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Characterization of Inhba knockdown in 4T1 cells.

Increased regulatory T cells (Treg) after radiotherapy have been reported, but the mechanisms of their induction remain incompletely understood. TGFβ is known to foster Treg differentiation within tumors and is activated following radiotherapy. Thus, we hypothesized that TGFβ blockade would result in decreased Tregs within the irradiated tumor microenvironment. We found increased Tregs in the tumors of mice treated with focal radiotherapy and TGFβ blockade. This increase was mediated by upregulation of another TGFβ family member, activin A. In vitro, activin A secretion was increased following irradiation of mouse and human breast cancer cells, and its expression was further enhanced upon TGFβ blockade. In vivo, dual blockade of activin A and TGFβ was required to decrease intratumoral Tregs in the context of radiotherapy. This resulted in an increase in CD8+ T-cell priming and was associated with a reduced tumor recurrence rate. Combination of immune checkpoint inhibitors with the dual blockade of activin A and TGFβ led to the development of tumor-specific memory responses in irradiated breast cancer. Supporting the translational value of activin A targeting to reduce Treg-mediated immunosuppression, retrospective analysis of a public dataset of patients with breast cancer revealed a positive correlation between activin A gene expression and Treg abundance. Overall, these results shed light on an immune escape mechanism driven by activin A and suggest that dual targeting of activin A and TGFβ may be required to optimally unleash radiation-induced antitumor immunity against breast cancer.

Supplementary Figure Legend

Proteomics profiles of UT-TEX and RT-TEX

Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA (dsDNA) in cancer cells, which activates type I IFN (IFN-I) via the cGAS/STING pathway. Cancer cell–derived IFN-I is required to recruit BATF3-dependent dendritic cells (DC) to poorly immunogenic tumors and trigger antitumor T-cell responses in combination with immune checkpoint blockade. We have previously demonstrated that the exonuclease TREX1 regulates radiation immunogenicity by degrading cytosolic dsDNA. Tumor-derived DNA can also activate cGAS/STING-mediated production of IFN-I by DCs infiltrating immunogenic tumors. However, how DNA from cancer cells is transferred to the cytoplasm of DCs remains unclear. Here, we showed that tumor-derived exosomes (TEX) produced by irradiated mouse breast cancer cells (RT-TEX) transfer dsDNA to DCs and stimulate DC upregulation of costimulatory molecules and STING-dependent activation of IFN-I. In vivo, RT-TEX elicited tumor-specific CD8+ T-cell responses and protected mice from tumor development significantly better than TEX from untreated cancer cells in a prophylactic vaccination experiment. We demonstrated that the IFN-stimulatory dsDNA cargo of RT-TEX is regulated by TREX1 expression in the parent cells. Overall, these results identify RT-TEX as a mechanism whereby IFN-stimulatory dsDNA is transferred from irradiated cancer cells to DCs. We have previously shown that the expression of TREX1 is dependent on the RT dose size. Thus, these data have important implications for the use of RT with immunotherapy. Cancer Immunol Res; 6(8); 910–20. ©2018 AACR.

Figure 1S: 1D11 treatment inhibits TGFβ pathway signaling in CD4 and CD8 T cells from TDLN.

Figure 2S: Local radiotherapy combined with TGFβ blockade induces immune-related canonical pathways.

Supplementary Figure Legend

Increased regulatory T cells (Treg) after radiotherapy have been reported, but the mechanisms of their induction remain incompletely understood. TGFβ is known to foster Treg differentiation within tumors and is activated following radiotherapy. Thus, we hypothesized that TGFβ blockade would result in decreased Tregs within the irradiated tumor microenvironment. We found increased Tregs in the tumors of mice treated with focal radiotherapy and TGFβ blockade. This increase was mediated by upregulation of another TGFβ family member, activin A. In vitro, activin A secretion was increased following irradiation of mouse and human breast cancer cells, and its expression was further enhanced upon TGFβ blockade. In vivo, dual blockade of activin A and TGFβ was required to decrease intratumoral Tregs in the context of radiotherapy. This resulted in an increase in CD8+ T-cell priming and was associated with a reduced tumor recurrence rate. Combination of immune checkpoint inhibitors with the dual blockade of activin A and TGFβ led to the development of tumor-specific memory responses in irradiated breast cancer. Supporting the translational value of activin A targeting to reduce Treg-mediated immunosuppression, retrospective analysis of a public dataset of patients with breast cancer revealed a positive correlation between activin A gene expression and Treg abundance. Overall, these results shed light on an immune escape mechanism driven by activin A and suggest that dual targeting of activin A and TGFβ may be required to optimally unleash radiation-induced antitumor immunity against breast cancer.

Radiotherapy is under investigation for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. We show that the DNA exonuclease Trex1 is induced by radiation doses above 12–18 Gy in different cancer cells, and attenuates their immunogenicity by degrading DNA that accumulates in the cytosol upon radiation. Cytosolic DNA stimulates secretion of interferon-β by cancer cells following activation of the DNA sensor cGAS and its downstream effector STING. Repeated irradiation at doses that do not induce Trex1 amplifies interferon-β production, resulting in recruitment and activation of Batf3-dependent dendritic cells. This effect is essential for priming of CD8+ T cells that mediate systemic tumour rejection (abscopal effect) in the context of immune checkpoint blockade. Thus, Trex1 is an upstream regulator of radiation-driven anti-tumour immunity. Trex1 induction may guide the selection of radiation dose and fractionation in patients treated with immunotherapy., Trex1 is an exonuclease that degrades cytosolic DNA and has been associated with modulation of interferon responses in autoimmunity and viral infections. Here, the authors show that Trex1 attenuates the immunogenicity of cancer cells treated with high radiation doses by degrading cytosolic DNA and preventing the activation of interferon response.

Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA (dsDNA) in cancer cells, which activates type I IFN (IFN-I) via the cGAS/STING pathway. Cancer cell–derived IFN-I is required to recruit BATF3-dependent dendritic cells (DC) to poorly immunogenic tumors and trigger antitumor T-cell responses in combination with immune checkpoint blockade. We have previously demonstrated that the exonuclease TREX1 regulates radiation immunogenicity by degrading cytosolic dsDNA. Tumor-derived DNA can also activate cGAS/STING-mediated production of IFN-I by DCs infiltrating immunogenic tumors. However, how DNA from cancer cells is transferred to the cytoplasm of DCs remains unclear. Here, we showed that tumor-derived exosomes (TEX) produced by irradiated mouse breast cancer cells (RT-TEX) transfer dsDNA to DCs and stimulate DC upregulation of costimulatory molecules and STING-dependent activation of IFN-I. In vivo, RT-TEX elicited tumor-specific CD8+ T-cell responses and protected mice from tumor development significantly better than TEX from untreated cancer cells in a prophylactic vaccination experiment. We demonstrated that the IFN-stimulatory dsDNA cargo of RT-TEX is regulated by TREX1 expression in the parent cells. Overall, these results identify RT-TEX as a mechanism whereby IFN-stimulatory dsDNA is transferred from irradiated cancer cells to DCs. We have previously shown that the expression of TREX1 is dependent on the RT dose size. Thus, these data have important implications for the use of RT with immunotherapy. Cancer Immunol Res; 6(8); 910–20. ©2018 AACR.

T cells directed to endogenous tumor antigens are powerful mediators of tumor regression. Recent immunotherapy advances have identified effective interventions to unleash tumor-specific T-cell activity in patients who naturally develop them. Eliciting T-cell responses to a patient's individual tumor remains a major challenge. Radiation therapy can induce immune responses to model antigens expressed by tumors, but it remains unclear whether it can effectively prime T cells specific for endogenous antigens expressed by poorly immunogenic tumors. We hypothesized that TGFβ activity is a major obstacle hindering the ability of radiation to generate an in situ tumor vaccine. Here, we show that antibody-mediated TGFβ neutralization during radiation therapy effectively generates CD8+ T-cell responses to multiple endogenous tumor antigens in poorly immunogenic mouse carcinomas. Generated T cells were effective at causing regression of irradiated tumors and nonirradiated lung metastases or synchronous tumors (abscopal effect). Gene signatures associated with IFNγ and immune-mediated rejection were detected in tumors treated with radiation therapy and TGFβ blockade in combination but not as single agents. Upregulation of programmed death (PD) ligand-1 and -2 in neoplastic and myeloid cells and PD-1 on intratumoral T cells limited tumor rejection, resulting in rapid recurrence. Addition of anti–PD-1 antibodies extended survival achieved with radiation and TGFβ blockade. Thus, TGFβ is a fundamental regulator of radiation therapy's ability to generate an in situ tumor vaccine. The combination of local radiation therapy with TGFβ neutralization offers a novel individualized strategy for vaccinating patients against their tumors. Cancer Res; 75(11); 2232–42. ©2015 AACR.

Meeting abstracts Exosomes are microvesicles (30-100nm) released from living cells that shuttle and transfer selected cellular biomolecules, including cytokines, cell surface molecules, growth factors, mRNA, and miRNA. Tumor-derived exosomes (TEX) allow for a sophisticated means of communication

Activin A is a member of the transforming growth factor beta (TGFβ) superfamily and a pleiotropic cytokine that regulates many processes, from reproduction and development to cancer and immunity. Activin A shares the smad2/3 signal transduction pathway with TGFβ and displays overlapping biological activities with the latter, including the ability to promote the differentiation of CD4 T cells to Th2 and regulatory T cells (Treg). Importantly, recent data indicate that activin A is expressed by some tumors, including breast cancer, suggesting that it could play a role in tumor escape from immune control. Radiotherapy (RT) delivered locally to a tumor induces the development of anti-tumor T cells, but its pro-immunogenic effects are hindered, in part, by concomitant activation of latent TGFβ and increase of Treg. While different mechanisms have been implicated in RT-induced Treg increase, the pathways responsible for this effect remain unclear. Here we tested the hypothesis that activin A is upregulated by RT in breast cancer cells and contributes to the generation of adaptive Treg. Three mouse breast cancer cell lines, 67NR, TSA and 4T1, which represent tumors of decreasing immunogenicity and increasing metastatic ability, were used. Expression of inhibin A (Inhba, the gene encoding activin A) was determined by qPCR. Secretion of activin A by untreated and irradiated tumor cells exposed to single dose (6Gy, 8Gy, 12Gy and 20Gy) or multifraction (5x6Gy; 3x8Gy) RT was quantified by ELISA. Transwell co-culture was used to assess the ability of activin A released by irradiated cancer cells to convert naive CD4 T cells into Treg. While 67NR, TSA and 4T1 cells expressed comparable levels of Inhba mRNA, only the most aggressive and metastatic 4T1 cells produced high levels of activin A (67NR: 37.1; TSA: 8.1; 4T1: 448.6 pg/mL for 105 cells/24h). RT significantly increased activin A secretion, with the largest increase seen after 3x8Gy RT regimen (67NR: 85.8; TSA: 55.1; 4T1: 993.1 pg/mL for 105 cells/24h; p

Tumor-derived exosomes (TEX) are constantly shed by cancer cells and have been shown to carry tumor-derived antigens to dendritic cells (DCs). However, presentation of tumor antigens derived from TEX in the absence of activation signals for DCs leads to immune tolerance. Our lab has demonstrated that ionizing radiotherapy (RT) can convert the irradiated tumor into an in situ vaccine, leading to anti-tumor immune responses. We hypothesized that TEX derived from irradiated cancer cells play a role in RT-induced anti-tumor immunity by delivering tumor antigens together with pro-inflammatory signals that activate DCs and prime tumor-specific effector T cells. To begin to address this hypothesis we first studied how RT modifies TEX molecular composition and their ability to induce DC activation in vitro. Mouse carcinoma cells TSA cultured in exosome-free media were treated in vitro with sham RT (control TEX), or 3 fractions of 8Gy (RT-TEX). TEX were isolated from supernatants 48 hr later using differential ultracentrifugation and purified with a sucrose gradient. Electron microscopy was used to confirm TEX size and morphology. Mass spectrometry (LFQ-MS) followed by MS/MS analyses was used to characterize protein composition. miRNA were analyzed by nanoString nCounter Mouse miRNA expression assay kit using a panel of 578 mouse miRNAs. Normalized results were analyzed with MultiExperiment Viewer. Mouse bone marrow-derived dendritic cells (BMDC) were cultured with TEX for 48 hours. Cells were analyzed by flow cytometry for expression of activation markers. Significant changes in microRNA and protein compositions were seen in RT-TEX compared to control TEX. Specifically, RT-TEX showed increase in proteins involved in the Antigen Processing and Presentation pathway. Additionally, 17 unique proteins were present only in RT-TEX and included proteins involved in T cell development, MHC class I peptide processing, and pro-inflammatory lipid signaling. Analysis of DCs cultured with RT-TEX, but not control TEX, revealed an increase in cell surface expression of CD80 (1428 MFI vs. 963 MFI) and CD86 (3487 MFI vs. 2578 MFI). Interestingly, culturing BMDC with RT-TEX also resulted in an increase in DCs expressing CD103 and CD8a (2% vs 1.2% of CD11c+ cells), suggesting that RT-TEX may influence differentiation of BMDC towards this subset, which is critical for cross-presentation of tumor antigens to T cells. Obtained data support the hypothesis that RT-TEX may switch tolerogenic DCs into activated DCs by providing activation signals together with tumor-derived antigens. Further in vivo experiments are ongoing to determine the ability of RT-TEX to stimulate anti-tumor immune responses. Citation Format: Julie M. Diamond, Jessica R. Chapman, Beatrix Ueberheide, Silvia C. Formenti, Sandra Demaria. Ionizing radiation switches the function of tumor-derived exosomes from messengers of tolerance to inducers of antitumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1648.

Metastatic breast cancer fosters an immunosuppressive tumor microenvironment (TME). Local ionizing radiotherapy (RT) has the potential to convert the tumor into an in situ vaccine by facilitating efficient uptake of tumor neoantigens by dendritic cells and enhancing antigen presentation to promote robust effector T cell responses. Exosomes are microvesicles (30-100nm) released from living cells that shuttle and transfer selected intracellular components, including cytokines, growth factors, mRNA, and miRNA. Tumor-derived exosomes (TEX) allow for a sophisticated means of communication with a variety of cells, including immune cells, within the TME. We hypothesized that TEX released from irradiated tumors may play a role in altering the susceptibility of tumor cells to immune-mediated rejection. To determine if RT changes TEX composition, we treated mouse mammary carcinoma cells TSA in vitro with sham RT, 1 dose of 20Gy, or 3 fractions of 8Gy given in three consecutive days (8Gyx3). Cells were transferred to exosome-depleted media following RT and cell-free supernatant was collected 48hr later. TEX were isolated using differential ultracentrifugation and purified with a sucrose gradient. Electron microscopy was used to confirm the expected size and morphology. TEX were lysed for identification of proteins using label-free quantitation mass spectrometry (LFQ-MS) followed by MS/MS analyses. To characterize miRNA signatures of TEX and their parent cells, RNA was isolated for nanoString miRNA expression assay, which includes a panel of 578 mouse miRNAs. Normalized results were analyzed with MultiExperiment Viewer. LFQ-MS revealed that TEX from 20Gy- and 8Gyx3-irradiated TSA shared 10 unique proteins not present in TEX from non-irradiated TSA. In addition, 17 unique proteins were present only in TEX from 8Gyx3-treated TSA. Among them were proteins involved in T cell development, MHC class I peptide processing, and proinflammatory lipid signaling. Radiation induced downregulation of 73% of miRNAs expressed in untreated TSA cells. Many of the changes in miRNA expression seen in irradiated cells were also seen in the TEX derived from these cells. Interestingly, unique expression patterns emerged with different miRNA subsets expressed in TEX depending on RT regimen used. Interesting candidates are being analyzed for functional relevance. This data suggests that irradiation of tumor cells results in changes in protein and miRNA composition of TEX, with differences dependent on the RT regimen used. We have previously shown that 8Gyx3 RT synergized with anti-CTLA-4 antibody and induced an abscopal effect in mice, while 20Gy did not (Dewan et al, Clin Cancer Res, 2009). We are testing whether TEX contain biomarkers that define the ability of RT regimens to synergize with immunotherapy. Exosome-mediated communication networks may provide a new therapeutic target for treatment of metastatic cancer. Citation Format: Julie M. Diamond, Jessica R. Chapman, Beatrix M. Ueberheide, Sandra Demaria. Ionizing radiation modifies the molecular composition of exosomes derived from breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2015-3329

Accumulating data support the concept that ionizing radiation therapy (RT) has the potential to convert the tumor into an in situ, individualized vaccine; however this potential is rarely realized by RT alone. Transforming growth factor β (TGFβ) is an immunosuppressive cytokine that is activated by RT and inhibits the antigen-presenting function of dendritic cells and the differentiation of effector CD8+ T cells. Here we tested the hypothesis that TGFβ hinders the ability of RT to promote anti-tumor immunity. Development of tumor-specific immunity was examined in two pre-clinical models of metastatic breast cancer and analyzed in patients with metastatic breast cancer treated with local radiotherapy and the TGFβ-neutralizing antibody Fresolimumab. Mice bearing established 4T1 and TSA mouse mammary carcinomas treated with pan-isoform specific TGFβ neutralizing antibody, 1D11, showed significantly improved control of the irradiated tumor and non-irradiated metastases, but no effect in the absence of RT. Notably, whole tumor transcriptional analysis demonstrated the selective upregulation of genes associated with immune-mediated rejection only in tumors of mice treated with RT+TGFβ blockade. Mice treated with RT+TGFβ blockade exhibited cross-priming of CD8+ T cells producing IFNγ in response to three tumor-specific antigens in tumor-draining lymph nodes, which was not evident for single modality treatment. Likewise, HLA-A2.1+ metastatic breast cancer patients (n=8) enrolled in NCT01401062 trial of local RT and fresolimumab were examined for CD8+ T cells specific for the tumor antigen survivin by tetramer staining. Three patients developed increased frequencies of survivin-specific CD8+ T cells in the blood during treatment, while two patients negative at baseline became positive. Analysis of the immune infiltrate in mouse tumors showed a significant increase in CD4+ and CD8+ T cells only in mice treated with the combination of RT+TGFβ blockade. Depletion of CD4+ or CD8+ T cells abrogated the therapeutic benefit of RT+TGFβ blockade. These data identify TGFβ as a master inhibitor of the ability of RT to generate an in situ tumor vaccine, which supports testing inhibition of TGFβ during radiotherapy to promote therapeutically effective anti-tumor immunity. Supported by DOD BCRP Multi-Team Award BC100481. Citation Format: Claire I. Vanpouille-Box, Julie M. Diamond, Jiri Zavadil, James Babb, Dörthe Schaue, Mary Helen Barcellos-Hoff, William H. McBride, Silvia C. Formenti, Sandra Demaria. Inhibition of TGFβ as a strategy to convert the irradiated tumor into in situ individualized vaccine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 633. doi:10.1158/1538-7445.AM2014-633

ligands, PDL-1/2, were upregulated by RT on TSA cells and tumor-infiltrating myeloid cells, suggesting PD-1’s interaction with its ligands may limit RT-activated antitumor T-cell activity. Consistent with this hypothesis, RMP1-14 alone had no effect on tumor growth; RT delayed growth (p < 0.01), but only 1/6 mice showed tumor regression, whereas all mice receiving RT+RMP114 completely rejected tumors by day 25. In spleen, RMP1-14 had no effect on AH1-specific CD8 + T-cells