Immunogenic oxidative therapy-based next-generation dendritic cell vaccines against high-grade glioma

High-grade glioma (HGG) represents a highly aggressive central nervous system (CNS) malignancy with mortality rates matching incidence rates. Dendritic cell vaccines aiming to stimulate the endogenous antiglioma immune response constitute a safe and promising treatment modality. Thus far, the efficacy of DC vaccines is considered suboptimal, in part owing to a lack of attention towards the immunogenicity of the tumor lysate used as an antigen source for loading the DC vaccines. Given the recent evidence that oxidation and the resultant oxidation-associated molecular patterns (OAMPs) can impact tumor cell immunogenicity, we applied two physico-chemical oxidizing therapies to obtain a more immunogenic tumor cell cargo: x-ray irradiation and hypericin-based photodynamic therapy (Hyp-PDT). DC vaccine immunogenicity was investigated in the orthotopic, syngeneic GL261 HGG model. Mice prophylactically immunized with DC vaccines loaded with x-ray irradiated freeze-thawed lysates survived significantly longer as compared to mice vaccinated with DCs loaded with freeze-thaw lysate only. This was associated, both in prophylactic and curative vaccination setups, with increased intrabrain infiltration of T helper-1 cells (Th1)/Cytotoxic T lymphocytes (CTLs) and reduced infiltration of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). Through further application of anti-oxidants and hydrogenperoxide, we found a striking correlation between the amount of lysate-associated protein carbonylation/OAMPs and the capacity of the DC vaccines to mediate glioma rejection. This result fostered the preclinical investigation of other strong oxidizers like Hyp-PDT, a potent type II inducer of ICD, in our HGG model. Hyp-PDT induced prototypical ICD in the GL261 cell line. In prophylactic as well as curative vaccination settings, both biologically and clinically-relevant versions of Hyp-PDT-based ICD-based DC vaccines provided strong anti-HGG survival benefit. The ability of DC vaccines to elicit HGG rejection was significantly blunted if cancer cell-associated reactive oxygen species (ROS) and emanating danger signals were blocked (either singly or concomitantly; showing hierarchical effect on immunogenicity i.e. extracellular-HMGB1>extracellular-ATP>ecto-calreticulin) or if DCs, the DCs-associated MyD88 signal or the adaptive immune system (especially CD8+ T cells) were depleted. In a curative setting, ICD-based DC vaccines synergized with standard-of-care chemotherapy (temozolomide) to increase survival of HGG-bearing mice by ~300% resulting in ~50% long-term survivors. Additionally, DC vaccines also induced an immunostimulatory shift in the brain immune-contexture from Tregs to Th1/CTLs/Th17 cells. Analysis of the TCGA glioblastoma-cohort confirmed that increased intra-tumor prevalence of Th1/CTLs/Th17 cells-linked genetic signatures associated with good patient prognosis. Our research demonstrates that combining DC vaccination with oxidizing treatment modalities can induce Th1-driven antitumor immune responses, enabling CTL-mediated glioma rejection. ICD-based DC vaccines exploiting ER-directed oxidative stress might be considered next generation DC vaccines.nrpages: 161status: publishe

Dendritic cell vaccines based on immunogenic cell death elicit danger signals and T cell-driven rejection of high-grade glioma

The promise of dendritic cell (DC)-based immunotherapy has been established by two decades of translational research. Of the four malignancies most targeted with clinical DC immunotherapy, high-grade glioma (HGG) has shown the highest susceptibility. HGG-induced immunosuppression is a roadblock to immunotherapy, but may be overcome by the application of T helper 1 (T(H)1) immunity-biased, next-generation, DC immunotherapy. To this end, we combined DC immunotherapy with immunogenic cell death (ICD; a modality shown to induce T(H)1 immunity) induced by hypericin-based photodynamic therapy. In an orthotopic HGG mouse model involving prophylactic/curative setups, both biologically and clinically relevant versions of ICD-based DC vaccines provided strong anti-HGG survival benefit. We found that the ability of DC vaccines to elicit HGG rejection was significantly blunted if cancer cell-associated reactive oxygen species and emanating danger signals were blocked either singly or concomitantly, showing hierarchical effect on immunogenicity, or if DCs, DC-associated MyD88 signal, or the adaptive immune system (especially CD8(+) T cells) were depleted. In a curative setting, ICD-based DC vaccines synergized with standard-of-care chemotherapy (temozolomide) to increase survival of HGG-bearing mice by ~300%, resulting in ~50% long-term survivors. Additionally, DC vaccines also induced an immunostimulatory shift in the brain immune contexture from regulatory T cells to T(H)1/cytotoxic T lymphocyte/T(H)17 cells. Analysis of the The Cancer Genome Atlas glioblastoma cohort confirmed that increased intratumor prevalence of T(H)1/cytotoxic T lymphocyte/T(H)17 cells linked genetic signatures was associated with good patient prognosis. Therefore, pending final preclinical checks, ICD-based vaccines can be clinically translated for glioma treatment.status: publishe

Preclinical efficacy of immune-checkpoint monotherapy does not recapitulate corresponding biomarkers-based clinical predictions in glioblastoma

Glioblastoma (GBM) is resistant to most multimodal therapies. Clinical success of immune-checkpoint inhibitors (ICIs) has spurred interest in applying ICIs targeting CTLA4, PD1 or IDO1 against GBM. This amplifies the need to ascertain GBM's intrinsic susceptibility (or resistance) toward these ICIs, through clinical biomarkers that may also "guide and prioritize" preclinical testing. Here, we interrogated the TCGA and/or REMBRANDT human patient-cohorts to predict GBM's predisposition toward ICIs. We exploited various broad clinical biomarkers, including mutational or predicted-neoantigen burden, pre-existing or basal levels of tumor-infiltrating T lymphocytes (TILs), differential expression of immune-checkpoints within the tumor and their correlation with particular TILs/Treg-associated functional signature and prognostic impact of differential immune-checkpoint expression. Based on these analyses, we found that predictive biomarkers of ICI responsiveness exhibited inconsistent patterns in GBM patients, i.e., they either predicted ICI resistance (as compared with typical ICI-responsive cancer-types like melanoma, lung cancer or bladder cancer) or susceptibility to therapeutic targeting of CTLA4 or IDO1. On the other hand, our comprehensive literature meta-analysis and preclinical testing of ICIs using an orthotopic GL261-glioma mice model, indicated significant antitumor properties of anti-PD1 antibody, whereas blockade of IDO1 or CTLA4 either failed or provided very marginal advantage. These trends raise the need to better assess the applicability of ICIs and associated biomarkers for GBM.peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=koni20status: publishe

Irradiation of necrotic cancer cells, employed for pulsing dendritic cells (DCs), potentiates DC vaccine-induced antitumor immunity against high-grade glioma

Dendritic cell (DC)-based immunotherapy has yielded promising results against high-grade glioma (HGG). However, the efficacy of DC vaccines is abated by HGG-induced immunosuppression and lack of attention toward the immunogenicity of the tumor lysate/cells used for pulsing DCs. A literature analysis of DC vaccination clinical trials in HGG patients delineated the following two most predominantly applied methods for tumor lysate preparation: freeze-thaw (FT)-induced necrosis or FT-necrosis followed by X-ray irradiation. However, from the available clinical evidence, it is unclear which of both methodologies has superior immunogenic potential. Using an orthotopic HGG murine model (GL261-C57BL/6), we observed that prophylactic vaccination with DCs pulsed with irradiated FT-necrotic cells (compared to FT-necrotic cells only) prolonged overall survival by increasing tumor rejection in glioma-challenged mice. This was associated, both in prophylactic and curative vaccination setups, with an increase in brain-infiltrating Th1 cells and cytotoxic T lymphocytes (CTL), paralleled by a reduced accumulation of regulatory T cells, tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC). Further analysis showed that irradiation treatment of FT-necrotic cells considerably increased the levels of carbonylated proteins - a surrogate-marker of oxidation-associated molecular patterns (OAMPs). Through further application of antioxidants and hydrogen peroxide, we found a striking correlation between the amount of lysate-associated protein carbonylation/OAMPs and DC vaccine-mediated tumor rejection capacity thereby suggesting for the first time a role for protein carbonylation/OAMPs in at least partially mediating antitumor immunity. Together, these data strongly advocate the use of protein oxidation-inducing modalities like irradiation for increasing the immunogenicity of tumor lysate/cells used for pulsing DC vaccines.peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=koni20status: publishe

Preclinical efficacy of immune-checkpoint monotherapy does not recapitulate corresponding biomarkers-based clinical predictions in glioblastoma

Glioblastoma (GBM) is resistant to most multimodal therapies. Clinical success of immune-checkpoint inhibitors (ICIs) has spurred interest in applying ICIs targeting CTLA4, PD1 or IDO1 against GBM. This amplifies the need to ascertain GBM's intrinsic susceptibility (or resistance) toward these ICIs, through clinical biomarkers that may also “guide and prioritize” preclinical testing. Here, we interrogated the TCGA and/or REMBRANDT human patient-cohorts to predict GBM's predisposition toward ICIs. We exploited various broad clinical biomarkers, including mutational or predicted-neoantigen burden, pre-existing or basal levels of tumor-infiltrating T lymphocytes (TILs), differential expression of immune-checkpoints within the tumor and their correlation with particular TILs/Treg-associated functional signature and prognostic impact of differential immune-checkpoint expression. Based on these analyses, we found that predictive biomarkers of ICI responsiveness exhibited inconsistent patterns in GBM patients, i.e. they either predicted ICI resistance (as compared with typical ICI-responsive cancer-types like melanoma, lung cancer or bladder cancer) or susceptibility to therapeutic targeting of CTLA4 or IDO1. On the other hand, our comprehensive literature meta-analysis and preclinical testing of ICIs using an orthotopic GL261-glioma mice model, indicated significant antitumor properties of anti-PD1 antibody, whereas blockade of IDO1 or CTLA4 either failed or provided very marginal advantage. These trends raise the need to better assess the applicability of ICIs and associated biomarkers for GBM.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Newcastle disease virotherapy induces long-term survival and tumor-specific immune memory in orthotopic glioma through the induction of immunogenic cell death

The oncolytic features of several naturally oncolytic viruses have been shown on Glioblastoma Multiforme cell lines and in xenotransplant models. However, orthotopic glioma studies in immunocompetent animals are lacking. Here we investigated Newcastle disease virus (NDV) in the orthotopic, syngeneic murine GL261 model. Seven days after tumor induction, mice received NDV intratumorally. Treatment significantly prolonged median survival and 50% of animals showed long-term survival. We demonstrated immunogenic cell death (ICD) induction in GL261 cells after NDV infection, comprising calreticulin surface exposure, release of HMGB1 and increased PMEL17 cancer antigen expression. Uniquely, we found absence of secreted ATP. NDV-induced ICD occurred independently of caspase signaling and was blocked by Necrostatin-1, suggesting the contribution of necroptosis. Autophagy induction following NDV infection of GL261 cells was demonstrated as well. In vivo, elevated infiltration of IFN-γ(+) T cells was observed in NDV-treated tumors, along with reduced accumulation of myeloid derived suppressor cells. The importance of a functional adaptive immune system in this paradigm was demonstrated in immunodeficient Rag2(-/-) mice and in CD8(+) T cell depleted animals, where NDV slightly prolonged survival, but failed to induce long-term cure. Secondary tumor induction with GL261 cells or LLC cells in mice surviving long-term after NDV treatment, demonstrated the induction of a long-term, tumor-specific immunological memory response by ND virotherapy. For the first time, we describe the therapeutic activity of NDV against GL261 tumors, evidenced in an orthotopic mouse model. The therapeutic effect relies on the induction of ICD in the tumor cells, which primes adaptive antitumor immunity. © 2014 Wiley Periodicals, Inc.status: publishe

Pathogen response-like recruitment and activation of neutrophils by sterile immunogenic dying cells drives neutrophil-mediated residual cell killing

Innate immune sensing of dying cells is modulated by several signals. Inflammatory chemokines-guided early recruitment, and pathogen-associated molecular patterns-triggered activation, of major anti-pathogenic innate immune cells like neutrophils distinguishes pathogen-infected stressed/dying cells from sterile dying cells. However, whether certain sterile dying cells stimulate innate immunity by partially mimicking pathogen response-like recruitment/activation of neutrophils remains poorly understood. We reveal that sterile immunogenic dying cancer cells trigger (a cell autonomous) pathogen response-like chemokine (PARC) signature, hallmarked by co-release of CXCL1, CCL2 and CXCL10 (similar to cells infected with bacteria or viruses). This PARC signature recruits preferentially neutrophils as first innate immune responders in vivo (in a cross-species, evolutionarily conserved manner; in mice and zebrafish). Furthermore, key danger signals emanating from these dying cells, that is, surface calreticulin, ATP and nucleic acids stimulate phagocytosis, purinergic receptors and toll-like receptors (TLR) i.e. TLR7/8/9-MyD88 signaling on neutrophil level, respectively. Engagement of purinergic receptors and TLR7/8/9-MyD88 signaling evokes neutrophil activation, which culminates into H2O2 and NO-driven respiratory burst-mediated killing of viable residual cancer cells. Thus sterile immunogenic dying cells perform 'altered-self mimicry' in certain contexts to exploit neutrophils for phagocytic targeting of dead/dying cancer cells and cytotoxic targeting of residual cancer cells.status: publishe