumor-associated Macrophage-mediated Immunosuppression to Produce a Therapeutic Effect for Liver Cancer

Abstract

Hepatocellular carcinoma (HCC) is a common malignant tumor in the digestive tract with limited therapeutic choices. Although sorafenib, an orally administered multikinase inhibitor, has produced survival benefits for patients with advanced HCC, favorable clinical outcomes are limited due to individual differences and resistance. The application of immunotherapy, a promising approach for HCC is urgently needed. Macrophage infiltration, mediated by the CCL2/CCR2 axis, is a potential immunotherapeutic target. Here, we report that a natural product from Abies georgei, named 747 and related in structure to kaempferol, exhibits sensitivity and selectivity as a CCR2 antagonist. The specificity of 747 on CCR2 was demonstrated via calcium flux, the binding domain of CCR2 was identified in an extracellular loop by chimera binding assay, and in vivo antagonistic activity of 747 was confirmed through a thioglycollate-induced peritonitis model. In animals, 747 elevated the number of CD8+ T cells in tumors via blocking tumor-infiltrating macrophage-mediated immunosuppression and inhibited orthotopic and subcutaneous tumor growth in a CD8+ T cell-dependent manner. Further, 747 enhanced the therapeutic efficacy of low-dose sorafenib without obvious toxicity, through elevating the numbers of intra-tumoral CD8+ T cells and increasing death of tumor cells. Thus, we have discovered a natural CCR2 antagonist and have provided a new perspective on development of this antagonist for treatment of HCC. In mouse models of HCC, 747 enhanced the tumor immunosuppressive microenvironment and potentiated the therapeutic effect of sorafenib, indicating that the combination of an immunomodulator with a chemotherapeutic drug could be a new approach for treating HCC.

Keywords: Combination therapy; Experimental therapeutics; Immunotherapy; Kaempferol; Preclinical models.

Graphical abstract

Fig. 1

747 is a promising CCR2 antagonist, as determined by tests in vitro and in mice. (A) The chemical structure of 747 is kaempferol 3-(2,4-di-E-p-coumaroylrhamnoside. (B) Inhibitory effect of 747 on chemotaxis of hCCL2-induced THP-1 cells and mCCL2-induced murine peritoneal macrophages. (C) Inhibitory effect of 747 on binding of ¹²⁵I-MCP-1 in CHO-K1-hCCR2/mCCR2 cells and binding of ¹²⁵I-MIP1α in CHO-K1-hCCR5/mCCR2 cells. (E) Inhibitory effect of 747 on CCL2-induced calcium flux in 293FT-Gcamp6-CCR2 cells and RANTES-induced calcium flux in 293FT-Gcamp6-CCR5 cells. (F) 747 inhibition of thioglycollate-induced infiltration of peritoneal cells. *p < 0.05.

Fig. 2

Mode of binding of 747 with CCR2. (A) Sequence alignment of CCR2 and CCR5, the transmembrane regions, intracellular and extracellular loops are marked. From the docking results, 747 bonds with CCR2 residues are marked in red. (B) Schematic diagram of CCR2, CCR5, and the chimeric receptors CCR2-CCR5 (C-term), CCR5-CCR2 (C-term), and CCR5-CCR2 (Extracellular loops). (C) Inhibitory effect of 747 on binding of ¹²⁵I-MCP-1 for CCR2, CCR5, and chimeric receptors expressed in CHO-K1 cells. (D) Inhibitory effect of 747 on binding of ¹²⁵I-MIP-1α for CCR2, CCR5, and chimeric receptors expressed in CHO-K1 cells. (E) Induced-fit docking of 747 in a homology model of CCR2 based on the C-X-C chemokine receptor-4 crystal structure. (F) Inhibitory effect of 747 on binding of ¹²⁵I-MIP-1α for CCR2 wild type and mutant receptors expressed in CHO-K1 cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

In an orthotopic liver cancer model, 747 suppresses liver cancer growth, reduces TAMs, and increases CD8 T cells. (A) The schedule for liver cancer treatment and imaging. (B) Representative bioluminescence images of mice treated with 747 intraperitoneally (low: 50 mg/kg, high: 100 mg/kg), sorafenib intragastrically (30 mg/kg), or the vehicle (n = 6–7 mice/group). Representative photographs of liver tumors after treatment; the black dotted lines indicate the tumor regions. (C) Relative luminescence of liver tumors depicted in (B). (D) Representative flow cytometry results showing the proportion of CD4 T cells, CD8 T cells, and TAMs (CD11b + F4/80 +) in Hepa1-6 orthotopic tumor tissues of C57BL/6 mice. (E) Quantification of immune cells by flow-cytometric analysis in tumor tissues. Data are shown as means ± SEM. *p < 0.05. (F) Body weight changes of mice during treatment. Data are shown as means ± SEM.

Fig. 4

Depletion of CD8 T cells eliminates the anti-tumor effect of 747. (A) The schedule of 747 treatment and depletion of macrophages and CD8 T cells in Hepa1-6 subcutaneous tumors (n = 5 mice/group). (B) Representative photographs of subcutaneous liver tumors after treatment with either 747. macrophage/CD8 T cell depletion, or both. (C) The tumors were measured and the proportion of TAMs, CD4 T cells, and CD8 T cells were quantified by FACS (D, E). Data are presented as means ± SEM. *p < 0.05.

Fig. 5

747 potentiates the therapeutic effect of sorafenib. (A) The schedule for 747 treatment and tumor measurement. (B) Representative photographs of subcutaneous liver tumors after treatment with 747, sorafenib, or the combination, and measured by tumor growth and weight. (C) Body weight changes of mice during treatment. (D, E) Proportions of TAMs, CD4 T cells, and CD8 T cells were quantified by FACS. Data are presented as means ± SEM. *p < 0.05.

Fig. 6

Treatment with the combination of 747 and sorafenib enhances tumor cell death and improves the cytotoxicity of CD8 T cells. (A) Representative hematoxylin and eosin staining of subcutaneous tumors after treatment with 747, sorafenib, or the combination (Scale Bar: 50 μm). (B) Representative immunohistochemistry of cleaved caspase-3 in tumor sections (Scale Bar: 50 μm). Representative immunohistochemistry of F4/80 (macrophage cells) in the tumor sections (C) and Arg-1 (M2 marker) in the tumor sections (D) (Scale Bar: 50 μm). (E) BMDM treated with vehicle, 747 (5 μM) for 24 h. qRT-PCR results indicating the expression of M1 and M2 macrophage markers in BMDM. (F) CD8 + T cells were sorted from tumor at the end point. qRT-PCR showing the expression of cytotoxic marker in CD8 + T cells.(G) BMDM treated with vehicle,747 (5 μM) for 24 h and subsequently co-cultured with mice primary CD8 + T cells for 72 h. Cytometric Bead Array kits were employed to measure cytokine expression in the culture supernatants.