Tina Verschuere

Dendritic Cell Therapy of High-Grade Gliomas

The prognosis of patients with malignant glioma is poor in spite of multimodal treatment approaches consisting of neurosurgery, radiochemotherapy and maintenance chemotherapy. Among innovative treatment strategies like targeted therapy, antiangiogenesis and gene therapy approaches, immunotherapy emerges as a meaningful and feasible treatment approach for inducing long‐term survival in at least a subpopulation of these patients. Setting up immunotherapy for an inherent immunosuppressive tumor located in an immune‐privileged environment requires integration of a lot of scientific input and knowledge of both tumor immunology and neuro‐oncology. The field of immunotherapy is moving into the direction of active specific immunotherapy using autologous dendritic cells (DCs) as vehicle for immunization. In the translational research program of the authors, the whole cascade from bench to bed to bench of active specific immunotherapy for malignant glioma is covered, including proof of principle experiments to demonstrate immunogenicity of patient‐derived mature DCs loaded with autologous tumor lysate, preclinical in vivo experiments in a murine orthotopic glioma model, early phase I/II clinical trials for relapsing patients, a phase II trial for patients with newly diagnosed glioblastoma (GBM) for whom immunotherapy is integrated in the current multimodal treatment, and laboratory analyses of patient samples. The strategies and results of this program are discussed in the light of the internationally available scientific literature in this fast‐moving field of basic science and translational clinical research.

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.

Galectin-1 in melanoma biology and related neo-angiogenesis processes.

Aggressiveness of advanced melanomas relates in part to their marked propensity to develop neoangiogenesis and metastases. Among its numerous pro-cancer roles, galectin (gal)-1 expressed and/or secreted by both cancer and endothelial cells stimulates proliferation and angiogenesis. This study first shows that gal-1 is more highly expressed at both mRNA and protein levels than its congeners in melanomas and particularly in advanced lesions. The roles of gal-1 were further investigated in vivo in the highly proliferating and vascularized pseudometastatic B16F10 mouse melanoma model using stable knockdown B16F10 cells and wild-type versus gal-1 knockout mice, and then in vitro in B16F10 tumoral and lung microvascular cells. Gal-1 depletion in the B16F10 tumor cells but not in the tumor-bearing mice significantly increased melanoma-bearing mice survival. Tumor-derived gal-1 thus seems to have more critical roles than the host-derived one. In fact, gal-1 displays distinct effects on the H-Ras-dependent p53/p21 pathways: in primary lung microvessel endothelial cells, gal-1 seems to be involved in the maintenance of senescent status through the induction of both p53 and p21 while it stimulates B16F10 cancer cell proliferation through a p53/p21 decrease. Altogether, these data point to gal-1 as a potential target to combat melanomas.

Glioma-derived galectin-1 regulates innate and adaptive antitumor immunity.

Galectin‐1 is a glycan‐binding protein, which is involved in the aggressiveness of glioblastoma (GBM) in part by stimulating angiogenesis. In different cancer models, galectin‐1 has also been demonstrated to play a pivotal role in tumor‐mediated immune evasion especially by modulating cells of the adaptive immune system. It is yet unknown whether the absence or presence of galectin‐1 within the glioma microenvironment also causes qualitative or quantitative differences in innate and/or adaptive antitumor immune responses. All experiments were performed in the orthotopic GL261 mouse high‐grade glioma model. Stable galectin‐1 knockdown was achieved via transduction of parental GL261 tumor cells with a lentiviral vector encoding a galectin‐1‐targeting miRNA. We demonstrated that the absence of tumor‐derived but not of host‐derived galectin‐1 significantly prolonged the survival of glioma‐bearing mice as such and in combination with dendritic cell (DC)‐based immunotherapy. Both flow cytometric and pathological analysis revealed that the silencing of glioma‐derived galectin‐1 significantly decreased the amount of brain‐infiltrating macrophages and myeloid‐derived suppressor cells (MDSC) in tumor‐bearing mice. Additionally, we revealed a pro‐angiogenic role for galectin‐1 within the glioma microenvironment. The data provided in this study reveal a pivotal role for glioma‐derived galectin‐1 in the regulation of myeloid cell accumulation within the glioma microenvironment, the most abundant immune cell population in high‐grade gliomas. Furthermore, the prolonged survival observed in untreated and DC‐vaccinated glioma‐bearing mice upon the silencing of tumor‐derived galectin‐1 strongly suggest that the in vivo targeting of tumor‐derived galectin‐1 might offer a promising and realistic adjuvant treatment modality in patients diagnosed with GBM.

Depletion of Regulatory T Cells in a Mouse Experimental Glioma Model through Anti-CD25 Treatment Results in the Infiltration of Non-Immunosuppressive Myeloid Cells in the Brain

The recruitment and activation of regulatory T cells (Tregs) in the micro-environment of malignant brain tumors has detrimental effects on antitumoral immune responses. Hence, local elimination of Tregs within the tumor micro-environment represents a highly valuable tool from both a fundamental and clinical perspective. In the syngeneic experimental GL261 murine glioma model, Tregs were prophylactically eliminated through treatment with PC61, an anti-CD25 mAb. This resulted in specific elimination of CD4+CD25hiFoxp3+ Treg within brain-infiltrating lymphocytes and complete protection against subsequent orthotopic GL261 tumor challenge. Interestingly, PC61-treated mice also showed a pronounced infiltration of CD11b+ myeloid cells in the brain. Phenotypically, these cells could not be considered as Gr-1+ myeloid-derived suppressor cells (MDSC) but were identified as F4/80+ macrophages and granulocytes.

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.

Galectin-1 and immunotherapy for brain cancer

The prognosis of patients diagnosed with high-grade glioma continues to be dismal in spite of multimodal treatment. Active specific immunotherapy by means of dendritic cell vaccination is considered to be a new promising concept that aims at generating an anti-tumoral immune response. However, it is now widely accepted that the success of immunotherapeutic strategies to promote tumor regression will rely not only on enhancing the effector arm of the immune response but also on downregulation of the counteracting tolerogenic signals. In this article, we summarize evidence that galectin-1, an evolutionarily conserved glycan-binding protein that is abundantly expressed in high-grade glioma, is an important player in glioma-mediated immune escape.

Immunosuppressive parameters in serum of ovarian cancer patients change during the disease course

ABSTRACT Neoplastic cells can escape immune control leading to cancer growth. Regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) are crucial in immune escape. TAM are divided based on their immune profile, M1 are immunostimulatory while M2 are immunosuppressive. Research so far has mainly focused on the intratumoral behavior of these cells. This study, on the other hand, explored the systemic changes of the key metabolites [IL-4 (interleukin), IL-13, arginase, IL-10, VEGF-A (vascular endothelial growth factor), CCL-2 (chemokine (C-C) motif ligand 2) and TGF-β (transforming growth factor)] linked to Treg, MDSC and TAM during the course of the disease in ovarian and fallopian tube cancer patients. Serum samples were therefore analyzed at diagnosis, after (interval)-debulking surgery and after chemotherapy (paclitaxel–carboplatin). We also determined galectin-1 (gal-1), involved in angiogenesis and tumor-mediated immune evasion. We found significantly lower levels of IL-10, VEGF-A, TGF-β and arginase and higher levels of gal-1 after chemotherapy compared to diagnosis. After debulking surgery, a decrease in IL-10 was significant. Gal-1 and CCL-2 appeared independent prognostic factors for progression-free and overall survival (OS) (multivariate analysis). These results will help us in the decision making of future therapies in order to further modulate the immune system in a positive way.

Characterization of PD-1 upregulation on tumor-infiltrating lymphocytes in human and murine gliomas and preclinical therapeutic blockade

Blockade of the immune checkpoint molecule programmed‐cell‐death‐protein‐1 (PD‐1) yielded promising results in several cancers. To understand the therapeutic potential in human gliomas, quantitative data describing the expression of PD‐1 are essential. Moreover, due the immune‐specialized region of the brain in which gliomas arise, differences between tumor‐infiltrating and circulating lymphocytes should be acknowledged. In this study we have used flow cytometry to quantify PD‐1 expression on tumor‐infiltrating T cells of 25 freshly resected glioma cell suspensions (10 newly and 5 relapsed glioblastoma, 10 lower grade gliomas) and simultaneously isolated circulating T cells. A strong upregulation of PD‐1 expression in the tumor microenvironment compared to the blood circulation was seen in all glioma patients. Additionally, circulating T cells were isolated from 15 age‐matched healthy volunteers, but no differences in PD‐1 expression were found compared to glioma patients. In the murine GL261 malignant glioma model, there was a similar upregulation of PD‐1 on brain‐infiltrating lymphocytes. Using a monoclonal PD‐1 blocking antibody, we found a marked prolonged survival with 55% of mice reaching long‐term survival. Analysis of brain‐infiltrating cells 21 days after GL261 tumor implantation showed a shift in infiltrating lymphocyte subgroups with increased CD8+ T cells and decreased regulatory T cells. Together, our results suggest an important role of PD‐1 in glioma‐induced immune escape, and provide translational evidence for the use of PD‐1 blocking antibodies in human malignant gliomas.

Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth

Glioma growth and progression are characterized by abundant development of blood vessels that are highly aberrant and poorly functional, with detrimental consequences for drug delivery efficacy. The mechanisms driving this vessel dysmorphia during tumor progression are poorly understood. Using longitudinal intravital imaging in a mouse glioma model, we identify that dynamic sprouting and functional morphogenesis of a highly branched vessel network characterize the initial tumor growth, dramatically changing to vessel expansion, leakage, and loss of branching complexity in the later stages. This vascular phenotype transition was accompanied by recruitment of predominantly pro‐inflammatory M1‐like macrophages in the early stages, followed by in situ repolarization to M2‐like macrophages, which produced VEGF‐A and relocate to perivascular areas. A similar enrichment and perivascular accumulation of M2 versus M1 macrophages correlated with vessel dilation and malignancy in human glioma samples of different WHO malignancy grade. Targeting macrophages using anti‐CSF1 treatment restored normal blood vessel patterning and function. Combination treatment with chemotherapy showed survival benefit, suggesting that targeting macrophages as the key driver of blood vessel dysmorphia in glioma progression presents opportunities to improve efficacy of chemotherapeutic agents. We propose that vessel dysfunction is not simply a general feature of tumor vessel formation, but rather an emergent property resulting from a dynamic and functional reorganization of the tumor stroma and its angiogenic influences.