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The ability of focal radiotherapy to promote priming of tumor-specific CD8+ T cells and increase responses to immunotherapy is dependent on infiltration of the tumor by Batf3-dependent conventional dendritic cell type 1 (cDC1) cells. Such infiltration is driven by radiotherapy-induced IFN type I (IFN-I). Other signals may also modulate cDC1 infiltration of irradiated tumors. Here we found increased expression of adenosine-generating enzymes CD38 and CD73 in irradiated mouse and human breast cancer cells and increased adenosine in mouse tumors following radiotherapy. CD73 blockade alone had no effect. CD73 blockade with radiotherapy restored radiotherapy-induced cDC1 infiltration of tumors in settings where radiotherapy induction of IFN-I was suboptimal. In the absence of radiotherapy-induced IFN-I, blockade of CD73 was required for rejection of the irradiated tumor and for systemic tumor control (abscopal effect) in the context of cytotoxic T-lymphocyte–associated protein 4 blockade. These results suggest that CD73 may be a radiation-induced checkpoint, and that CD73 blockade in combination with radiotherapy and immune checkpoint blockade might improve patient response to therapy.

Supplementary figures and tables

Proteomics profiles of UT-TEX and RT-TEX

Immune checkpoint inhibitors activate T cells to reject tumors. Unique tumor mutations are key T-cell targets, but a comprehensive understanding of the nature of a successful antitumor T-cell response is lacking. To investigate the T-cell receptor (TCR) repertoire associated with treatment success versus failure, we used a well-characterized mouse carcinoma that is rejected by CD8 T cells in mice treated with radiotherapy (RT) and anti–CTLA-4 in combination, but not as monotherapy, and comprehensively analyzed tumor-infiltrating lymphocytes (TILs) by high-throughput sequencing of the TCRΒ CDR3 region. The combined treatment increased TIL density and CD8/CD4 ratio. Assessment of the frequency of T-cell clones indicated that anti–CTLA-4 resulted in fewer clones and a more oligoclonal repertoire compared with untreated tumors. In contrast, RT increased the CD8/CD4 ratio and broadened the TCR repertoire, and when used in combination with anti–CTLA-4, these selected T-cell clones proliferated. Hierarchical clustering of CDR3 sequences showed a treatment-specific clustering of TCRs that were shared by different mice. Abundant clonotypes were commonly shared between animals and yet treatment-specific. Analysis of amino-acid sequence similarities revealed a significant increase in the number and richness of dominant CDR3 motifs in tumors treated with RT + anti–CTLA-4 compared with control. The repertoire of TCRs reactive with a single tumor antigen recognized by CD8+ T cells was heterogeneous but highly clonal, irrespective of treatment. Overall, data support a model whereby a diverse TCR repertoire is required to achieve tumor rejection and may underlie the synergy between RT and CTLA-4 blockade. Cancer Immunol Res; 6(2); 139–50. ©2017 AACR.

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 S1. Relative composition of TCRs in unsorted tumors within each treatment group. Figure S2. TCRβ CDR3 sequence distance among AH1-pentamer+/tetramer+CD8+ T cells. Table S1. Summary of TCR sequencing of whole 4T1 tumors. Table S2. Summary of TCR sequencing of AH1-pentamer+CD8+ T cells. Table S3. Abundance of AH1 clone signature (amino acid sequences) in CD4+ or CD8+ sorted T cells. Table S4. Three AH1 reactive T cell clones were shared between 4T1 tumors and AH1 peptide-vaccinated animals.

Immune-checkpoint inhibitors (ICI), although revolutionary in improving long-term survival outcomes, are mostly effective in patients with immune-responsive tumors. Most patients with cancer either do not respond to ICIs at all or experience disease progression after an initial period of response. Treatment resistance to ICIs remains a major challenge and defines the biggest unmet medical need in oncology worldwide. In a collaborative workshop, thought leaders from academic, biopharma, and nonprofit sectors convened to outline a resistance framework to support and guide future immune-resistance research. Here, we explore the initial part of our effort by collating seminal discoveries through the lens of known biological processes. We highlight eight biological processes and refer to them as immune resistance nodes. We examine the seminal discoveries that define each immune resistance node and pose critical questions, which, if answered, would greatly expand our notion of immune resistance. Ultimately, the expansion and application of this work calls for the integration of multiomic high-dimensional analyses from patient-level data to produce a map of resistance phenotypes that can be utilized to guide effective drug development and improved patient outcomes.

Radiation therapy (RT) increases tumor response to CTLA-4 inhibition (CTLA4i) in mice and in some patients, yet deep responses are rare. This raises the possibility that effector T cells are insufficiently activated by the combination. To address this question we use mouse models of triple negative breast cancer highly resistant to immunotherapy. We find that CTLA4i promotes the expansion of CD4+ T helper cells, whereas RT enhances T cell clonality and enriches for CD8+ T cells with an exhausted phenotype. Combination therapy decreases regulatory CD4+ T cells and increases CD8+ T cells with effector memory, early activation and precursor exhausted phenotype. A combined gene signature comprising these three CD8+ T cell clusters is associated with survival in triple negative breast cancer patients. Despite the expression of multiple immune checkpoints on intratumoral T cells, response to RT+CTLA4i is not increased by targeting additional checkpoints, including PD1. In contrast, agonistic CD40 therapy enhances responses in mice, indicating the need to target different immune compartments to increase responses to combinations of RT and CTLA4i.

Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)–expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited significantly elevated postirradiation CD47 levels. The combination of RT, anti-SIRPα, and anti–PD-1 reversed adaptive immune resistance and drove efficient TAA cross-presentation, resulting in robust TAA-specific CD8 T cell priming, functional activation of T effectors, and increased T cell clonality and clonal diversity. We observed significantly higher complete response rates to RT/anti-SIRPα/anti–PD-1 in both irradiated and abscopal tumors and prolonged survival in three distinct murine CRC models, including a cecal orthotopic model. The efficacy of triple combination therapy was STING dependent as knockout animals lost most benefit of adding anti-SIRPα and anti–PD-1 to RT. Despite activation across the myeloid stroma, the enhanced dendritic cell function accounts for most improvements in CD8 T cell priming. These data suggest ATR-mediated CD47 and PD-L1 up-regulation as a key mechanism restraining radiation-induced immune priming. RT combined with SIRPα and PD-1 blockade promotes robust antitumor immune priming, leading to systemic tumor regressions.

Most patients with non–small cell lung cancer (NSCLC) do not achieve durable clinical responses from immune checkpoint inhibitors, suggesting the existence of additional resistance mechanisms. Nicotinamide adenine dinucleotide (NAD)–induced cell death (NICD) of P2X7 receptor (P2X7R)–expressing T cells regulates immune homeostasis in inflamed tissues. This process is mediated by mono–adenosine 5′-diphosphate (ADP)–ribosyltransferases (ARTs). We found an association between membranous expression of ART1 on tumor cells and reduced CD8 T cell infiltration. Specifically, we observed a reduction in the P2X7R + CD8 T cell subset in human lung adenocarcinomas. In vitro, P2X7R + CD8 T cells were susceptible to ART1-mediated ADP-ribosylation and NICD, which was exacerbated upon blockade of the NAD + -degrading ADP-ribosyl cyclase CD38. Last, in murine NSCLC and melanoma models, we demonstrate that genetic and antibody-mediated ART1 inhibition slowed tumor growth in a CD8 T cell–dependent manner. This was associated with increased infiltration of activated P2X7R + CD8 T cells into tumors. In conclusion, we describe ART1-mediated NICD as a mechanism of immune resistance in NSCLC and provide preclinical evidence that antibody-mediated targeting of ART1 can improve tumor control, supporting pursuit of this approach in clinical studies.

Focal radiation therapy (RT) can increase tumor immunogenicity and T cell-mediated tumor rejection when combined with immune checkpoint blockade (ICB). Our prior findings in patients and in preclinical studies suggest that one mechanism whereby RT increases tumor immunogenicity is by enhancing the expression of neoantigens.1,2 In response to RT, cells rapidly transcribe and translate hundreds of genes associated with the DNA damage response (DDR), protein turnover and cellular stress. Genes in these pathways are frequently mutated in cancer. Thus, we hypothesized that the presence of immunogenic mutations in genes upregulated by radiation could help predict the benefits of RT used in combination with ICB. In order to develop such a predictor, we built an in vitro system to study the RT-induced transcriptional response of primary human tumors cultured as patient-derived tumor organoids (PDO). PDOs were established from 2 breast cancer (BC), 4 non-small cell lung cancer (NSCLC) and 3 colorectal cancer (CRC) patients. PDOs were irradiated with doses of 5 to 8 Gy daily for 3 days (n=4) or left untreated (UT, n=4) and cultured for 24h prior to RNA extraction. RNA sequencing was carried out using a NovaSeq6000 sequencer (Illumina) to a depth of 30 million reads. Differential expression analysis between RT and UT samples was performed using DESeq2 and significantly perturbed genes were defined as genes with fold change greater than 1.5 and adjusted p-value cutoff of 0.05. In total we detected an average of 15,000+/-800 protein coding genes per sample and the number of genes modulated by RT was found to vary with cancer type. This ranged from an average of 3.9+/-1.6% genes upregulated by RT in NSCLC to 12.1+/-2.5% in CRC organoids. There were 104 and 490 genes commonly upregulated by RT in NSCLC and CRC, respectively. One of the BC PDOs is a triple-negative BC and the other HR+ and despite this difference, we identified 24 commonly upregulated genes. Gene Set Enrichment Analysis (GSEA), revealed enrichments in DDR and pro-inflammatory signaling pathways among the upregulated genes. Whole exome sequencing and paired normal tissue was used to determine the variants and predicted neoantigens for these PDOs. In one of the EGFR mutated NSCLC PDOs we found that among the genes transcriptionally upregulated by RT, 18 carried somatic mutations predicted to be antigenic. The presentation of these neoantigens is being investigated by mass spectrometry analysis of the immunopeptidome eluted from surface MHC-I molecules. Similar investigations are being carried out in the other models. Overall, results support the feasibility of identifying a common signature of RT upregulated genes in PDOs. This will provide a system to predict RT- exposed neoantigens. [1] Formenti et al. Nat. Med. 2018;24(12):1845-1851, [2] Lhuillier et al. J. Clin. Invest. 2021;131(5):e138740 Citation Format: Samantha J. Van Nest, Jared Capuano, Bhavneet Bhinder, Martin G. Klatt, Tuo Zhang, M. Laura Martin, Silvia C. Formenti, Olivier Elemento, Nils-Petter Rudqvist, Sandra Demaria. Patient-derived tumor organoids as a platform to study radiation-exposed neoantigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1309.

Background Multiple genomics-based biomarkers of response to immune checkpoint inhibition have been reported or proposed, including tumor mutation/neoantigen frequency, PD-L1 expression, T cell receptor repertoire clonality, interferon gene signature expression, HLA expression, and others.1 Although genomics associations of response have been reported, the primary studies have used a variety of data generation and processing techniques. There is a need for data harmonization and assessment of generalizability of potential biomarkers across multiple datasets. Methods We acquired patient-level RNA sequencing FASTQ data files from 10 data sets reported in seven pan-cancer PD-1 and CTLA-4 immune checkpoint inhibition trials with matched clinical annotations.2–7 We applied a common bioinformatics workflow for quality control, mapping to reference (STAR), generating gene expression matrices (SALMON), T cell receptor repertoire inference (MiXCR), extraction of immune gene signatures and immune subtypes,8 and differential gene expression analysis (DESeq2). We analyzed i) immunogenomics features proposed as biomarkers, and ii) gene expression signatures built from each trial for association with overall survival across the set of trials using univariable Cox proportional hazards regression. In all, we assessed 9 total immunogenomics features/signatures. P-values were adjusted for multiple testing using the Benjamini-Hochberg method. Results Of the 9 immunogenomics features assessed, cytolytic activity score and expression of the Follicular Dendritic Cell Secreted Protein gene (FDCSP) were associated with survival in two of seven studies, respectively (adjusted p Conclusions No proposed biomarkers were highly generalizable across studies. We expect that integrated modeling incorporating multiple immunogenomics features will be required to build a robust and generalizable biomarker for ICI response. Further work is needed to analyze determinants of response and clinical benefit. Acknowledgements We would like to thank SITC for funding for this work as part of the Sparkathon TimIOS collaborative project. References Zappasodi R, Wolchok JD, Merghoub T. Strategies for Predicting Response to Checkpoint Inhibitors. Curr Hematol Malig Rep 2018;13(5):383–95. Liu D, Schilling B, Liu D, Sucker A, Livingstone E, Jerby-Arnon L, Zimmer L, Gutzmer R, Satzger I, Loquai C, Grabbe S, Vokes N, Margolis CA, Conway J, He MX, Elmarakeby H, Dietlein F, Miao D, Tracy A, Gogas H, Goldinger SM, Utikal J, Blank CU, Rauschenberg R, von Bubnoff D, Krackhardt A, Weide B, Haferkamp S, Kiecker F, Izar B, Garraway L, Regev A, Flaherty K, Paschen A, Van Allen EM, Schadendorf D. Integrative molecular and clinical modeling of clinical outcomes to PD1 blockade in patients with metastatic melanoma. Nat Med 2019;25(12):1916–27. Gide TN, Quek C, Menzies AM, Tasker AT, Shang P, Holst J, Madore J, Lim SY, Velickovic R, Wongchenko M, Yan Y, Lo S, Carlino MS, Guminski A, Saw RPM, Pang A, McGuire HM, Palendira U, Thompson JF, Rizos H, Silva IPD, Batten M, Scolyer RA, Long GV, Wilmott JS. distinct immune cell populations define response to anti-pd-1 monotherapy and Anti-PD-1/Anti-CTLA-4 Combined Therapy. Cancer Cell 2019;35(2):238–55 e6. Cloughesy TF, Mochizuki AY, Orpilla JR, Hugo W, Lee AH, Davidson TB, Wang AC, Ellingson BM, Rytlewski JA, Sanders CM, Kawaguchi ES, Du L, Li G, Yong WH, Gaffey SC, Cohen AL, Mellinghoff IK, Lee EQ, Reardon DA, O’Brien BJ, Butowski NA, Nghiemphu PL, Clarke JL, Arrillaga-Romany IC, Colman H, Kaley TJ, de Groot JF, Liau LM, Wen PY, Prins RM. Neoadjuvant anti-PD-1 immunotherapy promotes a survival benefit with intratumoral and systemic immune responses in recurrent glioblastoma. Nat Med. 2019;25(3):477–86. Riaz N, Havel JJ, Makarov V, Desrichard A, Urba WJ, Sims JS, Hodi FS, Martin-Algarra S, Mandal R, Sharfman WH, Bhatia S, Hwu WJ, Gajewski TF, Slingluff CL, Jr., Chowell D, Kendall SM, Chang H, Shah R, Kuo F, Morris LGT, Sidhom JW, Schneck JP, Horak CE, Weinhold N, Chan TA. Tumor and microenvironment evolution during immunotherapy with nivolumab. Cell 2017;171(4):934–49 e16. Hugo W, Zaretsky JM, Sun L, Song C, Moreno BH, Hu-Lieskovan S, Berent-Maoz B, Pang J, Chmielowski B, Cherry G, Seja E, Lomeli S, Kong X, Kelley MC, Sosman JA, Johnson DB, Ribas A, Lo RS. Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell 2016;165(1):35–44. Rosenberg JE, Hoffman-Censits J, Powles T, van der Heijden MS, Balar AV, Necchi A, Dawson N, O’Donnell PH, Balmanoukian A, Loriot Y, Srinivas S, Retz MM, Grivas P, Joseph RW, Galsky MD, Fleming MT, Petrylak DP, Perez-Gracia JL, Burris HA, Castellano D, Canil C, Bellmunt J, Bajorin D, Nickles D, Bourgon R, Frampton GM, Cui N, Mariathasan S, Abidoye O, Fine GD, Dreicer R. Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial. Lancet. 2016;387(10031):1909–20. Thorsson V, Gibbs DL, Brown SD, Wolf D, Bortone DS, Ou Yang TH, Porta-Pardo E, Gao GF, Plaisier CL, Eddy JA, Ziv E, Culhane AC, Paull EO, Sivakumar IKA, Gentles AJ, Malhotra R, Farshidfar F, Colaprico A, Parker JS, Mose LE, Vo NS, Liu J, Liu Y, Rader J, Dhankani V, Reynolds SM, Bowlby R, Califano A, Cherniack AD, Anastassiou D, Bedognetti D, Mokrab Y, Newman AM, Rao A, Chen K, Krasnitz A, Hu H, Malta TM, Noushmehr H, Pedamallu CS, Bullman S, Ojesina AI, Lamb A, Zhou W, Shen H, Choueiri TK, Weinstein JN, Guinney J, Saltz J, Holt RA, Rabkin CS, Cancer Genome Atlas Research N, Lazar AJ, Serody JS, Demicco EG, Disis ML, Vincent BG, Shmulevich I. The Immune Landscape of Cancer. Immunity 2018;48(4):812–30e14.

Exosomes are small extracellular vesicles released by prokaryotic and eukaryotic cells with a crucial role in cell-to-cell communication in both physiological and pathological conditions. Exosomes contain and transfer active biomolecules, including nucleic acids, proteins and lipids to target recipient cells. In the last decade, many methodologies have been developed for isolating specific exosomal components. In this chapter, we will detail methods to isolate exosomal DNA, considering the crucial role of exosomal DNA in regulating the behavior of recipient cells in multiple settings, including the response of malignant cells to chemo-, radio- and immunotherapy.

Tumor infiltration of conventional dendritic cells has been shown to be essential for triggering efficient antitumor immune responses. These findings have generated an increasing demand for reliable methods to accurately identify and quantify specific DC-subpopulations, both in immune monitoring of clinical trial samples as well as in preclinical mouse tumor models. Here, we describe a flow cytometric approach to assess percentages and absolute counts of conventional dendritic cells in solid mouse tumors.

BackgroundBackground: Radiotherapy of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically; however, incidences of abscopal tumor remission are extremely rare. We sought to unravel the post-irradiation immune escape mechanisms in CRC.MethodsMethodsFlow cytometry, gene knockdown, RNA and T cell receptor sequencing, and multiple murine syngeneic CRC models were used to interrogate mechanisms of CRC immune evasion following radiotherapy. Comparison of immunohistochemistry staining between pretreatment biopsy and post-irradiation surgical specimens was performed in rectal patients who underwent neoadjuvant radiotherapy with 5 Gy for 5 fractions.ResultsResultsWe find that CRC cells utilize a common DNA repair signaling pathway — ATR/Chk1/STAT3 — to upregulate both CD47 and PD-L1 in response to radiotherapy, which through engagement of SIRPα and PD-1 suppresses the capacity of antigen-presenting cells (APCs) to phagocytose them thereby preventing TAA cross-presentation. This post-irradiation CD47 and PD-L1 upregulation can be observed in CRC cells treated with either photon or proton radiotherapy and across a wide variety of human solid tumor cells. Concordantly, rectal cancer patients who responded poorly (tumor regression grade 4–5, n = 10) to neoadjuvant radiotherapy exhibited significantly elevated post-irradiation CD47 levels (P = 0.005). In murine CRC models, the combination of radiotherapy, αSIRPα, and αPD-1 (RSP) profoundly enhances TAA uptake, activation of innate immune sensors, and TAA cross-priming across various antigen-presenting myeloid populations in the irradiated tumor microenvironment and facilitates TAA-presenting APC migration to secondary lymphoid organs. Furthermore, we observed robust production of TAA-specific CD8 T cells, functional activation of effector T cells, and increased tumor-infiltrating T cell clonality and clonal diversity in mice treated with RSP. Importantly, radiotherapy coupled with phagocytosis checkpoint blockade significantly improves complete response rates in both irradiated and abscopal tumors and prolongs survival in three distinct murine CRC models, including a cecal orthotopic model. In addition, αSIRPα exerts superior tumoricidal efficacy than αCD47 in combination with RT and αPD-1. We find RSP efficacy to be STING dependent as knockout animals lose most benefit of phagocytosis checkpoint blockade.ConclusionATR-mediated CD47 and PD-L1 upregulation restrains radiation-induced immune priming in CRC. Blockade of the phagocytosis checkpoints SIRPα and PD-1 during radiotherapy promotes vigorous anti-CRC immune priming leading to systemic tumor regression.AcknowledgementsThis study is supported in part by NIH grant P30 CA16672, the MD Anderson Andrew Sabin Family Fellowship, and Chang Gung Memorial Hospital grant CMRPG3K1751. RCH was supported by the CPRIT Research Training Grant (RP170067) and Ralph B. Arlinghaus Ph.D. Scholarship. The authors are grateful to the members of the Advanced Cytometry & Sorting Facility at South Campus, Tissue Bank of Chang Gung Memorial Hospital at Linkou, and MHC Tetramer Core Facility at Baylor College of Medicine for their invaluable help.Ethics ApprovalThis study was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan; approval number: 202001191B0C601.

Table S1. qPCR validation of microarray data. (DOCX 14 kb)

Background 177Lu-octreotate is used for therapy of somatostatin receptor expressing neuroendocrine tumors with promising results, although complete tumor remission is rarely seen. Previous studies on nude mice bearing the human small intestine neuroendocrine tumor, GOT1, have shown that a priming injection of 177Lu-octreotate 24 h before the main injection of 177Lu-octreotate resulted in higher 177Lu concentration in tumor, resulting in increased absorbed dose, volume reduction, and time to regrowth. To our knowledge, the cellular effects of a priming treatment schedule have not yet been studied. The aim of this study was to identify transcriptional changes contributing to the enhanced therapeutic response of GOT1 tumors in nude mice to 177Lu-octreotate therapy with priming, compared with non-curative monotherapy. Results RNA microarray analysis was performed on tumor samples from GOT1-bearing BALB/c nude mice treated with a 5 MBq priming injection of 177Lu-octreotate followed by a second injection of 10 MBq of 177Lu-octreotate after 24 h and killed after 1, 3, 7, and 41 days after the last injection. Administered activity amounts were chosen to be non-curative, in order to facilitate the study of tumor regression and regrowth. Differentially regulated transcripts (RNA samples from treated vs. untreated animals) were identified (change ≥ 1.5-fold; adjusted p value

The immunostimulatory properties of radiation therapy (RT) have recently generated widespread interest due to preclinical and clinical evidence that tumor-localized RT can sometimes induce antitumor immune responses mediating regression of non-irradiated metastases (abscopal effect). The ability of RT to activate antitumor T cells explains the synergy of RT with immune checkpoint inhibitors, which has been well documented in mouse tumor models and is supported by observations of more frequent abscopal responses in patients refractory to immunotherapy who receive RT during immunotherapy. However, abscopal responses following RT remain relatively rare in the clinic, and antitumor immune responses are not effectively induced by RT against poorly immunogenic mouse tumors. This suggests that in order to improve the pro-immunogenic effects of RT, it is necessary to identify and overcome the barriers that pre-exist and/or are induced by RT in the tumor microenvironment. On the one hand, RT induces an immunogenic death of cancer cells associated with release of powerful danger signals that are essential to recruit and activate dendritic cells (DCs) and initiate antitumor immune responses. On the other hand, RT can promote the generation of immunosuppressive mediators that hinder DCs activation and impair the function of effector T cells. In this review, we discuss current evidence that several inhibitory pathways are induced and modulated in irradiated tumors. In particular, we will focus on factors that regulate and limit radiation-induced immunogenicity and emphasize current research on actionable targets that could increase the effectiveness of radiation-induced in situ tumor vaccination.