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Human cytomegalovirus (HCMV) antigens in glioblastoma (GBM) present opportunities for personalised immunotherapy. However, their presence in GBM tissue is still under debate, and evidence of their impact on functional immune responses and prognosis is sparse. Here, we investigated the presence of pp65 (UL83) and immediate early 1 (IE-1) HCMV antigens in a cohort of Norwegian GBM patients (n= 177), using qPCR, immunohistochemistry, and serology. HCMV status was then used to investigate whether viral antigens influenced immune cell phenotype, infiltration, activation and patient survival. Pp65 and IE-1 were detected by qPCR in 23% and 43% of GBM patients, respectively. Furthermore, there was increased seropositivity in GBM patients relative to donors (79%vs. 48%, respectively; Logistic regression, OR = 4.05, 95%CI [1.807-9.114],P= 0.001, also when adjusted for age (OR = 2.84, 95%CI [1.110-7.275],P= 0.029). Tissue IE-1-positivity correlated with increased CD3+CD8+T-cell infiltration (P< 0.0001), where CD8+effector memory T (TEM) cells accounted for the majority of CD8+T cells compared with peripheral blood of HCMV+patients (P< 0.0001), and HCMV+(P< 0.001) and HCMV−(P< 0.001) donors. HLA-A2/B8-restricted HCMV-specific CD8+T cells were more frequent in blood and tumor of HCMV+GBM patients compared with seronegative patients, and donors irrespective of their serostatus. In biopsies, the HCMV-specific CD8+TEMcells highly expressed CTLA-4 and PD-1 immune checkpoint protein markers compared with populations in peripheral blood (P< 0.001 andP< 0.0001), which expressed 3-fold greater levels of CD28 (P< 0.001 andP< 0.0001). These peripheral blood T cells correspondingly secreted higher levels of IFNγ in response to pp65 and IE-1 peptide stimulation (P< 0.001). Thus, despite apparent increased immunogenicity of HCMV compared with tumor antigens, the T cells were tolerised, and HCMV status did not impact patient survival (Log Rank3.53HR = 0.85 95%CI [0.564-1.290],P =0.45). Enhancing immune functionality in the tumor microenvironment thus may improve patient outcome. [ABSTRACT FROM AUTHOR]

Abstract Background The tumor microenvironment is pivotal in tumor progression. Thus, we aimed to develop a mammary tumor model to elucidate molecular characteristics in the stroma versus the tumor cell compartment by global gene expression. Secondly, since tumor hypoxia influences several aspects of tumor pathophysiology, we hypothesized that hyperoxia might have an inhibitory effect on tumor growth per se. Finally, we aimed to identify differences in gene expression and key molecular mechanisms, both in the native state and following treatment. Methods 4T1 dsRed breast cancer cells were injected into eGFP expressing NOD/SCID mice. Group 1 was exposed to 3 intermittent HBO treatments (Day 1, 4 and 7), Group 2 to 7 daily HBO treatments (both 2.5bar, 100% O2, à 90 min), whereas the controls were exposed to a normal atmosphere. Tumor growth, histology, vascularisation, cell proliferation, cell death and metastasis were assessed. Fluorescence-activated cell sorting was used to separate tumor cells from stromal cells prior to gene expression analysis. Results The purity of sorted cells was verified by fluorescence microscopy. Gene expression profiling demonstrated that highly expressed genes in the untreated tumor stroma included constituents of the extracellular matrix and matrix metalloproteinases. Tumor growth was significantly inhibited by HBO, and the MAPK pathway was found to be significantly reduced. Immunohistochemistry indicated a significantly reduced microvessel density after intermittent HBO, whereas daily HBO did not show a similar effect. The anti-angiogenic response was reflected in the expression trends of angiogenic factors. Conclusions The present in vivo mammary tumor model enabled us to separate tumor and stromal cells, and demonstrated that the two compartments are characterized by distinct gene expressions, both in the native state and following HBO treatments. Furthermore, hyperoxia induced a significant tumor growth-inhibitory effect, with significant down-regulation of the MAPK pathway. An anti-angiogenic effect after intermittent HBO was observed, and reflected in the gene expression profile.

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults and one of the most aggressive cancers in man. Despite technological advances in surgical management, combined regimens of radiotherapy with new generation chemotherapy, the median survival for these patients is 14.6 months. This is largely due to a highly deregulated tumour genome with opportunistic deletion of tumour suppressor genes, amplification and/or mutational hyper-activation of receptor tyrosine kinase receptors. The net result of these genetic changes is augmented survival pathways and systematic defects in the apoptosis signalling machinery. The only randomised, controlled phase II trial conducted targeting the epidermal growth factor receptor (EGFR) signalling with the small molecule inhibitor, erlotinib, has showed no therapeutic benefit. Survival signalling and apoptosis resistance in GBMs can be viewed as two sides of the same coin. Targeting increased survival is unlikely to be efficacious without at the same time targeting apoptosis resistance. We have critically reviewed the literature regarding survival and apoptosis signalling in GBM, and highlighted experimental, preclinical and recent clinical trials attempting to target these pathways. Combined therapies simultaneously targeting apoptosis and survival signalling defects might shift the balance from tumour growth stasis to cytotoxic therapeutic responses that might be associated with greater therapeutic benefits.

Abstract Background Establishing clinically relevant animal models of glioblastoma multiforme (GBM) remains a challenge, and many commonly used cell line-based models do not recapitulate the invasive growth patterns of patient GBMs. Previously, we have reported the formation of highly invasive tumour xenografts in nude rats from human GBMs. However, implementing tumour models based on primary tissue requires that these models can be sufficiently standardised with consistently high take rates. Methods In this work, we collected data on growth kinetics from a material of 29 biopsies xenografted in nude rats, and characterised this model with an emphasis on neuropathological and radiological features. Results The tumour take rate for xenografted GBM biopsies were 96% and remained close to 100% at subsequent passages in vivo, whereas only one of four lower grade tumours engrafted. Average time from transplantation to the onset of symptoms was 125 days ± 11.5 SEM. Histologically, the primary xenografts recapitulated the invasive features of the parent tumours while endothelial cell proliferations and necrosis were mostly absent. After 4-5 in vivo passages, the tumours became more vascular with necrotic areas, but also appeared more circumscribed. MRI typically revealed changes related to tumour growth, several months prior to the onset of symptoms. Conclusions In vivo passaging of patient GBM biopsies produced tumours representative of the patient tumours, with high take rates and a reproducible disease course. The model provides combinations of angiogenic and invasive phenotypes and represents a good alternative to in vitro propagated cell lines for dissecting mechanisms of brain tumour progression.

Background: Tumor burden assessment is essential for radiation therapy (RT), treatment response evaluation, and clinical decision-making. However, manual tumor delineation remains laborious and challenging due to radiological complexity. The objective of this study was to investigate the feasibility of the HD-GLIO tool, an ensemble of pre-trained deep learning models based on the nnUNet-algorithm, for tumor segmentation, response prediction, and its potential for clinical deployment., Methods: We analyzed the predicted contrast-enhanced (CE) and non-enhancing (NE) HD-GLIO output in 49 multi-parametric MRI examinations from 23 grade-4 glioma patients. The volumes were retrospectively compared to corresponding manual delineations by 2 independent operators, before prospectively testing the feasibility of clinical deployment of HD-GLIO-output to a RT setting., Results: For CE, median Dice scores were 0.81 (95% CI 0.71-0.83) and 0.82 (95% CI 0.74-0.84) for operator-1 and operator-2, respectively. For NE, median Dice scores were 0.65 (95% CI 0.56-0,69) and 0.63 (95% CI 0.57-0.67), respectively. Comparing volume sizes, we found excellent intra-class correlation coefficients of 0.90 ( P < .001) and 0.95 ( P < .001), for CE, respectively, and 0.97 ( P < .001) and 0.90 ( P < .001), for NE, respectively. Moreover, there was a strong correlation between response assessment in Neuro-Oncology volumes and HD-GLIO-volumes ( P < .001, Spearman's R 2 = 0.83). Longitudinal growth relations between CE- and NE-volumes distinguished patients by clinical response: Pearson correlations of CE- and NE-volumes were 0.55 ( P = .04) for responders, 0.91 ( P > .01) for non-responders, and 0.80 ( P = .05) for intermediate/mixed responders., Conclusions: HD-GLIO was feasible for RT target delineation and MRI tumor volume assessment. CE/NE tumor-compartment growth correlation showed potential to predict clinical response to treatment., Competing Interests: The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Therapy of recurrent glioblastoma (GBM) is challenging due to lack of standard treatment. We investigated physicians' treatment choice at recurrence and prognostic and predictive factors for survival in GBM patients from Norway's two largest regional hospitals. Clinicopathological data from n = 467 patients treated at Haukeland and Oslo university hospitals from January 2015 to December 2017 was collected. Data included tumour location, promoter methylation of O6 methylguanine-DNA methyltransferase (MGMT) and mutation of isocitrate dehydrogenase (IDH), patient age, sex, extent of resection at primary diagnosis and treatment at successive tumour recurrences. Cox-proportional hazards regression adjusting for multiple risk factors was used. Median overall survival (OS) was 12.1 months and 21.4% and 6.8% of patients were alive at 2 and 5 years, respectively. Median progression-free survival was 8.1 months. Treatment at recurrence varied but was not associated with difference in overall survival (OS) (p = 0.201). Age, MGMT hypermethylation, tumour location and extent of resection were independent prognostic factors. Patients who received 60 Gray radiotherapy with concomitant and adjuvant temozolomide at primary diagnosis had 16.1 months median OS and 9.3% were alive at 5 years. Patients eligible for gamma knife/stereotactic radiosurgery alone or combined with chemotherapy at first recurrence had superior survival compared to chemotherapy alone (p<0.001). At second recurrence, combination chemotherapy with or without bevacizumab were both superior to no treatment. Treatment at recurrence differed between the institutions but there was no difference in median OS, indicating that it is the disease biology that dictates patient outcome., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Blakstad et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Glioblastoma (GBM) is invariably resistant to temozolomide (TMZ) chemotherapy. Inhibiting the proteasomal pathway is an emerging strategy to accumulate damaged proteins and inhibit their lysosomal degradation. We hypothesized that pre-treatment of glioblastoma with bortezomib (BTZ) might sensitize glioblastoma to temozolomide by abolishing autophagy survival signals to augment DNA damage and apoptosis. Methods: P3 patient-derived glioblastoma cells, as well as the tumour cell lines U87, HF66, A172, and T98G were investigated for clonogenic survival after single or combined treatment with temozolomide and bortezomib in vitro . We investigated the requirement of functional autophagy machinery by utilizing pharmacological inhibitors or CRISPR-Cas9 knockout (KO) of autophagy-related genes -5 and -7 (ATG5 and ATG7) in glioblastoma cells and monitored changes in autophagic flux after temozolomide and/or bortezomib treatments. P3 wild-type and P3 ATG5-/- (ATG5 KO) cells were implanted orthotopically into NOD-SCID mice to assess the efficacy of bortezomib and temozolomide combination therapy with and without functional autophagy machinery. Results: The chemo-resistant glioblastoma cells increased autophagic flux during temozolomide treatment as indicated by increased degradation of long-lived proteins, diminished expression of autophagy markers LC3A/B-II and p62 (SQSTM1), increased co-localisation of LC3A/B-II with STX17, augmented and no induction of apoptosis. In contrast, bortezomib treatment abrogated autophagic flux indicated by the accumulation of LC3A/B-II and p62 (SQSTM1) positive autophagosomes that did not fuse with lysosomes and thus reduced the degradation of long-lived proteins. Bortezomib synergistically enhanced temozolomide efficacy by attenuating cell proliferation, increased DNA double-strand breaks, and apoptosis in an autophagy-dependent manner. Abolishing autophagy in ATG5 KOs reversed the bortezomib-induced toxicity, rescued glioblastoma cell death and reduced animal survival. Discussion: We conclude that bortezomib abrogates temozolomide induced autophagy flux through an ATG5 dependent pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rahman, Engelsen, Sarowar, Bindesbøll, Birkeland, Goplen, Lotsberg, Knappskog, Simonsen and Chekenya.)

Background: Glioblastoma cells assemble to a syncytial communicating network based on tumor microtubes (TMs) as ultra-long membrane protrusions. The relationship between network architecture and transcriptional profile remains poorly investigated. Drugs that interfere with this syncytial connectivity such as meclofenamate (MFA) may be highly attractive for glioblastoma therapy., Methods: In a human neocortical slice model using glioblastoma cell populations of different transcriptional signatures, three-dimensional tumor networks were reconstructed, and TM-based intercellular connectivity was mapped on the basis of two-photon imaging data. MFA was used to modulate morphological and functional connectivity; downstream effects of MFA treatment were investigated by RNA sequencing and fluorescence-activated cell sorting (FACS) analysis., Results: TM-based network morphology strongly differed between the transcriptional cellular subtypes of glioblastoma and was dependent on axon guidance molecule expression. MFA revealed both a functional and morphological demolishment of glioblastoma network architectures which was reflected by a reduction of TM-mediated intercellular cytosolic traffic as well as a breakdown of TM length. RNA sequencing confirmed a downregulation of NCAM and axon guidance molecule signaling upon MFA treatment. Loss of glioblastoma communicating networks was accompanied by a failure in the upregulation of genes that are required for DNA repair in response to temozolomide (TMZ) treatment and culminated in profound treatment response to TMZ-mediated toxicity., Conclusion: The capacity of TM formation reflects transcriptional cellular heterogeneity. MFA effectively demolishes functional and morphological TM-based syncytial network architectures. These findings might pave the way to a clinical implementation of MFA as a TM-targeted therapeutic approach., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glioblastoma (GBM) is the most prevalent, aggressive primary brain tumour with a dismal prognosis. Treatment at diagnosis has limited efficacy and there is no standardised treatment at recurrence. New, personalised treatment options are under investigation, although challenges persist for heterogenous tumours such as GBM. Gene editing technologies are a game changer, enabling design of novel molecular-immunological treatments to be used in combination with chemoradiation, to achieve long lasting survival benefits for patients. Here, we review the literature on how cutting-edge molecular gene editing technologies can be applied to known and emerging tumour-associated antigens to enhance chimeric antigen receptor T and NK cell therapies for GBM. A tight balance of limiting neurotoxicity, avoiding tumour antigen loss and therapy resistance, while simultaneously promoting long-term persistence of the adoptively transferred cells must be maintained to significantly improve patient survival. We discuss the opportunities and challenges posed by the brain contexture to the administration of the treatments and achieving sustained clinical responses.

Background: Glioblastoma (GBM) is an aggressive malignant brain tumor where median survival is approximately 15 months after best available multimodal treatment. Recurrence is inevitable, largely due to O 6 methylguanine DNA methyltransferase (MGMT) that renders the tumors resistant to temozolomide (TMZ). We hypothesized that pretreatment with bortezomib (BTZ) 48 hours prior to TMZ to deplete MGMT levels would be safe and tolerated by patients with recurrent GBM harboring unmethylated MGMT promoter. The secondary objective was to investigate whether 26S proteasome blockade may enhance differentiation of cytotoxic immune subsets to impact treatment responses measured by radiological criteria and clinical outcomes., Methods: Ten patients received intravenous BTZ 1.3 mg/m 2 on days 1, 4, and 7 during each 4th weekly TMZ-chemotherapy starting on day 3 and escalated from 150 mg/m 2 per oral 5 days/wk via 175 to 200 mg/m 2 in cycles 1, 2, and 3, respectively. Adverse events and quality of life were evaluated by CTCAE and EQ-5D-5L questionnaire, and immunological biomarkers evaluated by flow cytometry and Luminex enzyme-linked immunosorbent assay., Results: Sequential BTZ + TMZ therapy was safe and well tolerated. Pain and performance of daily activities had greatest impact on patients' self-reported quality of life and were inversely correlated with Karnofsky performance status. Patients segregated a priori into three groups, where group 1 displayed stable clinical symptoms and/or slower magnetic resonance imaging radiological progression, expanded CD4 + effector T-cells that attenuated cytotoxic T-lymphocyte associated protein-4 and PD-1 expression and secreted interferon γ and tumor necrosis factor α in situ and ex vivo upon stimulation with PMA/ionomycin. In contrast, rapidly progressing group 2 patients exhibited tolerised T-cell phenotypes characterized by fourfold to sixfold higher interleukin 4 (IL-4) and IL-10 Th-2 cytokines after BTZ + TMZ treatment, where group 3 patients exhibited intermediate clinical/radiological responses., Conclusion: Sequential BTZ + TMZ treatment is safe and promotes Th1-driven immunological responses in selected patients with improved clinical outcomes (Clinicaltrial.gov (NCT03643549))., (© 2020 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Background: Resistance to temozolomide (TMZ) is due in part to enhanced DNA repair mediated by high expression of O 6 -methyl guanine DNA methyltransferase (MGMT) that is often characterised by unmethylated promoter. Here, we investigated pre-treatment of glioblastoma (GBM) cells with the 26S-proteasome inhibitor bortezomib (BTZ) as a strategy to interfere with MGMT expression and thus sensitise them to TMZ., Methods: Cell lines and patient GBM-derived cells were examined in vitro, and the latter also implanted orthotopically into NOD-SCID C.B.-Igh-1b/lcrTac-Prkdc mice to assess efficacy and tolerability of BTZ and TMZ combination therapy. MGMT promoter methylation was determined using pyrosequencing and PCR, protein signalling utilised western blotting while drug biodistribution was examined by LC-MS/MS. Statistical analysis utilised Analysis of variance and the Kaplan-Meier method., Results: Pre-treatment with BTZ prior to temozolomide killed chemoresistant GBM cells with unmethylated MGMT promoter through MGMT mRNA and protein depletion in vitro without affecting methylation. Chymotryptic activity was abolished, processing of NFkB/p65 to activated forms was reduced and corresponded with low MGMT levels. BTZ crossed the blood-brain barrier, diminished proteasome activity and significantly prolonged animal survival., Conclusion: BTZ chemosensitized resistant GBM cells, and the schedule may be amenable for temozolomide refractory patients with unmethylated MGMT promoter.

Background: Natural killer (NK) cells are potential effectors in anti-cancer immunotherapy; however only a subset potently kills cancer cells. Here, we examined whether pretreatment of glioblastoma (GBM) with the proteasome inhibitor, bortezomib (BTZ), might sensitize tumour cells to NK cell lysis by inducing stress antigens recognized by NK-activating receptors. Methods: Combination immunotherapy of NK cells with BTZ was studied in vitro against GBM cells and in a GBM-bearing mouse model. Tumour cells were derived from primary GBMs and NK cells from donors or patients. Flow cytometry was used for viability/cytotoxicity evaluation as well as in vitro and ex vivo phenotyping. We performed a Seahorse assay to assess oxygen consumption rates and mitochondrial function, Luminex ELISA to determine NK cell secretion, protein chemistry and LC-MS/MS to detect BTZ in brain tissue. MRI was used to monitor therapeutic efficacy in mice orthotopically implanted with GBM spheroids. Results: NK cells released IFNγ, perforin and granzyme A cytolytic granules upon recognition of stress-ligand expressing GBM cells, disrupted mitochondrial function and killed 24-46% of cells by apoptosis. Pretreatment with BTZ further increased stress-ligands, induced TRAIL-R2 expression and enhanced GBM lysis to 33-76% through augmented IFNγ release ( p < 0.05). Blocking NKG2D, TRAIL and TRAIL-R2 rescued GBM cells treated with BTZ from NK cells, p = 0.01. Adoptively transferred autologous NK-cells persisted in vivo ( p < 0.05), diminished tumour proliferation and prolonged survival alone (Log Rank 10.19 , p = 0.0014, 95%CI 0.252-0.523) or when combined with BTZ (Log Rank 5.25 , p = 0.0219, 95%CI 0.295-0.408), or either compared to vehicle controls (median 98 vs. 68 days and 80 vs. 68 days, respectively). BTZ crossed the blood-brain barrier, attenuated proteasomal activity in vivo ( p < 0.0001; p < 0.01 compared to vehicle control or NK cells only, respectively) and diminished tumour angiogenesis to promote survival compared to vehicle-treated controls (Log Rank 6.57 , p = 0.0104, 95%CI 0.284-0.424, median 83 vs. 68 days). However, NK ablation with anti-asialo-GM1 abrogated the therapeutic efficacy. Conclusions: NK cells alone or in combination with BTZ inhibit tumour growth, but the scheduling of BTZ in vivo requires further investigation to maximize its contribution to the efficacy of the combination regimen.

Glioblastoma (GBM) is the most aggressive brain malignancy in adults, where survival is approximately 14.6 months. Novel therapies are urgently needed and immunotherapy has hailed a new dawn for treatment of solid tumors. Natural killer (NK) cells may be amenable therapeutic effectors against heterogeneous GBM, since they also do not require co-stimulation and antigen specificity. However, it is unclear how culture media routinely used in pre-clinical studies affect GBM cell responses to NK-mediated cytotoxicity. We hypothesized that the culture medium would affect GBM cell phenotype, proliferation, and responses to NK cytotoxicity. We investigated in paired analyses n  = 6 patient-derived primary GBM cells propagated in stem cell or serum-containing medium for morphology, proliferation, as well as susceptibility to NK cytolysis and related this to expression of surface and intracellular lineage markers, as well as ligands for NK cell activating and inhibitory receptors. We genotyped the GBM cells for human leukocyte antigen (HLA) as well as the killer immunoglobulin-like receptors (KIR) of the n  = 6 allogeneic NK cells used as effector cells. Culture in serum-containing medium induced a switch in GBM cell morphology from suspension neuropsheres to adherent epithelial-mesenchymal-like phenotypes, which was partially reversible. The differentiated cells diminished expression of nestin, CD133 (prominin-1), and A2B5 putative glioma stem-cell markers, attenuated growth, diminished expression of ligands for activating NK cell receptors, while upregulating class I HLA ligands for NK cell inhibitory receptors. When maintained in serum-containing medium, fewer GBM cells expressed intercellular cell adhesion molecule-1 (ICAM-1) and were less susceptible to lysis by NK cells expressing α L β 2 integrin receptor (LFA-1), mediated through combination of inhibitory KIR-HLA ligand mismatch and diminished activation receptor-ligand interactions compared to cells maintained in stem cell media. We conclude that development of preclinical immunotherapy strategies against GBM should not use cells propagated in serum-containing media to avoid misinterpretation of potential therapeutic responses.

By affecting immunological presentation, the presence of cytomegalovirus in some glioblastomas may impact progression. In this study, we examined a hypothesized role for natural killer (NK) cells in impacting disease progression in this setting. We characterized 108 glioblastoma patients and 454 healthy controls for HLA-A,-B,-C, NK-cell KIR receptors, and CMV-specific antibodies and correlated these metrics with clinical parameters. Exome sequences from a large validation set of glioblastoma patients and control individuals were examined from in silico databases. We demonstrated that the KIR allele KIR2DS4*00101 was independently prognostic of prolonged survival. KIR2DS4*00101 displayed 100% concordance with cognate HLA-C1 ligands in glioblastoma patients, but not controls. In the context of both HLA-C1/C2 ligands for the KIR2DS4 receptor, patient survival was further extended. Notably, all patients carrying KIR2DS4*00101 alleles were CMV seropositive, but not control individuals, and exhibited increased NK-cell subpopulations, which expressed the cytotoxicity receptors CD16, NKG2D, and CD94/NKG2C. Finally, healthy controls exhibited a reduced risk for developing glioblastoma if they carried two KIR2DS4*00101 alleles, where protection was greatest among Caucasian individuals. Our findings suggest that KIR2DS4*00101 may offer a molecular biomarker to identify intrinsically milder forms of glioblastoma. Cancer Res; 76(18); 5326-36. ©2016 AACR., (©2016 American Association for Cancer Research.)

Natural killer (NK) cells are innate lymphoid cells that hold tremendous potential for effective immunotherapy for a broad range of cancers. Due to the mode of NK cell killing, requiring one-to-one target engagement and site-directed release of cytolytic granules, the therapeutic potential of NK cells has been most extensively explored in hematological malignancies. However, their ability to precisely kill antibody coated cells, cancer stem cells, and genotoxically altered cells, while maintaining tolerance to healthy cells makes them appealing therapeutic effectors for all cancer forms, including metastases. Due to their release of pro-inflammatory cytokines, NK cells may potently reverse the anti-inflammatory tumor microenvironment (TME) and augment adaptive immune responses by promoting differentiation, activation, and/or recruitment of accessory immune cells to sites of malignancy. Nevertheless, integrated and coordinated mechanisms of subversion of NK cell activity against the tumor and its microenvironment exist. Although our understanding of the receptor ligand interactions that regulate NK cell functionality has evolved remarkably, the diversity of ligands and receptors is complex, as is their mechanistic foundations in regulating NK cell function. In this article, we review the literature and highlight how the TME manipulates the NK cell phenotypes, genotypes, and tropism to evade tumor recognition and elimination. We discuss counter strategies that may be adopted to augment the efficacy of NK cell anti-tumor surveillance, the clinical trials that have been undertaken so far in solid malignancies, critically weighing the challenges and opportunities with this approach.

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., (Copyright © 2014 by The American Association of Immunologists, Inc.)

There are currently no established radiological parameters that predict response to immunotherapy. We hypothesised that multiparametric, longitudinal magnetic resonance imaging (MRI) of physiological parameters and pharmacokinetic models might detect early biological responses to immunotherapy for glioblastoma targeting NG2/CSPG4 with mAb9.2.27 combined with natural killer (NK) cells. Contrast enhanced conventional T1-weighted MRI at 7±1 and 17±2 days post-treatment failed to detect differences in tumour size between the treatment groups, whereas, follow-up scans at 3 months demonstrated diminished signal intensity and tumour volume in the surviving NK+mAb9.2.27 treated animals. Notably, interstitial volume fraction (ve), was significantly increased in the NK+mAb9.2.27 combination therapy group compared mAb9.2.27 and NK cell monotherapy groups (p = 0.002 and p = 0.017 respectively) in cohort 1 animals treated with 1 million NK cells. ve was reproducibly increased in the combination NK+mAb9.2.27 compared to NK cell monotherapy in cohort 2 treated with increased dose of 2 million NK cells (p<0.0001), indicating greater cell death induced by NK+mAb9.2.27 treatment. The interstitial volume fraction in the NK monotherapy group was significantly reduced compared to mAb9.2.27 monotherapy (p<0.0001) and untreated controls (p = 0.014) in the cohort 2 animals. NK cells in monotherapy were unable to kill the U87MG cells that highly expressed class I human leucocyte antigens, and diminished stress ligands for activating receptors. A significant association between apparent diffusion coefficient (ADC) of water and ve in combination NK+mAb9.2.27 and NK monotherapy treated tumours was evident, where increased ADC corresponded to reduced ve in both cases. Collectively, these data support histological measures at end-stage demonstrating diminished tumour cell proliferation and pronounced apoptosis in the NK+mAb9.2.27 treated tumours compared to the other groups. In conclusion, ve was the most reliable radiological parameter for detecting response to intralesional NK cellular therapy.

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 + CCR2 low macrophages that stimulated ED1 + ED2 low MHCII high microglial cells to exert robust cytotoxicity. Our findings demonstrate the therapeutic potential of targeting salient tumor associated-antigens.

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.

We characterized GBM patients' tumor and systemic immune contexture with aim to reveal the mechanisms of immunological escape, their impact on patient outcome, and identify targets for immunotherapy. Increased CD3(+) T-cell infiltration was associated with prolonged survival independent of age, MGMT promoter methylation and post-operative treatment that implies potential for immunotherapy for GBM. Several mechanisms of escape were identified: within the tumor microenvironment: induced CD8(+)CD28(-)Foxp3(+) Tregs that may tolerize antigen presenting cells, elevated CD73 and CD39 ectonucleotidases that suppress T-cell function, and at the systemic level: elevated IL-10 levels in serum, diminished helper T-cell counts, and upregulated inhibitory CTLA-4., (© 2013. Published by Elsevier B.V. All rights reserved.)

Glioblastoma (GBM) is the most malignant brain tumor where patients' survival is only 14.6 months, despite multimodal therapy with debulking surgery, concurrent chemotherapy and radiotherapy. There is an urgent, unmet need for novel, effective therapeutic strategies for this devastating disease. Although several immunotherapies are under development for the treatment of GBM patients, the use of natural killer (NK) cells is still marginal despite this being a promising approach to treat cancer. In regard of our knowledge on the role of NG2/CSPG4 in promoting GBM aggressiveness we investigated the potential of an innovative immunotherapeutic strategy combining mAb9.2.27 against NG2/CSPG4 and NK cells in preclinical animal models of GBM. Multiple immune escape mechanisms maintain the tumor microenvironment in an anti-inflammatory state to promote tumor growth, however, the distinct roles of resident microglia versus recruited macrophages is not elucidated. We hypothesized that exploiting the cytokine release capabilities of activated (NK) cells to reverse the anti-inflammatory axis combined with mAb9.2.27 targeting the NG2/CSPG4 may favor tumor destruction by editing pro-GBM immune responses. Combination treatment with NK+mAb9.2.27 diminished tumor growth that was associated with reduced tumor proliferation, increased cellular apoptosis and prolonged survival compared to vehicle and monotherapy controls. The therapeutic efficacy was mediated by recruitment of CCR2low macrophages into the tumor microenvironment, increased ED1 and MHC class II expression on microglia that might render them competent for GBM antigen presentation, as well as elevated IFN-γ and TNF-α levels in the cerebrospinal fluid compared to controls. Depletion of systemic macrophages by liposome-encapsulated clodronate decreased the CCR2low macrophages recruited to the brain and abolished the beneficial outcomes. Moreover, mAb9.2.27 reversed tumor-promoting effects of patient-derived tumor-associated macrophage/microglia(TAM) ex vivo.Taken together, these findings indicate thatNK+mAb9.2.27 treatment may be an amenable therapeutic strategy to treat NG2/CSPG4 expressing GBMs. We provide a novel conceptual approach of combination immunotherapy for glioblastoma. The results traverse beyond the elucidation of NG2/CSPG4 as a therapeutic target, but demonstrate a proof of concept that this antibody may hold potential for the treatment of GBM by activation of tumor infiltrated microglia/macrophages.

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.

Object: Gamma knife surgery (GKS) may be used for recurring glioblastomas (GBMs). However, patients have then usually undergone multimodal treatment, which makes it difficult to specifically validate GKS independent of established treatments. Thus, we developed an experimental brain tumor model to assess the efficacy and radiotoxicity associated with GKS., Methods: GBM xenografts were implanted intracerebrally in nude rats, and engraftment was confirmed with MRI. The rats were allocated to GKS, with margin doses of 12 Gy or 18 Gy, or to no treatment. Survival time was recorded, tumor sections were examined, and radiotoxicity was evaluated in a behavioral open field test., Results: In the first series, survival from the time of implantation was 96 days in treated rats and 72 days in controls (P < 0.001). In a second experiment, survival was 72 days in the treatment group versus 54 days in controls (P < 0.006). Polynuclear macrophages and fibrosis was seen in groups subjected to GKS. Untreated rats with GBM xenografts displayed less mobility than GKS-treated animals in the open field test 4 weeks after treatment (P = 0.04)., Conclusion: GKS administered with clinically relevant doses prolongs survival in rats harboring GBM xenografts, and the associated toxicity is mild.

A method is proposed for quantitative description of blood-vessel trees, which can be used for tree classification and/or physical parameters indirect monitoring. The method is based on texture analysis of 3D images of the trees. Several types of trees were defined, with distinct tree parameters (number of terminal branches, blood viscosity, input and output flow). A number of trees were computer-simulated for each type. 3D image was computed for each tree and its texture features were calculated. Best discriminating features were found and applied to 1-NN nearest neighbor classifier. It was demonstrated that (i) tree images can be correctly classified for realistic signal-to-noise ratio, (ii) some texture features are monotonously related to tree parameters, (iii) 2D texture analysis is not sufficient to represent the trees in the discussed sense. Moreover, applicability of texture model to quantitative description of vascularity images was also supported by unsupervised exploratory analysis. Eventually, the experimental confirmation was done, with the use of confocal microscopy images of rat brain vasculature. Several classes of brain tissue were clearly distinguished based on 3D texture numerical parameters, including control and different kinds of tumours - treated with NG2 proteoglycan to promote angiogenesis-dependent growth of the abnormal tissue. The method, applied to magnetic resonance imaging e.g. real neovasculature or retinal images can be used to support noninvasive medical diagnosis of vascular system diseases., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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 O(6)-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.

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.

Natural killer (NK) cells are important effectors of both innate and adaptive immune responses. Although human and mouse NK cells are extensively characterized, much less is known about the rat cells, partly because of the current lack of reliable isolation techniques. We aimed to develop a method for isolating highly pure 'untouched' rat NK cells by negative selection from splenocytes. Thereafter, we characterized them phenotypically and functionally in comparison with those isolated by positive selection targeting the NKR-P1 receptor. Our novel method isolated highly pure untouched NK cells reproducibly with 97 ± 0.7% (n = 7), 96.6 ± 0.8% (n = 3) and 88.3 ± 1.5% (n = 9) in LEWIS, Fischer and athymic nude rats, respectively. The positively selected NK cells were less homogeneous and exhibited undesired method-related activation profiles. Resting negatively selected NK cells were less proliferative and less robust compared with positively selected NK cells. Although resting positively selected NK cells were more cytotoxic, interleukin-2 (IL-2) activation increased the cytotoxicity of negatively selected cells three-fold. The negatively selected NK cells responded to cross-linking of the NKR-P1 receptor by calcium mobilization from intracellular stores. However, combined IL-2 and IL-12 activation resulted in significantly more interferon-γ release from positively selected NK cells. This new NK-cell isolation method will allow a deeper insight into rat NK-cell phenotypes and the roles of their receptors in the biology of these cells., (© 2010 The Authors. Immunology © 2010 Blackwell Publishing Ltd.)

The combination of irradiation with angiogenic inhibition is increasingly being investigated for treatment of glioblastoma multiforme (GBM). We investigated whether vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitor DC101 affects morbidity and tumor growth in irradiated and non-irradiated intracerebral GBM-bearing mice, controlled with sham treatments. End-points were toxicity, morbidity and histology. Irradiation either or not combined, reduced tumor size strongly, whereas DC101 mono-treatment reduced tumor size by 64%. Irradiation delayed morbidity from 5.8 weeks in sham-treated mice to 10.3 weeks. Morbidity after combined treatment occurred after 5.9 weeks. Treatment with angiogenesis inhibitor DC101 delays tumor growth but it induces morbidity, by itself or combined with irradiation.

Tumor hypoxia is relevant for tumor growth, metabolism and epithelial-to-mesenchymal transition (EMT). We report that hyperbaric oxygen (HBO) treatment induced mesenchymal-to-epithelial transition (MET) in a dimethyl-alpha-benzantracene induced mammary rat adenocarcinoma model, and the MET was associated with extensive coordinated gene expression changes and less aggressive tumors. One group of tumor bearing rats was exposed to HBO (2 bar, pO(2) = 2 bar, 4 exposures à 90 minutes), whereas the control group was housed under normal atmosphere (1 bar, pO(2) = 0.2 bar). Treatment effects were determined by assessment of tumor growth, tumor vascularisation, tumor cell proliferation, cell death, collagen fibrils and gene expression profile. Tumor growth was significantly reduced (approximately 16%) after HBO treatment compared to day 1 levels, whereas control tumors increased almost 100% in volume. Significant decreases in tumor cell proliferation, tumor blood vessels and collagen fibrils, together with an increase in cell death, are consistent with tumor growth reduction and tumor stroma influence after hyperoxic treatment. Gene expression profiling showed that HBO induced MET. In conclusion, hyperoxia induced MET with coordinated expression of gene modules involved in cell junctions and attachments together with a shift towards non-tumorigenic metabolism. This leads to more differentiated and less aggressive tumors, and indicates that oxygen per se might be an important factor in the "switches" of EMT and MET in vivo. HBO treatment also attenuated tumor growth and changed tumor stroma, by targeting the vascular system, having anti-proliferative and pro-apoptotic effects.

Malignant primary brain tumors are characterized by a short median survival and an almost 100% tumor-related mortality. Despite the addition of new chemotherapy regimes, the overall survival has improved marginally, and radiotherapy is only transiently effective, illustrating the profound impact of treatment resistance on prognosis. Recent studies suggest that a small subpopulation of cancer stem cells (CSCs) has the capacity to repopulate tumors and drive malignant progression and mediate radio- and chemoresistance. This implies that future therapies should turn from the elimination of the rapidly dividing, but differentiated tumor cells, to specifically targeting the minority of tumor cells that repopulate the tumor. Although there exists some support for the CSC hypothesis, there remain many uncertainties regarding theoretical, technical, and interpretational aspects of the data supporting it. If correct, the CSC hypothesis could have profound implications for the way tumors are classified and treated. In this review of the literature, we provide original data and hypotheses supporting alternative explanations and outline some of the therapeutic implications that can be derived.

Neuroepithelial cells of the central nervous system constitute neuroglia (astrocytes, oligodendrocytes, and microglia), ependyma, and neurons, which make up the stromal cells of the brain. The stromal tissue organization of the brain is tightly regulated, but occasionally the signals that define the normal contexts become disrupted and result in cancer. Malignant progression is then maintained by cross-talks between the tumor and its stroma, where the activated stroma nurtures the proliferative and invasive neoplastic cells, by providing neovasculature, extracellular matrix components, and stimulatory growth factors. The NG2/HMP plays a major role in tumor-stroma activation through alterations in cellular adhesion, migration, proliferation, and vascular morphogenesis. Therapeutic strategies specifically targeting NG2/HMP may be useful in normalizing the tumor stroma and may reduce the toxic side effects when used in combination with conventional treatments.

In this work, highly infiltrative brain tumors with a stem-like phenotype were established by xenotransplantation of human brain tumors in immunodeficient nude rats. These tumors coopted the host vasculature and presented as an aggressive disease without signs of angiogenesis. The malignant cells expressed neural stem cell markers, showed a migratory behavior similar to normal human neural stem cells, and gave rise to tumors in vivo after regrafting. Serial passages in animals gradually transformed the tumors into an angiogenesis-dependent phenotype. This process was characterized by a reduction in stem cells markers. Gene expression profiling combined with high throughput immunoblotting analyses of the angiogenic and nonangiogenic tumors identified distinct signaling networks in the two phenotypes. Furthermore, proinvasive genes were up-regulated and angiogenesis signaling genes were down-regulated in the stem-like tumors. In contrast, proinvasive genes were down-regulated in the angiogenesis-dependent tumors derived from the stem-like tumors. The described angiogenesis-independent tumor growth and the uncoupling of invasion and angiogenesis, represented by the stem-like cancer cells and the cells derived from them, respectively, point at two completely independent mechanisms that drive tumor progression. This article underlines the need for developing therapies that specifically target the stem-like cell pools in tumors.

During embryogenesis, the NG2 proteoglycan is expressed on immature capillary vessels, but as the vessels mature they lose this expression. NG2 is up-regulated in high-grade gliomas, but it is not clear to what extent it contributes to malignant progression. Using a combination of high spatial and temporal resolution functional magnetic resonance imaging and histopathological analyses, we show here that overexpression of NG2 increases tumor initiation and growth rates, neovascularization, and cellular proliferation, which predisposes to a poorer survival outcome. By confocal microscopy and cDNA gene array expression profiles, we also show that NG2 tumors express lower levels of hypoxia inducible factor-1a, vascular endothelial growth factor, and endogenous angiostatin in vivo compared with wild-type tumors. Moreover, we demonstrate that NG2-positive cells bind, internalize, and coimmunoprecipitate with angiostatin. These results indicate a unique role for NG2 in regulating the transition from small, poorly vascularized tumors to large, highly vascular gliomas in situ by sequestering angiostatin.