Justyna Kmiecik

Expression of the progenitor marker NG2/CSPG4 predicts poor survival and resistance to ionising radiation in glioblastoma

Glioblastoma (GBM) is a highly aggressive brain tumour, where patients respond poorly to radiotherapy and exhibit dismal survival outcomes. The mechanisms of radioresistance are not completely understood. However, cancer cells with an immature stem-like phenotype are hypothesised to play a role in radioresistance. Since the progenitor marker neuron-glial-2 (NG2) has been shown to regulate several aspects of GBM progression in experimental systems, we hypothesised that its expression would influence the survival of GBM patients. Quantification of NG2 expression in 74 GBM biopsies from newly diagnosed and untreated patients revealed that 50% express high NG2 levels on tumour cells and associated vessels, being associated with significantly shorter survival. This effect was independent of age at diagnosis, treatment received and hypermethylation of the O6-methylguanine methyltransferase (MGMT) DNA repair gene promoter. NG2 was frequently co-expressed with nestin and vimentin but rarely with CD133 and the NG2 positive tumour cells harboured genetic aberrations typical for GBM. 2D proteomics of 11 randomly selected biopsies revealed upregulation of an antioxidant, peroxiredoxin-1 (PRDX-1), in the shortest surviving patients. Expression of PRDX-1 was associated with significantly reduced products of oxidative stress. Furthermore, NG2 expressing GBM cells showed resistance to ionising radiation (IR), rapidly recognised DNA damage and effectuated cell cycle checkpoint signalling. PRDX-1 knockdown transiently slowed tumour growth rates and sensitised them to IR in vivo. Our data establish NG2 as an important prognostic factor for GBM patient survival, by mediating resistance to radiotherapy through induction of ROS scavenging enzymes and preferential DNA damage signalling.

Targeting the NG2/CSPG4 Proteoglycan Retards Tumour Growth and Angiogenesis in Preclinical Models of GBM and Melanoma

Aberrant expression of the progenitor marker Neuron-glia 2 (NG2/CSPG4) or melanoma proteoglycan on cancer cells and angiogenic vasculature is associated with an aggressive disease course in several malignancies including glioblastoma multiforme (GBM) and melanoma. Thus, we investigated the mechanism of NG2 mediated malignant progression and its potential as a therapeutic target in clinically relevant GBM and melanoma animal models. Xenografting NG2 overexpressing GBM cell lines resulted in increased growth rate, angiogenesis and vascular permeability compared to control, NG2 negative tumours. The effect of abrogating NG2 function was investigated after intracerebral delivery of lentivirally encoded shRNAs targeting NG2 in patient GBM xenografts as well as in established subcutaneous A375 melanoma tumours. NG2 knockdown reduced melanoma proliferation and increased apoptosis and necrosis. Targeting NG2 in two heterogeneous GBM xenografts significantly reduced tumour growth and oedema levels, angiogenesis and normalised vascular function. Vascular normalisation resulted in increased tumour invasion and decreased apoptosis and necrosis. We conclude that NG2 promotes tumour progression by multiple mechanisms and represents an amenable target for cancer molecular therapy.

NK Cells in Central Nervous System Disorders

NK cells are important players in immunity against pathogens and neoplasms. As a component of the innate immune system, they are one of the first effectors on sites of inflammation. Through their cytokine production capacities, NK cells participate in the development of a potent adaptive immune response. Furthermore, NK cells were found to have regulatory functions to limit and prevent autoimmunity via killing of autologous immune cells. These paradoxical functions of NK cells are reflected in CNS disorders. In this review, we discuss the phenotypes and functional features of peripheral and brain NK cells in brain tumors and infections, neurodegenerative diseases, acute vascular and traumatic damage, as well as mental disorders. We also discuss the implication of NK cells in neurotoxicity and neuroprotection following CNS pathology, as well as the crosstalk between NK cells and brain-resident immune cells.

Natural killer cells in intracranial neoplasms: presence and therapeutic efficacy against brain tumours.

Natural killer (NK) cells are lymphocytes that play an important role in anti-tumour immunity. Their potential against brain cancer has been demonstrated in vitro and in vivo, both as a direct anti-tumour agent and in experimental therapies stimulating endogenous NK cell cytotoxicity. However, the clinical translation of these promising results requires detailed knowledge about the immune status of brain tumour patients, with focus on the NK cell population. In this report, we provide an overview of the studies investigating NK cell infiltration into the tumour, emphasizing the need of revision of the methodologies and further research in this field. We also discuss the potential of using autologous or allogeneic NK cells as effector cells in cellular therapy against brain cancer and developing immunotherapies stimulating endogenous NK cell-mediated anti-tumour response, such as blocking inhibitory killer immunoglobulin-like receptors. Combination of NK cell adoptive transfer with targeted therapies, such as anti-EGFR therapeutic antibody (CetuximAb) could also be a potent strategy.

NK Cells with KIR2DS2 Immunogenotype Have a Functional Activation Advantage To Efficiently Kill Glioblastoma and Prolong Animal Survival

Glioblastomas (GBMs) are lethal brain cancers that are resistant to current therapies. We investigated the cytotoxicity of human allogeneic NK cells against patient-derived GBM in vitro and in vivo, as well as mechanisms mediating their efficacy. We demonstrate that KIR2DS2 immunogenotype NK cells were more potent killers, notwithstanding the absence of inhibitory killer Ig–like receptor (KIR)-HLA ligand mismatch. FACS-sorted and enriched KIR2DS2+ NK cell subpopulations retained significantly high levels of CD69 and CD16 when in contact with GBM cells at a 1:1 ratio and highly expressed CD107a and secreted more soluble CD137 and granzyme A. In contrast, KIR2DS2− immunogenotype donor NK cells were less cytotoxic against GBM and K562, and, similar to FACS-sorted or gated KIR2DS2− NK cells, significantly diminished CD16, CD107a, granzyme A, and CD69 when in contact with GBM cells. Furthermore, NK cell–mediated GBM killing in vitro depended upon the expression of ligands for the activating receptor NKG2D and was partially abrogated by Ab blockade. Treatment of GBM xenografts in NOD/SCID mice with NK cells from a KIR2DS2+ donor lacking inhibitory KIR-HLA ligand mismatch significantly prolonged the median survival to 163 d compared with vehicle controls (log-rank test, p = 0.0001), in contrast to 117.5 d (log-rank test, p = 0.0005) for NK cells with several inhibitory KIR-HLA ligand mismatches but lacking KIR2DS2 genotype. Significantly more CD56+CD16+ NK cells from a KIR2DS2+ donor survived in nontumor-bearing brains 3 wk after infusion compared with KIR2DS2− NK cells, independent of their proliferative capacity. In conclusion, KIR2DS2 identifies potent alloreactive NK cells against GBM that are mediated by commensurate, but dominant, activating signals.

Combining NK cells and mAb9.2.27 to combat NG2-dependent and anti-inflammatory signals in glioblastoma

Glioblastoma is a deadly brain cancer with limited treatment options. Targeting chondroitin sulfate proteoglycan 4 (CSPG4, best known as NG2) with the monoclonal antibody mAb9.2.27 and activated natural killer (NK) cells abrogated the tumor growth and prolonged the survival of glioblastoma-bearing animals by favoring the establishment of a pro-inflammatory microenvironment. The combination of NK cells and mAb9.2.27 recruited ED1+CCR2low macrophages that stimulated ED1+ED2lowMHCIIhigh microglial cells to exert robust cytotoxicity. Our findings demonstrate the therapeutic potential of targeting salient tumor associated-antigens.

Abstract 3542: Interplay between cancer and the immune system: Beneficial role of TILs and mechanisms of tumor immune escape in GBM patients

Glioblastoma multiforme (GBM) is the most malignant brain cancer and despite advances in present treatment regimen, the patients’ survival remains low, emphasizing importance of continued research to develop novel therapies. One of the promising treatment strategies is immunotherapy. However, detailed studies of the immune status of GBM patients are required to design appropriate immunotherapy tailored to these patients. Our preliminary characterization of 63 GBM patients’ biopsies using IHC staining for CD3, CD4 and CD8 markers revealed a great heterogeneity in the amounts of T-cell infiltrates between and within the patients’ tumors. The amounts of CD3 + , CD4 + and CD8 + T-cells were quantified and correlated with patients’ survival outcomes. Reduced CD3 + T-cell infiltration into the tumor ( 5% T-cells (p=0.0029). Similarly, significant (p=0.0062) positive correlation of the level of CD8 + cells infiltration with longer patients’ survival was observed. In further studies we focused on detailed characterization of tumor infiltrating and peripheral blood lymphocytes and based on these results we propose several mechanisms impairing the patients’ immune system. Within the tumor microenvironment immunosuppression is driven by increased expression of CD39 and CD73 molecules that leads to high levels of extracellular adenosine resulting in T-cell suppression. Additionally, we observed modulation of Fas expression on tumor cells and FasL concentration in patients’ plasma dependent on the immune cells’ status. This may result in either tumor9s escape from Fas-mediated cytotoxicity or apoptosis of the immune cells. A subpopulation of the patients had elevated CD8 + regulatory T-cells that are known to suppress APCs - a major immune cell population infiltrating the tumor. However, in contrast to other published reports, we didn9t detect CD4 + Tregs among TILs. In addition, we demonstrated a systemic immunosuppression due to decreased numbers of T-helper cells in the peripheral blood compared to healthy donors (p=0.0391) and their up-regulated expression of the inhibitory receptor CD152 (p=0.0039). Moreover, increased concentration of IL-10 (p=0.0039) and decreased concentration of IL-2 and IL-12 in patients’ plasma comparing to healthy donors’ indicates that the immune response balance is shifted towards Th2 anti-inflammatory response.In conclusion, our preliminary results showing that the increased T-cells infiltration to the tumor correlates with patients’ longer survival, suggest that there is a potential for developing immunotherapies against GBM. However, further studies revealed a large repertoire of immunosuppressive mechanisms developed by the tumor that need to be taken into consideration when designing an immunotherapy tailored to GBM patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3542. doi:1538-7445.AM2012-3542