Targeting the anti-inflammatory interplay promoting glioblastoma progression with combined natural killer cells and mab9.2.27 against NG2/CSPG4. A novel strategy to reveal glioblastoma to the immune system
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Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults. The patients’ median survival is only 14.6 months despite multimodal treatment, including surgery and concomitant radiation and chemotherapy. Thus, there is a stark need for the development of new, potent therapeutic strategies and targets. For a decade our group investigated the implication of the cell surface neuroglial-2 proteoglycan, NG2/CSPG4, in the development and growth of glioma. These studies revealed that NG2/CSPG4 is critical in multiple mechanisms that favor tumor survival and expansion. Indeed, GBM cells expressing NG2/CSPG4 were characterized to have a higher proliferation rate and migratory capacity in vitro. In addition, in vivo implantation of GBM cell lines expressing this proteoglycan resulted in a bigger tumor mass, with a higher neovascularization compared to its negative counterpart. Furthermore, these NG2/CSPG4 positive neoplastic cells were more resistant to chemotherapy and radiotherapy in vitro and in vivo. We further demonstrated that 50 % of GBM patients expressed NG2/CSPG4, and that this expression independently correlated with a poorer survival. Taken together, this proteoglycan might be an amenable target for immunotherapy. On the other hand, our research team has high expertise on natural killer (NK) cell biology. We observed that despite the promising results described for NK cell based immunotherapy to treat hematological malignancies, there is sparse data on their use to treat GBM. The main goal of this thesis was the study and the validation of a novel combination treatment using mAb9.2.27 targeted against NG2/CSPG4 and activated NK cells to treat GBM in rat orthotopic xenograft models. Thus, we first investigated the presence and phenotype of NK cells in the healthy brain. We further characterized the immunological status of GBM patients, both within the tumor microenvironment and their peripheral blood. Then we designed a novel purification method to obtain highly pure “untouched” NK cells from rat. This last method was developed to be able to finally investigate the efficacy of the combined NK+mAb9.2.27 treatment in GBM -bearing rats. All this work led to 4 original publications. The first publication of this thesis revealed that NK cells could be found in the brain of naïve mice as well as in the cerebrospinal fluid obtained from non-pathological brain of patients. Phenotypically, brain NK cells displayed immature CD11blowCD27+ phenotypes in mice and they were mostly CD56bright in patients. In this publication we discussed these new findings in relation to the existing literature on systemic and brain NK cells in the context of central nervous system (CNS) disorders, such as brain tumors, infections, neurodegenerative diseases and mental disorders. We observed that the knowledge on brain NK cells is limited in the context of CNS disorders, though more information about the presence and role of NK cells in the peripheral blood of patients with such disorders is readily available. Nevertheless, this work distinguished that NK cells could be either implicated in neuroprotection or neurodegeneration. This review highlights the potential of a better comprehension of NK cells in brain pathogenesis that could further help to delineate new therapeutic targets for the treatment of CNS disorders. In the second paper, we performed the characterization of the immunological status of GBM patients, in order to delineate the interplay between the immune system and tumor that can favor tumor growth. Immunohistochemistry staining on 65 GBM biopsies revealed that the patients’ survival correlated with their infiltration by CD3+ cells as well as CD8+ cells. We further observed that following tumor infiltration the T lymphocytes as well as antigen presenting cells down modulated their activation molecules and up regulated their inhibitory molecules. Furthermore, we characterized for the first time the presence of infiltrated regulatory T cells with CD8+CD28-Foxp3+ phenotype that may further propagate the anti-inflammatory environment created by the tumor. Despite these findings, further analyses indicated potential for immunomodulatory therapies for GBM management. The third paper of this thesis detailed a novel protocol to obtain highly pure “untouched” rat NK cells, based on magnetic-bead purification method. As currently no commercial kits to get untouched rat NK cells exist, many research teams used positive selection to purify them. Our method permits the separation of NK cells from athymic nude, Lewis and Fisher rat strains with high purity, ease, and cost affectivity and without bias of method-related activation artifacts. Indeed, we observed that positive selection of NK cells modified their proliferative and functional capacities. We conclude that for future fundamental studies, negative purification should be preferred to limit activation bias induced by separation methods. This method was further applied to purify NK cells in order to investigate their efficacy as monotherapy or in combination with mAb9.2.27 against NG2/CSPG4 as a new strategy to treat GBM. This NK+mAb9.2.27 treatment was evaluated in several orthotropic models of rats bearing GBM tumors. We observed that this combined therapy prolonged animal survival compared to monotherapy controls. This was associated with a diminution of tumor mass, associated with increased cellular apoptosis and diminished proliferation in the tumor bed. The levels of pro-inflammatory IFN-g and TNF-a, were increased in the brain of treated animals, while in contrast anti-inflammatory IL-10 molecules were reduced. We further identified tumor recruited pro-inflammatory macrophages as the mechanism that mediated the anti-tumor immune responses as their depletion by clodronate abolished tumor destruction. All together, we propose that targeting the anti-inflammatory interplay promoting GBM progression with combined NK cells and mAb9.2.27 against NG2/CSPG4 could be amenable to treat GBM patients, by reversing the anti-inflammatory tumor microenvironment to a proinflammatory one in order to reveal GBM to the immune system.
Has partsPaper I: NK Cells in Central Nervous System Disorders. Poli A, Kmiecik J, Domingues O, Hentges F, Bléry M, Chekenya M, Boucraut J and Zimmer J. J Immunol. 2013 Jun 1;190(11):5355-62. The article is not available in BORA due to publisher restrictions. The published version is available at: http://www.jimmunol.org/content/190/11/5355
Paper II: Elevated CD3+ and CD8+ tumor–infiltrating immune cells correlate with prolonged survival in glioblastoma patients despite integrated immunosuppressive mechanisms in the tumor and peripheral microenvironment. Kmiecik J, Poli A, Brons NHC, Waha A, Eide G, Enger PO, Zimmer J and Chekenya M. J Neuroimmunol. 2013 Nov 15;264(1-2):71-83. The article is available at: http://hdl.handle.net/1956/7668
Paper III: Novel method for isolating untouched rat natural killer cells with higher purity compared with positive selection and fluorescence-activated cell sorting. Poli A, Brons NH, Ammerlaan W, Michel T, Hentges F, Chekenya M, Zimmer J. Immunology. 2010 Nov;131(3):386-94. The article is available at: http://hdl.handle.net/1956/7865
Paper IV: Targeting glioblastoma with NK cells and mAb against NG2/CSPG4 prolongs animal survival. Poli A, Wang J, Domingues O, Rygh CB, Yan T, Thorsen F, Planagumà J, McCormack E, Hentges F, Pedersen PH, Zimmer J, Enger PO and Chekenya M. Oncotarget. 2013 Sep;4(9):1527-46. The article is available at: http://hdl.handle.net/1956/7864