Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells

Abstract

Impressive responses have been observed in patients treated with checkpoint inhibitory anti-programmed cell death-1 (PD-1) or anti-cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibodies. However, immunotherapy against poorly immunogenic cancers remains a challenge. Here we report that treatment with both anti-PD-1 and anti-CTLA-4 antibodies was unable to eradicate large, modestly immunogenic CT26 tumors or metastatic 4T1 tumors. Cotreatment with epigenetic-modulating drugs and checkpoint inhibitors markedly improved treatment outcomes, curing more than 80% of the tumor-bearing mice. Functional studies revealed that the primary targets of the epigenetic modulators were myeloid-derived suppressor cells (MDSCs). A PI3K inhibitor that reduced circulating MDSCs also eradicated 4T1 tumors in 80% of the mice when combined with immune checkpoint inhibitors. Thus, cancers resistant to immune checkpoint blockade can be cured by eliminating MDSCs.

Keywords: 5-azacytidine; HDAC; entinostat; exome; methyltransferase.

Fig. 1.

Therapeutic response of tumor-bearing mice. BALB/c mice bearing different tumors were treated with various therapeutic modalities as indicated. C, anti–CTLA-4 antibody; IgG, IgG control; P, anti–PD-1 antibody. Tumor volumes (A, C, and E) and animal survival (B, D, and F) were recorded. (A and B) BALB/c mice with CT26 tumors of moderate sizes. (C and D) BALB/c mice with large CT26 tumors. (E and F) BALB/c mice with metastatic 4T1 tumors. (G) 4T1 tumor-bearing mice were treated as indicated and euthanized 6 wk after tumor implantation. The primary tumor from each mouse was measured and metastatic lesions in different organs were counted. Means and SDs are shown. The number of animals used in each experimental arm and P values are also indicated. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Fig. 2.

Response of immune cells following immune checkpoint blockade and epigenetic modulation. BALB/c mice bearing metastatic 4T1 tumors were treated with the indicated therapeutic modalities, followed by FACS and immunohistofluorescence analyses to assess tumor-infiltrating and circulating immune cells. Means and SDs are shown, with P values indicated. (A) FACS result for tumor-infiltrating CD8⁺ T cells. (B) Representative immunohistofluorescence staining of tumor-infiltrating CD8⁺ T cells. (Scale bars, 50 μm.) (C) FACS result for tumor-infiltrating CD4⁺CD25⁺FoxP3⁺ Tregs. (D) Representative FACS data showing percentages of FoxP3 and CD25 double positive cells in CD45⁺CD3⁺CD4⁺ gated tumor-infiltrating cells. (E) FACS result for circulating G-MDSCs. (F) Representative FACS data showing percentages of Ly6G⁺Ly6C^lo cells (G-MDSCs) in CD45⁺CD11b⁺F4/80⁻MHC-II⁻ gated circulating cells. (G) FACS result for tumor-infiltrating G-MDSCs. (H) Representative immunohistofluorescence staining of tumor-infiltrating Ly6G⁺ cells. (Scale bars, 50 μm.) *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Fig. 3.

Myeloid-derived Ly6G⁺ cells are responsible for resistance to immune checkpoint blockade. (A) BALB/c mice bearing 4T1 tumors were treated with various antibodies or antibody combinations as indicated and tumor volumes were recorded over time. αCD25, anti-CD25 antibody; αLy6G, anti-Ly6G antibody. (B) FACS result for circulating G-MDSCs after treatment with different antibodies or antibody combinations. (C) 4T1 tumor-bearing mice were treated with anti–PD-1/anti–CTLA-4 antibodies plus epigenetic modulators with or without adoptive transfer of MDSCs isolated by affinity purification from the 4T1 tumor-bearing animals. Tumor volumes were recorded following the treatments. Means and SDs are shown, with P values indicated. *P < 0.05, **P < 0.01; ns, not significant.

Fig. 4.

Direct effects of epigenetic modulators on cultured cells. (A and B) 4T1 cells, purified CD8⁺ T cells, or G-MDSCs were treated with different concentrations of ENT (A) or AZA (B). Cell viability was assessed using a metabolism-based colorimetric assay. (C) Conditioned media from cocultures of G-MDSCs and CD8⁺ T cells at different ratios were analyzed for IFN-γ concentration. (D) Conditioned media from cocultures at a G-MDSC-to-CD8⁺ T-cell ratio of 1:1 were collected after treatment with ENT at increasing doses for 24 h and analyzed for IFN-γ concentration. Means and SDs of data from at least triplicate wells are shown, with P values indicated. *P < 0.05, **P < 0.01; ns, not significant.