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

doi:10.21203/rs.3.rs-2366313/v1

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

https://doi.org/10.21203/rs.3.rs-2366313/v1

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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.

abscopal effect

immune checkpoint inhibitors

MDSC

erythroid progenitor cells

erythropoietin

The abscopal effect is an intriguing phenomenon describing regression of distant metastatic tumors following local radiation of the primary tumor target[1]. However, this effect is rarely reported in clinical practice after treatment with radiotherapy alone[2, 3], inconsistent to observations made in animal models[4–6]. An increasing number of studies have documented abscopal effects in patients receiving combined radiation and immunotherapy, especially with immune checkpoint inhibitors (ICIs)[7–10], prompting us to hypothesize that abscopal effects observed in human settings require the breakdown of immunosuppressive barriers surrounding metastatic tumor sites[11, 12]. Metastatic disease remains the principal cause of cancer-related death and treatment failure, so finding new therapeutic strategies to control and eliminate metastases represents a major unmet medical need.

To date, the mechanisms of abscopal effects have been characterized and triggered by the release of damage-associated molecular patterns, high-mobility group protein B1, double-stand DNA fragments, and tumor-associated antigens after irradiation[13, 14]. Dendritic cells recognize these signals and then present antigens to prime cytotoxic T cells, killing irradiated tumors as well as out-of-field metastases. The tumor microenvironment (TME) of both local and distant tumors is comprised by the interaction of tumor cells, immune cells, stromal cells, cancer-associated fibroblasts, vascular endothelial cells, pericytes and other cells[15, 16]. To evade from immune surveillance, tumor cells create an immunosuppressive network to orchestrate induction of negative co-stimulatory receptors, release of immunosuppressive factors, impairment of antigen presentation, and recruitment of regulatory immune cells[17]. Immunosuppressive populations involved in this network include tumor-associated macrophages (TAM), tumor-associated neutrophils, cancer-associated fibroblasts, regulatory T cells, myeloid-derived suppressor cells (MDSC), and erythroid progenitor cells (EPC)[18–21]. Radiation-activated T cells are supposed to migrate to distant tumor sites and execute their cytotoxic tasks; however, the TME of distant lesions is infiltrated by immunosuppressive cells causing T cell exhaustion, which is likely to explain the low incidence of abscopal effects in clinical routine settings, unless the inhibitory modulation on T cells is relieved by ICI therapy.

Among immunosuppressive cells, MDSC are known to play a major role in tumor progression by inhibiting T cells[22, 23]. Cancer cells stimulate myeloid cells with an immature phenotype known as MDSC and sustain emergency myelopoiesis in the bone marrow[24, 25]. Apart from immature myeloid cells, immature red blood cells called erythroid progenitor cells (EPC) were also reported to have robust immunosuppressive properties in newborns and patients with advanced cancer[26, 27]. Tumor growth-associated extramedullary hematopoiesis is the formation of blood cells outside the bone marrow, including myeloid, megakaryocytic cells and erythroid progenitor cells[28, 29]. Extramedullary hematopoiesis not only contributes to replenish blood cells but also pro-tumorigenic immune responses via production of immunosuppressive cells, such as MDSC and EPC. EPC are defined as TER119⁺CD71⁺-expressing cells and mainly accumulate in the spleen, liver, and peripheral blood[30]. The early stage EPC inhibit T cell-mediated cytotoxicity by producing reactive oxygen species, whereas the late stage EPC (Ter-cells) promote tumor progression via secretion of artemin. After blocking ROS secretion and artemin, improved tumor control and prolonged survival were observed[31, 32]. Zhao L et al. showed that the early stage EPC mainly attributed to splenomegaly during tumorigenesis and were 3- and 30-fold higher than MDSC and Treg in the spleen, respectively[32]. Reports from Han Y group indicated the late stage EPC behaving as potent immunosuppressors to cause systemic impairment of CD8⁺ T cell function in murine orthotopic hepatocellular carcinoma and T lymphoma-bearing mouse models[33]. High dosage of radiotherapy (20 Gy) was demonstrated to decrease tumor-induced Ter-cell accumulation in the spleen of several subcutaneous mouse models independent of tumor types[34]. Therefore, targeting ineffective erythropoiesis and the tumor-promoting effects of EPC are promising strategies in novel cancer therapies.

In this study, we used a mouse model of lung metastases originating from colorectal cancer and melanoma cells to dissect the mechanisms underlying the abscopal effect[35]. We hypothesized that the improved efficacy of abscopal effects observed in combinational radiotherapy with ICI was through additional inhibition of immunosuppressive cells such as MDSC originating from the bone marrow. Thus, we performed focal radiotherapy on the bone marrow of one extremity, named Single Limb Irradiation (SLI). We set out to conduct experiments to examine whether SLI alone or in combination with ICI could reduce metastatic tumor growth in a mouse model of metastatic lung cancer. Our data demonstrated that combinational therapy of SLI and ICI elicited therapeutic abscopal responses by activating the host’s immune system and transiently eliminating immunosuppressive barriers posed by MDSC and EPC in the TME. Importantly, combinational treatment of EPO blockade and SLI mimicked and prolonged the treatment efficacy observed for SLI and ICI. Our findings emphasize the favorable role of focal exposure of radiation on the bone marrow to induce abscopal anti-tumor effects and allow for the development of a new therapeutic combinational strategy to treat metastatic malignancies.

Cell culture

The CT26 murine colorectal adenocarcinoma and B16-F10 murine melanoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). CT26 and B16-F10 cells were cultured in RPMI-1640 and DMEM medium (Invitrogen, Carlsbad, CA, USA), respectively, with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT, USA) and 2 mM L-glutamine (Invitrogen). Stable clones of CT26 cells expressing luciferase (CT26-Luc) were generated by lentiviral transduction with the North American firefly luciferase gene under the control of the SV40 promoter. CT26-Luc cells were further passaged in the previously-mentioned medium containing G418 (400 µg/ml; Sigma-Aldrich, St. Louis, MO, USA).

Lung metastatic mouse model and treatments

Four-week-old male BALB/c mice or C57BL/6 mice were acquired from the Animal Resource Center of the National Science Council of Taiwan (Taipei, Taiwan). All animal experiments have been approved by the Animal Ethics Committee of China Medical University, Taiwan (CMUIACUC-2021-018). We intravenously administered CT26-Luc (2 ⋅ 10⁶) into the tail veins of mice to induce lung metastasis and assessed lung metastatic tumor growth by the IVIS 200 imaging system (Xenogen Biosciences, Cranbury, NJ) at the indicated time points. Alternatively, B16-F10 (1 ⋅ 10⁶) cells were intravenously injected into the tail veins of mice to induce pulmonary dissemination and scored the number of black lung melanoma nodules on the surface of each lobe using a dissecting microscope (Motic China Group Co., Ltd). Before irradiation, tumor-bearing mice were intraperitoneally anesthetized using a mixed solution of Zoletil™ (4 mg/kg) and Ropum™ (1 mg/kg), then single limb irradiation (SLI) treatment was performed by a linear accelerator (Clinac 1800, Varian Associates, Inc., Palo Alto, CA, USA) with 25x25 cm field size and an SSD of 100 cm for electron beams with energies of 6 MeV. Left hind limbs of tumor-bearing mice were exposed to 5 Gy with a bolus of 0.5 cm to improve dose homogeneity. For immune checkpoint inhibitor therapy, the anti-mouse PD-1 mAb (200 µg/mouse; clone RMP1-14, BioXcell), anti-mouse PD-L1 mAb (150 µg/mouse, clone 10F.9G2, BioXcell) were intraperitoneally administered on days 0, 2, and 4 post-SLI treatment. For EPO experiments, mouse EPO protein (10 µg/kg, #587602, BioLegend, San Diego, CA, USA) was retro-orbitally injected on days 0, 2, and 4 post-SLI treatment, and anti-EPO mAb (7.5 mg/kg, clone 148438, R&D Systems, Minneapolis, MN, USA) was intraperitoneally administered on days 0 post-SLI treatment. Mice were sacrificed on day 4, 8, and 17 for TME assessment.

Hemogram and biochemistry analyses

Murine blood samples were obtained by facial vein bleed and collected in tubes containing 20 IU/ml of heparin. Plasma was further separated by spinning at 1,000 x g at room temperature for 20 min. White blood cell (WBC) counts were quantified by an automatic Coulter counter (HEMAVET HV950; Drew Scientific, Inc., Dallas, TX). Functional indicators including alanine aminotransferase and creatinine were colorimetrically assessed using Fuji Dri-Chem Slide and analyzed by Fuji Dri-Chem 3500i Biochemistry Analyzer (Fujifilm Ltd, Japan) according to instructions of the manufacturer.

Flow cytometric analysis

Mice with lung metastasis were euthanized via intraperitoneal injection of a mixed solution of Zoletil™ (4 mg/kg) and Ropum™ (1 mg/kg). Whole lungs were removed and digested with collagenase A (1.5 mg/ml) and DNase I (0.4 mg/ml) at 37°C for 30 min[36]. Whole spleens were mashed through a 70-µm cell strainer to obtain single-cell suspensions. Whole livers were mashed through a 70-µm cell strainer and centrifuged at 55 x g for 3 min to remove hepatocytes. Bone marrow from left and right hind limbs were isolated as described previously[37]. Leukocytes isolated from each organ were further incubated with ACK (ammonium-chloride-potassium) solution for red blood cell lysis except for the erythrocyte progenitor cell detection. Prior to staining with cell surface markers, cells were treated with an Fc receptor block (1 µg/1 × 10⁶ cells; BD Bioscience, San Diego, CA) for the reduction of nonspecific binding. Prior to staining, Zombie Green™ or Zombie UV™ Fixable Viability Kits were used to discriminate between dead and live cells according to instructions of the manufacturer (BioLegend, San Diego, CA). Single immune cells were stained with antibodies conjugated with the indicated fluorochromes for 20 min on ice, including anti-CD3-Alexa488, anti-CD11c-Alexa488, anti-NKG2D-PE, anti-PD-L1-PE/Dazzle 594, anti-Ly6G-PE/Cy7, anti-PD-1-APC, anti-F4/80-APC/Cy7, anti-SiglecF-APC/Cy7, anti-Ly6C-BV421, anti-CD45-BV510, anti-CD11b-BV605, anti-CD4-BV650, anti-MHCII-BV650, anti-CD8-BV785, and anti-CD3-BV785 (BioLegend, San Diego, CA). After washing, the stained cell samples were run on the CytoFLEX 13-color cytometer (Beckman Coulter, Brea, CA, USA) and quantified using the CytExpert analysis software. Immune cell populations were characterized as follows: PMN-MDSC (CD11b⁺/Ly6G⁺), eosinophils (CD11b⁺/F4/80⁺/SiglecF⁺), alveolar macrophages (MHCII⁻/CD11b⁻/SiglecF⁺), interstitial macrophages (MHCII⁺/CD11b⁺/F4/80⁺), splenic macrophages (MHCII⁺/CD11b⁻/F4/80⁺), M-MDSC (CD11b⁺/Ly6C⁺⁺), natural killer cells (CD3⁻/F4/80⁻/NKG2D⁺), dendritic cells (MHCII⁺/CD11c⁺), CD4⁺ T cells (CD3⁺/CD11b⁻/CD4⁺), CD8⁺ T cells (CD3⁺/CD11b⁻/CD8⁺), natural killer T cells (CD3⁺/CD11b⁻/Nkp46⁺), and B cells (MHCII⁺/CD11b⁻/CD11c⁻). Each population of immune cells was delineated by different colors: PMN-MDSC (brown), eosinophils (yellow), M-MDSC (light blue), dendritic cells (pink), interstitial macrophages (blue), alveolar macrophages (orange), NK cells (green), B cells (grey). CD4⁺ T cells (light red), CD8⁺ T cells (red).

Cytokine detection

Murine blood samples were collected in tubes containing 5 mM EDTA. Plasma was further obtained with 1,000 x g at room temperature for 20 min. Cytokine concentrations in the murine plasma were measured using the LEGENDplex™ Mouse Inflammation Panel (13-plex; BioLegend) according to the manufacturer’s recommendations. Levels of GM-CSF, IL-6, TPO, M-CSF, IL-5, IL-34, CXCL12, EPO, SCF, LIF, IL-3, TGF-β1, IL-15 were analyzed using the CytoFLEX 13-color cytometer (Beckman Coulter, Brea, CA, USA) and BioLegend LEGENDplex software (BioLegend, San Diego, CA).

Western blot analysis

Tissue homogenization was performed by using the Miltenyi gentleMACS dissociator

instrumentation (GentleMACS, Miltenyi Biotec). Proteins were lysed in RIPA buffer (Millpore # 20–188) and quantified by Assay Dye Reagent Concentrate (Bio-Rad #5000006). Gel electrophoresis was separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) by using Mini Gel Tank (Invitrogen™, cat. #A25977, Waltham, MA) and gels were transferred to a polyvinyl difluoride membrane (PVDF, 0.45 mm; Immobilon, Millipore, Eschborn, Germany) using the Mini Blot Module (Invitrogen™, cat. #B1000, Waltham, MA). Membranes were further blocked in blocking buffer (3% BSA, 0.1% Tween-20 in PBS, pH 7.4) for 1 hour at room temperature. The primary antibodies against EPO (Sion #59099-T42), ARGINASE 1 (Cell Signaling # 93668T), GRANZYME B (Cell Signaling #44153), TUBULIN (Elabscience #E-AB-20036), and ACTIN (Millipore #MAB1501) were diluted in the blocking buffer and incubated overnight at 4°C with gentle agitation. Membranes were washed with PBST (0.1% Tween 20), and probed with goat anti-mouse (Elabscience # E-AB-1001) or anti-rabbit secondary antibodies (Elabscience # E-AB-1003). Chemiluminescent detection was performed using ECL Plus reagent (PerkinElmer Life Sciences) and detected using a ChemiDoc XRS imaging system (Bio-Rad, Hercules, CA, USA).

Real-time reverse transcription polymerase chain reaction

Total RNA was extracted using TRIzol (Invitrogen) and quantified by Synergy HTX (BioTek). Two µg of total RNA were reversed to cDNA using TOOLS Easy Fast RT Kit (BioTOOLS #KRT-BA06-2) with T100 Thermal Cycler (BIO-RAD). Real-time polymerase chain reaction was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme #Q711) using CFX Connect Real-Time PCR Detection System (BIO-RAD). The amplification of cDNA was performed under the following conditions: 3 min at 94°C followed by cycles of 15 s/94°C, 20 s/60°C, 20 s/72°C. Primer sequences for the genes of interest including Arg1, Cd8, Ccl2, B2m, Hprt were shown in Table 1. Transcripts for B2m and Hprt served as reference genes.

Table 1
Forward Primer	Sequences	Reverse Primer	Sequences
Arg1-F	5’-GCCTATCTTACAGAGAAGGT-3’	Arg1-R	5’-GTCCCTGGCTTATGGTTAC-3’
Cd8-F	5’-CAGAGACCAGAAGATTGTCG-3’	Cd8-R	5’-TGATCAAGGACAGCAGAAGG-3’
Ccl2-F	5’-GCTCAGCCAGATGCAGTTAA-3’	Ccl2-R	5’-TCTTGAGCTTGGTGACAAAAACT-3’
B2m-F	5’-ACAGTTCCACCCGCCTCACATT-3’	B2m-R	5’-TAGAAAGACCAGTCCTTGCTGAAG-3’
Hprt-F	5’-CTGGTGAAAAGGACCTCTCGAAG-3’	Hprt-R	5’-CCAGTTTCACTAATGACACAAACG-3’

Statistical analysis

Results were expressed as the mean ± standard error of the mean (SEM). Statistical comparisons were analyzed by two-tailed Student t-test or one-way ANOVA. Differences were considered significant at *P < 0.05, **P < 0.01, ***P < 0.001. The graphs were generated and analyzed using GraphPad Prism (Version 9.0 GraphPad Software, San Diego, CA).

Single limb irradiation (SLI) induces abscopal effects and enhances ICI efficacy

We employed a lung metastasis mouse model using colon cancer cells stably expressing luciferase (CT26-Luc cells) that we described previously[38]. One week after tail vein injection of tumor cells, tumor burden in the lung was examined using the IVIS image system and treatment was initiated. Single Limb Irradiation (SLI) was performed by irradiating the left limb once with a dose of 5 Gy of electron beam radiation (Day 0). Anti-PD-1 or anti-PD-L1 inhibitors were then administered three times (day 0, 2, 4) with or without SLI treatment (Fig. 1a). In BALB/c mice bearing lung metastasis, SLI treatment elicited significant abscopal effects on distant tumors in the lung and improved the tumor-inhibitory efficacy compared to ICI monotherapy (Fig. 1b). Combining SLI treatment with anti-PD-1 further enhanced the abscopal effect against distant lung metastases with 60% (three out of five) of mice showing complete tumor eradication rated as complete remission (CR) (Fig. 1b). In line with these results, SLI combined with anti-PD-L1 resulted in a CR of 40% (two out of five) of mice. Thus, comparing single ICI treatments with combinational therapy administering SLI, prior SLI treatment enhanced the therapeutic efficacy of PD-1/PD-L1 inhibitor therapies (Fig. 1c). In terms of side effects, there was no significant difference in the body weight of mice in each group (Fig. 1d). Furthermore, there was consistent and mild suppression of white blood cells observed on day 5 and 10 with all ICI and SLI therapies but within the normal ranges (Fig. 1e). No significant changes in liver and kidney functions were noted within 15 days post-treatment in the SLI monotherapy or combined groups (data not shown). These data indicated that, in the lung metastasis model, SLI treatment is more effective than ICI monotherapy and combined SLI and anti-PD-1/PD-L1 treatment maximize tumor control without significant side effects.

Single limb irradiation (SLI) modulates the tumor microenvironment

Next, we performed immune cell profiling of tumor-infiltrating, tumor-resident, and circulating immune cells in lung metastatic CT26-Luc-bearing mice after SLI treatment with or without ICIs by multi-color flow cytometric analysis. Different populations of immune cells were defined as described in material and methods (Fig. 2a). In the TME, SLI treatment, alone or as combinational treatment with PD-1/PD-L1, dramatically suppressed myeloid-lineage cells such as PNM-MDSC and interstitial macrophages, whereas local-resident macrophages such as alveolar macrophages were significantly increased (Fig. 2b). Interstitial macrophages are thought to emerge from bone marrow-derived M-MDSC[39, 40]. Besides, interstitial macrophages positively correlated with the volumes of lung metastatic tumor burden in our system; thus, we defined interstitial macrophages as tumor promoting M2-like macrophages in the TME. For lymphoid-lineage cells, NK cells, B cells and CD4⁺ T cells were all induced by SLI (Fig. 2c). Although the relative number of CD8⁺ T cells was not altered, combinational treatment of SLI and ICI reduced the expression level of the inhibitory checkpoint molecule PD-1 in CD8⁺ T cells (CD8⁺/PD-1⁺) (Fig. 2d, e). Consistent with the immune cell profile in the TME, SLI treatment resulted in a decrease of PMN-MDSC and increase of NK cells and CD4⁺ T cells in the spleen (Fig. 2f). Quantification of the results indicated that SLI treatment, alone or as combinational treatment with ICIs, significantly reduced PMN-MDSC, while macrophages in the spleen were not affected, or even increased, on day 17 post-SLI (Fig. 2g). Likewise, SLI treatment, alone or as combinational treatment with ICIs, increased both NK cells in the spleen on day 17 post-SLI (Fig. 2h).

Single limb irradiation (SLI) reduces MDSC in the irradiated bone marrow and TME

After examining alterations of the immune cell profiling in the lung TME at a late time point, we set out to elucidate the early response of immunosuppressive cells and T cells in the irradiated bone marrow and in the TME. After 1 and 10 days of SLI treatment, we obtained the irradiated bone marrow, spleen, and lungs from lung-metastatic CT26-Luc-bearing mice to isolate single cells for the staining of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC, CD11b⁺/Ly6G⁺), monocytic polymorphonuclear myeloid-derived suppressor cells (M-MDSC, CD11b⁺/Ly6C⁺⁺), and T cells. The results of multi-color flow analysis showed that 1 day after SLI irradiation, M-MDSC in the bone marrow of the irradiated bone marrow, spleen, and most importantly the lung metastases, were significantly decreased (Fig. 3a-d). Ten days after SLI treatment, M-MDSC in the irradiated bone marrow and lung metastases were still suppressed down to the same level they showed at day 1 (Fig. 3b, d). For PMN-MDSC, there was an increase in the bone marrow after SLI, however, PMN-MDSC were significantly decreased in the lung TME 16 days after SLI. On the contrary, T cells were decreased in the lung TME as tumors grew larger, and SLI treatment showed a trend to increase the number of T cells at the metastatic tumor sites (Fig. 3d).

Single limb irradiation (SLI) reduces systemic EPO and erythrocyte progenitor cells

After analyzing the immune cell profile in the irradiated bone marrow and TME at early time points, we dissected the differences of crucial cytokines for hematopoiesis. Among 13 cytokines, macrophage colony stimulating factor (M-CSF) and EPO were suppressed on day 4 and day 7 post-SLI treatment (Fig. 4a, b). Another cytokine, CXC motif chemokine ligand 12 (CXCL12), involved in the mobilization of hematopoietic stem cells required for extramedullary hematopoiesis, also showed a reduced trend after SLI exposure, but not reached significance. Next, we evaluated the level of immature red blood cells named erythroid progenitor cells (EPC, CD71⁺/TER119⁺) in the spleen, liver, and in lung metastases in mice on day 3 post-SLI. Our data indicated that early stages of EPC were increased in the spleen, liver, and lung metastases of tumor-bearing mice, and SLI treatment significantly reduced EPC in these organs (Fig. 4c, d). Exploring the level of late stage EPC in the spleen after combination of SLI and ICI therapies, we observed that mono- and combination treatments of SLI and ICI exhibited significant suppression of EPC in splenocytes (Fig. 4e). Our data suggest that SLI monotherapy or combination therapy with ICI reduces the level of immunosuppressive EPC, thus contributing to the therapeutic efficacy.

Combination of SLI and anti-EPO suppresses tumor-associated macrophages and increases NKT cells in the TME

Previous studies have found that administration of EPO protein can stimulate type-2 macrophage polarization in the presence of red blood cells in vitro[41]. To explore the role of EPO signaling for the abscopal effect after SLI therapy, we divided lung metastatic colon cancer-bearing mice into six groups: control, SLI (on day 0), EPO (recombinant murine EPO protein using 10 µg/kg on day 0, 2, 4), SLI + EPO, anti-EPO (anti-EPO antibody using 7.5 mg/kg on day 0), and SLI + anti-EPO groups (Fig. 5a). In vivo bioluminescence analysis showed that administration of EPO protein promoted tumor growth both in single and combinational treatment groups (Fig. 5b, c). On the contrary, administration of EPO antibody after SLI therapy further enhanced the inhibitory effect of SLI on lung metastases (Fig. 5b, c). Consistently, we observed similar results using the lung metastatic B16-F10 melanoma model (Fig. 5d, e). Analysis of the composition of immune cells in the lung metastatic tumor microenvironment revealed that EPO antibody combined with SLI therapy decreased M2-type interstitial macrophages, while increasing type M1 alveolar macrophages (Fig. 5f, h). Most importantly, administration of EPO antibody combined with SLI therapy significantly increased NKT cells in lung metastases (Fig. 5g, h). Thus, these data suggest that SLI treatment inhibited the differentiation of pro-tumorigenic M2-type interstitial macrophages from the supply of M-MDSC from the bone marrow. The relationship between the increase of alveolar macrophages and NKT cells in the TME and the induction of NK cells in the bone marrow after the combination of SLI and anti-EPO treatments needs further investigation.

Combination of SLI and anti-EPO reduced immunosuppressive barriers in the TME of lung metastases

Next, we evaluated functional markers of M2 macrophages and T cells in single and combined treatments of SLI and EPO. Our data showed that administration of EPO protein in combination with SLI therapy increased arginase 1 (Arg1) protein expression, a marker for M2-type macrophages, in the TME, while administration of EPO antibody in combination with SLI therapy decreased Arg1 protein expression in the TME on day 4 post-treatments (Fig. 6a). The level of granzyme B, a cytotoxic enzyme released by activated memory T cells, NK and NKT cells, was significantly elevated in lung metastases of mice treated with SLI combined with anti-EPO (Fig. 6a, b). To further corroborate this notion, we tested the efficacy of the SLI and anti-EPO combination in the B16-F10 melanoma lung metastatic model showing similar results (Fig. 6c, d). To determine the initial response to SLI treatment, we examined mRNA expression levels on day 1 post-treatments. Consistently, Arg1 mRNA levels were significantly increased in response to EPO protein treatment, while being strongly suppressed by anti-EPO treatment alone or in combination with SLI (Fig. 6e). Moreover, anti-EPO used as mono- or combinational treatment with SLI significantly reduced the levels of Ccl2 mRNA, indicative for the expression of the monocyte-attracting chemokine CC-chemokine ligand 2 (Fig. 6f). In line with these results, Cd8 mRNA levels were significantly elevated after single and combined treatment of anti-EPO and SLI (Fig. 6g). These results suggest that anti-EPO treatment alone or in combination with SLI increased the infiltration of CD8⁺ cytotoxic lymphocytes mediated through reduction of M2-type tumor-associated macrophages.

Over the last decades, radiation-induced abscopal effects in cancer patients have been elusive. In recent years, the development of immune checkpoint inhibitors combined with radiation therapy has increased chances to observe abscopal effects in clinical practice, indicating that blockade of the inhibitory signals of checkpoint molecules in T cells is pivotal for the abscopal effect. In contrast to previous studies investigating the abscopal effect, our study employed focal radiation of the bone marrow to study the out-of-field (abscopal) effect of radiotherapy on the immune system of a host bearing distant lung metastases. This procedure allowed us to study abscopal effects without inducing potentially irreversible radiation-induced damage on neighboring healthy tissue. Our data revealed that bone-marrow radiation significantly reduced the growth of metastatic lung tumors, an effect that could be further enhanced by the implementation of ICI. Mechanistically, we demonstrated that SLI reduced the number of M-MDSC in the bone marrow resulting in reduced type-2 tumor-associated macrophages in the tumor microenvironment of lung metastases. In turn, this allowed infiltration of NK cells, B cells, and CD4⁺ T cells in combination with ICI. EPO treatment alone demonstrated strong pro-tumorigenic capacities in our model, while SLI in combination with EPO antibody mimicked the abscopal effect noticed in mice treated with dual therapy of SLI and ICI.

Our study showed that the interruption of the tumor-promoting signals through irradiation on the bone marrow is required for the induction of the abscopal effect. At sublethal radiation doses used for SLI treatment, the number of mature blood cells in the bone marrow were temporarily reduced, especially B cells and M-MDSC. In addition to irradiated bone marrow, M-MDSC were significantly decreased in the spleen and lung after SLI exposure. Compared with the subcutaneous tumor mouse models, the lung metastasis mouse model develops a more translatable tumor microenvironment, which are composed of abundant lymphocytes in the TME suitable for observing the immune response induced by abscopal effects[42, 43]. Mirjolet C. et al. also suggested the choice of more immunogenic organs including liver and lung should be favored for the induction of abscopal effects[44]. Using tail vein injection to induce lung metastases, we cannot rule out the presence of a small number of tumor cells were caught within the bone marrow, which may also increase tumor-associated antigens after SLI treatment. In the TME, alveolar macrophages are tissue-resident M1-type macrophages with an inherent self-renewal capacity rendering these cells independent of the supply from infiltrating monocytes. The combination of SLI treatment with anti-EPO increased the level of alveolar macrophages. On the other hand, SLI therapy significantly reduced interstitial macrophages accompanied by the reduction of M-MDSC and circulating M-CSF levels. Consistent to our observation, Gilboa D. et al. found that administration of EPO protein in mice resulted in the proliferation of Kupffer cells and elevated production of the monocyte chemoattractant CCL2, corresponding to the egress of M-MDSC from the bone marrow[45]. With respect to M2 macrophages polarization, both GM-CSF and M-CSF can prime interstitial macrophages for M2 polarization in response to IL-4 stimulation[46]. As EPOR expression is required for direct response to EPO, Wang J. et al. demonstrated EPOR was expressed not only on EPC but also on a subset of macrophages tightly connected with EPC[47]. Chen Y. et al. also revealed that EPOR expression was restricted to splenic immature stress erythroid progenitors and erythroblastic island macrophages[48]. Since erythroid cells and macrophages are in tight contact with each other, macrophages may be altered by cytokines or chemokines secreted by erythroblasts[47]. Thus, the communication of erythroblast and macrophage through direct cell-cell contacts or indirect paracrine cytokine signaling might play a crucial role in the differentiation of immunosuppressive macrophages in the TME.

Beyond erythropoiesis, EPO is a multifunctional cytokine with anti-inflammatory properties binding to a heterodimeric EPOR and EPO common-β receptor (CD131). In steady state, EPOR and CD131 are localized within intracellular compartments and rapidly translocated to the cell membrane in response to hypoxia and proinflammatory cytokines[49]. Under inflammatory conditions, EPO protein can be produced by hepatic stellate cells and high concentrations of EPO further binds to the EPOR/CSF2RB heterocomplex to inhibit inflammation[50, 51]. Hypoxia induces the expression of EPO via the induction of hypoxia-inducible factor 2A, while proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, inhibit EPO production[52, 53]. In our lung metastases mouse model, SLI significantly induced TNF-α in the TME (Additional file 1: Fig. S1), which may explain the suppression of EPO and M2 macrophages after SLI treatment. Consequently, the combination of SLI and anti-EPO generated the lowest level of interstitial macrophages and the best tumor control.

In animal models, the importance of NK cells and T cells on abscopal effects has been extensively studied. Caetano M.S. et al. designed triple therapies consisting of radiation (12 Gy x 3 fractions), anti-PD-1 and MerTK inhibitors, and demonstrated that the presence of NK and CD8⁺ T cells was required for the stimulation of abscopal effects in immunocompetent mice bearing subcutaneous tumors[54]. Barsoumian H.B. et al. stated that high-dose irradiation (12 Gy x 3 fractions) on the primary tumor site activated T cells, while low-dose irradiation (1 Gy x 2 fractions) directed to metastatic tumors showed modulation of stromal responses. Combining checkpoint inhibitors in lung cancer-bearing subcutaneous mouse models maximized systemic outcomes by favoring M1 macrophage polarization, enhancing NK cell infiltration, and reducing TGF-β levels. Importantly, depletion of CD4⁺ T cells and NK cells abrogated the observed anti-tumor effect[55]. The induction of CD4⁺ T cells and NK cells was also observed in the TME of our lung metastases, implying the involvement of bone marrow responses triggered by irradiation. Shiraishi K. et al. used human macrophage inflammatory protein-1alpha variant to potentiate the abscopal effect of radiation in a subcutaneous mouse model, which involved CD4⁺, CD8⁺ T cells and NK cells[56], consistent with our observation that T cells and NK cells were significantly induced by SLI treatment in the bone marrow.

In cancer patients, few cases of abscopal effects were reported after single radiotherapy. Sakaguchi T. et al. revealed that irradiation (3 Gy x 10 fractions) on iliac bone metastases of lung cancer reduced distant non-irradiated osteolytic lesions[57]. Ishikawa Y. et al. showed that radiation therapy (3 Gy x 12 fractions) performed on the thoracic vertebra diminished all of the pulmonary and osseous metastatic lesions[58]. Ellerin B.E. et al. reported that irradiating parotid gland cancer (2.5 Gy x 20 fractions) completely resolved non-irradiated foci in the lungs[59]. Guo Z. et al. demonstrated that radiotherapy (2 Gy x 30 fractions) on metastatic cervical lymph nodes reduced abdominal mass[60]. These reports imply that irradiation of metastases located in lymphatic organs such bone marrow and lymph nodes increase the incidence of abscopal effects in patients. Other special cases include reports on the radiation of the brain mitigating bone metastases (3 Gy x 12 fractions)[9], and lung mass (3 Gy x 10 fractions)[61], indicating the requirement of alterations in the microenvironment of brain to initiate the activation of immune system to induce abscopal effects.

In this study, abscopal effects were induced by single limb irradiation (SLI), which enhanced the tumor infiltration of T cells and NK cells and attenuated immunosuppressive barriers including type 2-macrophages and erythroid progenitor cells, potent immunosuppressive cells inducing T cell dysfunction[62]. Prolonged inhibition of erythropoietin after SLI through combination with anti-EPO strengthened the abscopal effect induced by SLI and reversed tumor-induced T cell dysfunction (Fig. 7).

Our study found combinational therapy consisting of SLI and EPO antibody treatment is an attractive novel therapeutic strategy to enhance immune activation, while decreasing immunosuppressive barriers to achieve control of tumor growth in colorectal cancer-derived lung metastases. Activation of the immune system by radiotherapy and blocking of erythroblast-macrophage communication could be a potential therapeutic strategy for metastatic advanced cancers.

ICI: immune checkpoint inhibitors

Radio-ICI: radiotherapy and ICI

TME: tumor microenvironment

SLI: single limb irradiation

EPO: erythropoietin

MDSC: myeloid-derived suppressor cells

PMN-MDSC: polymorphonuclear myeloid-derived suppressor cells

M-MDSC: monocytic polymorphonuclear myeloid-derived suppressor cells

EPC: erythroid progenitor cells

TAM: tumor-associated macrophages

Tnfa: tumor necrosis factor-α

M-CSF: Macrophage Colony Stimulating Factor

CXCL12: CXC motif chemokine ligand 12

Arg1: arginase 1

Ccl2: monocyte-attracting chemokine CC-chemokine ligand 2

Acknowledgements

We would like to acknowledge the services and supports from the Drug Development Center of China Medical University and the Center for Translational Genomics & Regenerative Medicine Research of China Medical University Hospital, Taichung, Taiwan.

Funding

This study was supported in part by Ministry of Science and Technology (MOST 109-2314-B-039-059, MOST 109-2314-B-195-003-MY3); the China Medical University (CMU109-N-26), the Taitung MacKay Memorial Hospital (TTMMH-109-03 and TTMMH-110-02), and the Mackay Memorial Hospital (MMH-E-111-11), Taiwan, R.O.C. Figures were created using Biorender.com.

Author information

Authors and Affiliations

Department of Biomedical Imaging and Radiological Science, China Medical University, Taichung, Taiwan

Shin-Yi Liu & I-Fang Wu

Division of Nephrology, Department of Internal Medicine, Taitung MacKay Memorial Hospital, Taitung, Taiwan

Feng-Chi Kuo

Department of Radiology, Taitung MacKay Memorial Hospital, Taitung, Taiwan

Wan-Zu Liou

Department of Radiation Oncology, Chung Shan Medical University Hospital, Taichung City, Taiwan

Ying-Hsiang Chou

Precision Immunotherapy Group, Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan

Alexandra Aicher & Chi-Pin Lee

Department Medical Research, MacKay Memorial Hospital, New Taipei City, Taiwan

Yu-Jen Chen

Contributions

YJC and SYL conceived and designed the study; SYL, IFW, and CPL performed the experiments and finished the analyses, and the analysis processes were checked by FCK, WZL and YHC; YJC, SYL and AA interpreted the results and wrote the manuscript. All authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Yu-Jen Chen or Shin-Yi Liu.

Ethics declarations

Ethics approval and consent to participate

All animal experiments have been approved by the Animal Ethics Committee of China Medical University, Taiwan (CMUIACUC-2021-018).

Consent for publication

All authors have read the manuscript and agree to publish.

Competing interests

The authors declare that they have no competing interests.

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

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Abstract

Figures

Background

Methods

Cell culture

Lung metastatic mouse model and treatments

Hemogram and biochemistry analyses

Flow cytometric analysis

Cytokine detection

Western blot analysis

Real-time reverse transcription polymerase chain reaction

Statistical analysis

Results

Single limb irradiation (SLI) induces abscopal effects and enhances ICI efficacy

Single limb irradiation (SLI) modulates the tumor microenvironment

Single limb irradiation (SLI) reduces MDSC in the irradiated bone marrow and TME

Single limb irradiation (SLI) reduces systemic EPO and erythrocyte progenitor cells

Combination of SLI and anti-EPO suppresses tumor-associated macrophages and increases NKT cells in the TME

Combination of SLI and anti-EPO reduced immunosuppressive barriers in the TME of lung metastases

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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