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BackgroundOncolytic viruses are considered part of immunotherapy and have shown promise in preclinical experiments and clinical trials. Results from these studies have suggested that tumor microenvironment remodeling is required to achieve an effective response in solid tumors. Here, we assess the extent to which targeting specific mechanisms underlying the immunosuppressive tumor microenvironment optimizes viroimmunotherapy.MethodsWe used RNA-seq analyses to analyze the transcriptome, and validated the results using Q-PCR, flow cytometry, and immunofluorescence. Viral activity was analyzed by replication assays and viral titration. Kyn and Trp metabolite levels were quantified using liquid chromatography–mass spectrometry. Aryl hydrocarbon receptor (AhR) activation was analyzed by examination of promoter activity. Therapeutic efficacy was assessed by tumor histopathology and survival in syngeneic murine models of gliomas, including Indoleamine 2,3-dioxygenase (IDO)-/- mice. Flow cytometry was used for immunophenotyping and quantification of cell populations. Immune activation was examined in co-cultures of immune and cancer cells. T-cell depletion was used to identify the role played by specific cell populations. Rechallenge experiments were performed to identify the development of anti-tumor memory.ResultsBulk RNA-seq analyses showed the activation of the immunosuppressive IDO-kynurenine-AhR circuitry in response to Delta-24-RGDOX infection of tumors. To overcome the effect of this pivotal pathway, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors. The combination therapy increased the frequency of CD8+ T cells and decreased the rate of myeloid-derived suppressor cell and immunosupressive Treg tumor populations in animal models of solid tumors. Functional studies demonstrated that IDO-blockade-dependent activation of immune cells against tumor antigens could be reversed by the oncometabolite kynurenine. The concurrent targeting of the effectors and suppressors of the tumor immune landscape significantly prolonged the survival in animal models of orthotopic gliomas.ConclusionsOur data identified for the first time the in vivo role of IDO-dependent immunosuppressive pathways in the resistance of solid tumors to oncolytic adenoviruses. Specifically, the IDO-Kyn-AhR activity was responsible for the resurface of local immunosuppression and resistance to therapy, which was ablated through IDO inhibition. Our data indicate that combined molecular and immune therapy may improve outcomes in human gliomas and other cancers treated with virotherapy.

BackgroundOsteosarcoma is an aggressive bone tumor, primarily arising in the pediatric age. Despite years of intensive research, outcome for metastatic and non-responder patients is very poor and has not improved in the last 30 years. These tumors harbor a highly immunosuppressive environment, making the existing immunotherapies ineffective. Inhibition of galectin-3 (Gal3), a protein involved in immunosuppression,1–3 adhesion of tumor cells,4 5 and metastases,6–9 has been demonstrated to reduce tumor progression in different tumor models, including osteosarcoma.10–12 On the other hand, virotherapy based on recombinant Semliki Forest Virus (SFV), a self-replicating RNA virus, has shown therapeutic effect in orthotopic osteosarcoma mouse models.13MethodsWe generated SFV vectors expressing truncated forms of Gal3, including its carboxy-terminal domain (SFV-Gal3-C) and its amino-terminal domain alone (SFV-Gal3-N) or fused to the Gal3 inhibitor peptide C12 (SFV-Gal3-N-C12). An additional construct expressed the C12 peptide14 15 (SFV-C12). We analyzed Gal3 expression in different murine and human osteosarcoma cell lines. Orthotopic osteosarcoma tumors, induced by intratibial injection of K7M2 murine cells, which showed high expression of Gal3, were treated with SFV vectors expressing Gal3 inhibitors or luciferase or with PBS (control). Animals were maintained under standard conditions, and all procedures were approved by the Institutional Ethical Committee (CEEA) in accordance with the guidelines of the University of Navarra, approval number 044-21.ResultsTreatment with the SFV-Gal3-N-C12 vector showed the highest antitumor activity, significantly reducing tumor growth compared to control mice that received PBS. In fact, this vector prolonged animal survival, leading to 47% of complete regressions. Among the other vectors, SFV-Gal3-N and SFV-C12 were also able to transiently decrease tumor growth, although they had no impact on animal survival. Moreover, the number of spontaneous lung metastasis were reduced in mice treated with SFV vectors expressing Gal3 inhibitors. Preliminary mechanistic studies showed an increase of CD3 cells infiltration in tumors treated with SFV-Gal3-N-C12 and SFV-Gal3-N vectors. Despite the antitumor effect observed with SFV-Gal3-N-C12, no protection against tumor rechallenge was observed in cured mice, indicating the lack or insufficient memory immune response generation. These data suggested that this therapeutic approach might benefit from combination with other immunodulatory strategies. We are currently characterizing the underpinnings of the mechanisms underlying this strategy.ConclusionsIn summary, we believe that inhibition of Gal3 using SFV vectors could constitute a potential approach to explore as therapy for pediatric osteosarcoma.ReferencesFarhad M, Rolig AS, Redmond WL. The role of Galectin-3 in modulating tumor growth and immunosuppression within the tumor microenvironment. Oncoimmunology 2018;7(6):p.e1434467.Ruvolo PP. Galectin 3 as a guardian of the tumor microenvironment. Biochim Biophys Acta 2016;1863(3):427–437.Guo Y, et al. Roles of galectin3 in the tumor microenvironment and tumor metabolism (Review). Oncol Rep 2020;44(5):1799–1809.Cao Z, et al. Endogenous and exogenous galectin-3 promote the adhesion of tumor cells with low expression of MUC1 to HUVECs through upregulation of N-cadherin and CD44. Lab Invest 2018;98(12):1642–1656.Kim SJ, Chun KH. Non-classical role of Galectin-3 in cancer progression: translocation to nucleus by carbohydrate-recognition independent manner. BMB Rep 2020;53(4):173–180.Pereira JX, et al. Galectin-3 regulates the expression of tumor glycosaminoglycans and increases the metastatic potential of breast cancer. J Oncol 2019;2019:9827147.Song M, et al. Galectin-3 favours tumour metastasis via the activation of beta-catenin signalling in hepatocellular carcinoma. Br J Cancer 2020;123(10):1521–1534.Fortuna-Costa A, et al. Extracellular galectin-3 in tumor progression and metastasis. Front Oncol 2014;4:138.Nakajima K, et al. Galectin-3 in bone tumor microenvironment: a beacon for individual skeletal metastasis management. Cancer Metastasis Rev 2016;35(2):333–46.Nakajima K, Balan V, Raz A. Galectin-3: an immune checkpoint target for musculoskeletal tumor patients. Cancer Metastasis Rev 2021;40(1):297–302.Lei P, et al. Small interfering RNA-induced silencing of galectin-3 inhibits the malignant phenotypes of osteosarcoma in vitro. Mol Med Rep 2015;12(4):6316–22.Park GB, et al. Silencing of galectin-3 represses osteosarcoma cell migration and invasion through inhibition of FAK/Src/Lyn activation and beta-catenin expression and increases susceptibility to chemotherapeutic agents. Int J Oncol 2015;46(1):185–94.Ketola A, et al. Oncolytic Semliki forest virus vector as a novel candidate against unresectable osteosarcoma. Cancer Res 2008;68(20):8342–50.Sun W, et al. G3-C12 peptide reverses galectin-3 from Foe to friend for active targeting cancer treatment. Mol Pharm 2015;12(11):4124–36.Sun W, et al. Two birds, one stone: dual targeting of the cancer cell surface and subcellular mitochondria by the galectin-3-binding peptide G3-C12. Acta Pharmacol Sin 2017;38(6):806–822.Ethics ApprovalAnimals were maintained under standard conditions, and all procedures were approved by the Institutional Ethical Committee (CEEA) in accordance with the guidelines of the University of Navarra, approval number 044-21.

BackgroundGlioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising strategy for these tumors. Our group constructed a new OV named Delta-24-ACT, which was based on the Delta-24-RGD platform armed with 4-1BB ligand (4-1BBL). In this study, we evaluated the antitumor effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor (ICI) in preclinical models of glioma.MethodsThe in vitro effect of Delta-24-ACT was characterized through analyses of its infectivity, replication and cytotoxicity by flow cytometry, immunofluorescence (IF) and MTS assays, respectively. The antitumor effect and therapeutic mechanism were evaluated in vivo using several immunocompetent murine glioma models. The tumor microenvironment was studied by flow cytometry, immunohistochemistry and IF.ResultsDelta-24-ACT was able to infect and exert a cytotoxic effect on murine and human glioma cell lines. Moreover, Delta-24-ACT expressed functional 4-1BBL that was able to costimulate T lymphocytes in vitro and in vivo. Delta-24-ACT elicited a more potent antitumor effect in GBM murine models than Delta-24-RGD, as demonstrated by significant increases in median survival and the percentage of long-term survivors. Furthermore, Delta-24-ACT modulated the tumor microenvironment, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by increases in the expression of Programmed Death 1 (PD-1) on T cells and Programmed Death-ligand 1 (PD-L1) on different myeloid cell populations. Because Delta-24-ACT did not induce an immune memory response in long-term survivors, as indicated by rechallenge experiments, we combined Delta-24-ACT with an anti-PD-L1 antibody. In GL261 tumor-bearing mice, this combination showed superior efficacy compared with either monotherapy. Specifically, this combination not only increased the median survival but also generated immune memory, which allowed long-term survival and thus tumor rejection on rechallenge.ConclusionsIn summary, our data demonstrated the efficacy of Delta-24-ACT combined with a PD-L1 inhibitor in murine glioma models. Moreover, the data underscore the potential to combine local immunovirotherapy with ICIs as an effective therapy for poorly infiltrated tumors.

BackgroundDiffuse Midline Gliomas (DMG) are aggressive pediatric brain tumors that arise in the brainstem of children between 5–10 years old. DMGs are the leading cause of pediatric death caused by a brain tumor, with a median survival of only 9 months.1 2 We have previously shown that the administration of the oncolytic adenovirus Delta-24-RGD is safe and lead to an increase in long-term survivors in murine models.3 4 In order to further increase the antitumor effect of Delta-24-RGD by boosting the immune response, we have constructed a new adenovirus, Delta-24-ACT, which incorporates the 4-1BBL (CD137L) into its backbone. 4-1BB is a costimulatory receptor that promotes the survival and expansion of activated T cells and NK cells and the generation and maintenance of memory CD8+ T cells, among other functions.5 6MethodsMurine and human DMG cell lines were used. 4-1BBL expression was assessed in infected cells by flow cytometry and immunofluorescence. Viral protein expression was measured by western blot, viral replication was analyzed using a method based on hexon detection and the oncolytic effect by MTS assay. For in vivo experiments, cells were injected in the pons of mice using a screw-guided system.7 A single administration of the adenovirus was injected intratumorally using the same procedure. The tumor immune populations were analyzed by flow cytometry.ResultsWe first confirmed by flow cytometry that DMG cells infected with Delta-24-ACT expressed 4-1BBL in their membrane in a dose-dependent manner. Afterwards, we analyzed the oncolytic effect of Delta-24-ACT in vitro. Delta-24-ACT was able to express viral early and late proteins in murine and human DMG cell lines and to replicate efficiently in human cells. In addition, the virus caused cell death in a dose-dependent manner. In vivo, Delta-24-ACT administration demonstrated to be safe and to produce a significant survival benefit in murine DMG models, obtaining 30–50% of long-term survivors depending on the model. More importantly, Delta-24-ACT generated immune memory, as long-term survivors were disease-free after cell rechallenge. On the other hand, we analyzed immune infiltration 7 or 10 days after the viral administration into the tumor and observed a significant increase of tumor infiltration in treated mice, which showed an activated state.ConclusionsDelta-24-ACT administration into DMG murine tumor models significantly increases the recruitment and activation of immune cells, which leads to long term survivors and immunological memory.ReferencesCooney T, Lane A, Bartels U, Bouffet E, Goldman S, Leary S, Foreman NK, Packer RJ, Broniscer A, Minturn JE, Shih C, Chintagumpala M, Hassall T, Gottardo NG, Dholaria H, Hoffman L, Chaney B, Baugh J, Doughman R, Leach JL, Jones BV, Fouladi M, Warren KE, Monje M. Contemporary survival endpoints: an International diffuse Intrinsic pontine glioma registry study. Neuro Oncol 2017;19(9):1279–1280.Grasso CS, Tang Y, Truffaux N, Berlow NE, Liu L, Debily MA, Quist MJ, Davis LE, Huang EC, Woo PJ, Ponnuswami A, Chen S, Johung TB, Sun W, Kogiso M, Du Y, Qi L, Huang Y, Hütt-Cabezas M, Warren KE, Le Dret L, Meltzer PS, Mao H, Quezado M, van Vuurden DG, Abraham J, Fouladi M, Svalina MN, Wang N, Hawkins C, Nazarian J, Alonso MM, Raabe EH, Hulleman E, Spellman PT, Li XN, Keller C, Pal R, Grill J, Monje M. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. Nat Med 2015;21(6):555–9.Martínez-Vélez N, Garcia-Moure M, Marigil M, González-Huarriz M, Puigdelloses M, Gallego Pérez-Larraya J, Zalacaín M, Marrodán L, Varela-Guruceaga M, Laspidea V, Aristu JJ, Ramos LI, Tejada-Solís S, Díez-Valle R, Jones C, Mackay A, Martínez-Climent JA, García-Barchino MJ, Raabe E, Monje M, Becher OJ, Junier MP, El-Habr EA, Chneiweiss H, Aldave G, Jiang H, Fueyo J, Patiño-García A, Gomez-Manzano C, Alonso MM. The oncolytic virus Delta-24-RGD elicits an antitumor effect in pediatric glioma and DIPG mouse models. Nat Commun 2019;10(1):2235.Garcia-Moure M, Gonzalez-Huarriz M, Labiano S, Guruceaga E, Bandres E, Zalacain M, Marrodan L, de Andrea C, Villalba M, Martinez-Velez N, Laspidea V, Puigdelloses M, Gallego Perez-Larraya J, Iñigo-Marco I, Stripecke R, Chan JA, Raabe EH, Kool M, Gomez-Manzano C, Fueyo J, Patiño-García A, Alonso MM. Delta-24-RGD, an oncolytic adenovirus, increases survival and promotes proinflamatory immune landscape remodeling in models of AT/RT and CNS-PNET. Clin Cancer Res 2021;27(6):1807–1820.Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1):49–57.Yonezawa A, Dutt S, Chester C, Kim J, Kohrt HE. Boosting cancer immunotherapy with anti-CD137 antibody therapy. Clin Cancer Res 2015;21(14):3113–20.Marigil M, Martinez-Velez N, Domínguez PD, Idoate MA, Xipell E, Patiño-García A, Gonzalez-Huarriz M, García-Moure M, Junier MP, Chneiweiss H, El-Habr E, Diez-Valle R, Tejada-Solís S, Alonso MM. Development of a DIPG orthotopic model in mice using an implantable guide-screw system. PLoS One 2017;12(1):e0170501.

BackgroundDiffuse Midline Gliomas (DMGs), encompassing Diffuse Intrinsic Pontine Gliomas (DIPGs), are the most aggressive pediatric brain tumors. Their meager survival has not changed despite the combination of radiotherapy with targeted therapies emphasizing the urgent need for effective treatments. Recent research suggested that the DIPG tumor microenvironment is neither highly immunosuppressive nor inflammatory.1 These analyses showed the lack of infiltrating lymphocytes and the abundance of CD11b+ cells. TIM-3 is a member of the T-cell immunoglobulin and mucin domain protein family expressed on multiple immune cell types, including T cells, Treg, NK cells, monocytes, dendritic cells, and microglia, where it potently regulates not only adaptive immunity but also innate immunity.2–3 Therefore, TIM-3 inhibitors could challenge several components in the tumor microenvironment, thereby providing potentially effective treatment for DMGs.MethodsNP53 and XFM murine DIPG cell lines were used for animal experiments in immunocompetent orthotopic models. The tumors were processed by mechanical and enzymatic digestion and immune populations were analyzed by a flow cytometry panel. Antibodies against NK cells (NK1.1), CD4 (GK1.5), CD8 (CD8β) were used for animal depletion experiments alone or in combination.ResultsIn silico assessment of TIM-3 expression in DIPG datasets showed a robust expression of this gene. Moreover, single-cell sequencing analyses of DIPG biopsies uncover its expression in the myeloid compartment (especially in microglia). In vivo efficacy studies showed that treatment with anti-TIM-3 antibody significantly increased the overall survival in two DIPG immunocompetent orthotopic animal models (doubling the median), lead to long-term survivors free of disease (50%) and showed immune memory. Analyses of CD45+ populations in the tumor microenvironment showed a significant increase in microglia, granulocytes, NK and CD8+ cells corresponding with a NK and T-cell activate phenotypes in treated-mice. In addition, we have a substantial decrease in the Treg population, which causes an increase in the CD8/Treg ratio. CD4 and CD8 T-cell depletion led to a significant but not total loss of treatment efficacy. NK cells depletion also reduced the effectiveness of this therapy, albeit to a lesser extent than CD4-CD8 depletion. We are currently investigating the role of microglia in the outcome of the treatment.ConclusionsOur data uncovered TIM-3 as a potential target for the treatment of DIPG tumors. Inhibition of this molecule led to a potent antitumor effect mediated by a profound tumor microenvironment remodelling.ReferencesLieberman NAP, DeGolier K, Kovar HM, et al. Characterization of the immune microenvironment of diffuse intrinsic pontine glioma: implications for development of immunotherapy. Neuro Oncol 2019;21(1):83–94. doi:10.1093/neuonc/noy145.Acharya N, Sabatos-Peyton C, Anderson AC. Tim-3 finds its place in the cancer immunotherapy landscape. J Immunother Cancer 2020;8(1):e000911. doi:10.1136/jitc-2020-000911.Wolf Y, Anderson AC, Kuchroo VK. TIM3 comes of age as an inhibitory receptor. Nat Rev Immunol 2020;20(3):173–185. doi:10.1038/s41577-019-0224-6

Cancer virotherapy is a paradigm-shifting treatment modality based on virus-mediated oncolysis and subsequent antitumor immune responses. Clinical trials of currently available virotherapies showed that robust antitumor immunity characterizes the remarkable and long-term responses observed in a subset of patients. These data suggest that future therapies should incorporate strategies to maximize the immunotherapeutic potential of oncolytic viruses. In this review, we highlight the recent evidence that the antiviral immunity of the patients may limit the immunotherapeutic potential of oncolytic viruses and summarize the most relevant approaches to strategically redirect the immune response away from the viruses and toward tumors to heighten the clinical impact of viro-immunotherapy platforms.