Carolien Koks

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.

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

Combining an immunogenic cell death inducer with dendritic cell immunotherapy treats high-grade glioma in a preclinical model. DAMPening Glioma Dendritic cell (DC)–based vaccines have shown promise for treating high-grade glioma (HGG), but efficacy has been limited by antigenic heterogeneity of the tumors. Now, Garg et al. combine DC vaccines with hypericin-based photodynamic therapy–induced immunogenic cell death (ICD) to treat HGG in an animal model. They found that ICD-based DC vaccines improved survival, and that this effect was dependent on the cell-associated reactive oxygen species and release of damage-associated molecular patterns (DAMPs) acting as danger signals. These ICD-based DC vaccines synergized with standard-of-care therapy to further improve survival in HGG-bearing mice and shifted the tumor immune signature from regulatory to TH1/TH17, which is associated with positive outcome in patients. 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 (TH1) immunity–biased, next-generation, DC immunotherapy. To this end, we combined DC immunotherapy with immunogenic cell death (ICD; a modality shown to induce TH1 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 TH1/cytotoxic T lymphocyte/TH17 cells. Analysis of the The Cancer Genome Atlas glioblastoma cohort confirmed that increased intratumor prevalence of TH1/cytotoxic T lymphocyte/TH17 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.

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

ABSTRACT 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.

Immune Suppression during Oncolytic Virotherapy for High-Grade Glioma; Yes or No?

Oncolytic viruses have been seriously considered for glioma therapy over the last 20 years. The oncolytic activity of several oncolytic strains has been demonstrated against human glioma cell lines and in in vivo xenotransplant models. So far, four of these stains have additionally completed the first phase I/II trials in relapsed glioma patients. Though safety and feasibility have been demonstrated, therapeutic efficacy in these initial trials, when described, was only minor. The role of the immune system in oncolytic virotherapy for glioma remained much less studied until recent years. When investigated, the immune system, adept at controlling viral infections, is often hypothesized to be a strong hurdle to successful oncolytic virotherapy. Several preclinical studies have therefore aimed to improve oncolytic virotherapy efficacy by combining it with immune suppression or evasion strategies. More recently however, a new paradigm has developed in the oncolytic virotherapy field stating that oncolytic virus-mediated tumor cell death can be accompanied by elicitation of potent activation of innate and adaptive anti-tumor immunity that greatly improves the efficacy of certain oncolytic strains. Therefore, it seems the three-way interaction between oncolytic virus, tumor and immune system is critical to the outcome of antitumor therapy. In this review we discuss the studies which have investigated how the immune system and oncolytic viruses interact in models of glioma. The novel insights generated here hold important implications for future research and should be incorporated into the design of novel clinical trials.

Irradiation of necrotic tumor cells used to pulse dendritic cells (DCs) potentiates DC vaccine-induced anti-tumor immunity in a mouse model of high-grade glioma

The prognosis of high-grade glioma (HGG) is poor despite advancements in neurosurgery, radio-and chemotherapy. DC-based immunotherapy has emerged as a promising and feasible treatment approach due to its high degree of selectivity and its ability to induce an antigen-specific immune-memory response. Our research group investigates the efficacy of vaccinating both primary and relapsed HGG patients with DC vaccines pulsed with autologous whole tumor lysate. The method of preparing autologous whole tumor lysate is, however, not standardized yet with most groups applying multiple freeze-thaw (FT) cycles to induce necrosis of tumor cells. As irradiation is known to induce oxidation-associated molecular patterns (OAMPs) like oxidized or carbonylated proteins, potent enablers of danger signaling, we hypothesized that irradiation could increase the immunogenicity of FT-treated necrotic tumor cells used to pulse DC vaccines in the context of HGG.