Blockade of Erythropoietin Enhances the Abscopal Effects of Radiotherapy Restraining Lung Metastasis by Inducing an Immunopermissive Tumor Microenvironment

Abstract

Abstract Background Distant out-of-field, so-called abscopal, anti-metastatic effects of local radiation are rarely observed in cancer patients treated with radiotherapy alone. However, the era of immune checkpoint inhibitors (ICI) has increased abscopal effects following combinational treatment using radiotherapy and ICI (Radio-ICI). Hence, ICI-induced activation of cytotoxic T cells in the metastatic tumor microenvironment (TME) was instrumental in mediating the abscopal effect. Here, we hypothesized to improve the efficacy of abscopal effects observed in Radio-ICI through additional inhibition of immunosuppressive cells originating from the bone marrow. Therefore, we employed focal radiotherapy on the bone marrow of a single limb combined with ICI as alternative treatment for the induction of anti-metastatic abscopal responses. Methods We established lung metastatic mouse models by intravenous injection of colorectal cancer and melanoma cells, followed by single limb irradiation (SLI) treatment with 5 Gy to trigger abscopal effects. Tumor control, adverse effects, and composition of immune cells in the TME were monitored after radiotherapy as monotherapy or combinational therapy with ICI. Suppression of erythropoietin (EPO) using a neutralizing antibody was combined with SLI treatment to dissect the contribution of EPO signaling for the induction of abscopal effects. Functional markers for lymphoid and myeloid lineage cells, including subsets of immunosuppressive myeloid-derived suppressor cells (MDSC) and erythroid progenitor cells (EPC), were determined by flow cytometry, western blotting, and real-time PCR. Results SLI treatment alone induced a significant abscopal effect against lung metastases and enhanced the therapeutic efficacy of anti-PD-1. MDSC and EPC were suppressed after SLI exposure, accompanied by the reduction of M-CSF and EPO in the plasma of lung metastatic mice. Addition of EPO protein neutralized the SLI-induced antitumor response, while treatment with EPO antibody alone or in combination with SLI effectively inhibited tumor growth. Suppression of arginase 1 protein with concomitant increase of CD8 mRNA expression in the TME was observed after SLI treatment combined with EPO antibody. These effects were abrogated when SLI was combined with EPO protein. Conclusion SLI treatment induced an abscopal anti-metastatic tumor effect mitigating immunosuppressive barriers provided by MDSC and EPC, thus reversing the tumor-induced T cell dysfunction in the TME.