Host STING-dependent MDSC mobilization drives extrinsic radiation resistance

Abstract

Radiotherapy induces and promotes innate and adaptive immunity in which host STING plays an important role. However, radioresistance in irradiated tumors can also develop, resulting in relapse. Here we report a mechanism by which extrinsic resistance develops after local ablative radiation that relies on the immunosuppressive action of STING. The STING/type I interferon pathway enhances suppressive inflammation in tumors by recruiting myeloid cells in part via the CCR2 pathway. Germ-line knockouts of CCR2 or treatment with an anti-CCR2 antibody results in blockade of radiation-induced MDSC infiltration. Treatment with anti-CCR2 antibody alleviates immunosuppression following activation of the STING pathway, enhancing the anti-tumor effects of STING agonists and radiotherapy. We propose that radiation-induced STING activation is immunosuppressive due to (monocytic) M-MDSC infiltration, which results in tumor radioresistance. Furthermore, the immunosuppressive effects of radiotherapy and STING agonists can be abrogated in humans by a translational strategy involving anti-CCR2 antibody treatment to improve radiotherapy.

Fig. 1

Monocytic-MDSCs are accumulated in tumors following radiation in WT but not in CCR2^−/− mice. Tumors were harvested 3 days post-IR and subjected to flow cytometry analysis. a Gating strategy and b flow analysis of Ly6C^hi populations in control (NonIR) and irradiated (IR) tumors grown in WT or CCR2^−/− hosts (n = 4). b Left, cell percentage; Right, absolute cell number. ***P = 0.0001; *P < 0.05, as calculated by two-sided Student’s t-test. The experiments were repeated 3 times. Data are presented as mean ± s.e.m

Fig. 2

CCR2 expression in the host is crucial for monocytic-MDSC mediated tumor resistance to radiation. CCR2 knockout sensitized tumor to radiation treatment. Tumors grown in CCR2^−/− mice were more radiosensitive than tumors in WT mice, in both the a MC38, and b LLC tumor models. c CCR2 deficiency in hosts led to eradication of 60% of tumors by IR. d Neutralization of CCL2 could not achieve better tumor control by IR. e CD31 staining (left) and quantification (right) of tumors grown in WT or CCR2^−/− mice. Tumors were harvested and frozen sections were fixed and stained. CD31 positive cells in three DAPI positive fields from three mice were counted. Scale bar = 100 µm; **P < 0.01; *P < 0.05. Data are presented as mean ± s.e.m

Fig. 3

Depletion of CCR2 cells enhances tumor radiosensitivity. a Scheme of treatment. b Treated tumor was measured every 3-4 days for 33 days starting from the day of radiation (n = 10). c Percentage of mice still bearing tumor at the end of the treatments (n = 10), P < 0.05. d Flow cytometry profile of CCR2⁺Ly6C^hi in tumors 3 days post the start of treatments. Left, representative flow graph, gated on CD45⁺CD11b⁺cells; Middle, percentage of CD45⁺; Right, absolute number of cells (n = 4). NS, non-significant.*P < 0.05; **P < 0.01; ***P < 0.001. Each experiment was repeated 3 times. Data are presented as mean ± s.e.m

Fig. 4

Depletion of CCR2⁺Ly6C^hi cells enhances adaptive T cell response, which is pivotal for the efficacy of IR + CCR2 ab treatment. a CD8⁺ T cells were purified from draining lymph nodes from WT and CCR2^−/− tumor bearing mice or WT mice that received indicated treatments, and b subjected to ELISPOT assay. c CCR2⁺Ly6c^hi cells were sorted from excised tumors and subjected to suppression assay. Ratio indicates CCR2⁺Ly6c^hi: CD8. d Depleting T cells abolished the therapeutic effect of the IR + anti-CCR2 treatment. e Mice that completely rejected MC38 tumors were re-challenged two months later by five million of MC38 cells in the flanks (n = 5); *P < 0.05; **P < 0.01; ***P < 0.001; Each experiment was repeated three times. Data are presented as mean ± s.e.m

Fig. 5

Monocytic MDSCs mobilization is dependent on STING and type I IFN signaling after irradiation. a Representative flow gating chart. b Quantification of Ly6c^hi population in tumors in WT and STING^−/− mice. c Activation of STING pathway by IR or by cGAMP administration can increase the recruitment of CCR2⁺Ly6c^hi cells. d Quantification of the CCR2⁺Ly6c^hi population in tumors grown in WT control, IR and IR + anti-IFNaR1 treated mice. e Type I IFN induces expression of CCL2 and CCL7 in tumor cells and CCL12 in host cells (n = 4); *P < 0.05; **P < 0.01; ***P < 0.001. Each experiment was repeated four times. Data are presented as mean ± s.e.m

Fig. 6

MDSC depletion enhances tumor response to radiation and STING agonist cGAMP treatment. a Flow cytometry analysis of immune CCR2⁺Ly6c^hi cells 3 days after IR (n = 3). b Tumor growth curves; n = 6; c percentage of tumor bearing mice ending on 33 days after starting of the treatments. d, e ratios of percentage of CD8 over CCR2⁺Ly6c^hi and Treg cells in treated tumors, respectively. n = 3. *P < 0.05; **P < 0.01. The experiments were repeated three times. Data are presented as mean ± s.e.m