Glioblastoma Multiforme Therapy Research Articles

Abstract 5583: Plasma levels of circulating MACC1 transcripts co-segregate with other prognostic markers and treatment response in glioblastoma patients

Abstract IDH1-mutation and MGMT-promoter methylation are established prognostic markers in Glioblastoma multiforme (GBM); however, invasive procedures are needed to obtain the required tumor tissue. Metastasis-associated in colon cancer-1 (MACC1) can easily be measured in peripheral blood and has been established as a prognostic plasma marker for several solid cancer entities. Further, MACC1 has been identified by us and others as a promising biomarker for prognostication for GBM, when detected in the tumor tissue. Here, we evaluated whether circulating MACC1 transcripts could be useful to predict clinical outcome and response to therapy in GBM. Plasma samples were collected from GBM patients (IDH1 wildtype (wt), IDH1R132H, and unspecified) before surgery. Samples from healthy volunteers served as controls. MACC1-transcript levels, determined by qRT-PCR, were correlated with patient overall survival (OS) and clinical data of known prognostic significance. Cut-off values for Kaplan-Meier analyses were determined by ROC calculations. Cluster analyses were performed. MACC1 transcripts were higher in plasma of GBM patients compared to healthy controls (P<0.0001), yet differentially distributed within the cohort. Low MACC1 levels clustered together with all other favorable markers, i.e. small tumor volume, young age, IDH1R132H and MGMT promoter methylation. MACC1 levels correlated with patient prognosis in conjunction with the IDH mutation status: patients with IDH1R132H mutation and low MACC1 levels showed the most favorable outcome (mean OS 54.0 months (m), SD=9.8; 95% CI 34.7-73.3), those with IDH1wt and high MACC1 had the worst prognosis (mean OS 10.5 m), while those with IDH1wt and low MACC1 were intermediate (mean OS 20.3 m). No patients in this cohort displayed IDH1R132H and high MACC1 expression. Interestingly, patients with low MACC1 levels receiving standard therapy (operation and radiochemotherapy according to Stupp) survived longest (mean OS 36.9 m, SD=6.5; 95% CI 24.1-49.7). Patients with high MACC1 showed shorter OS (mean OS 13.0 m). The worst prognosis had patients not receiving the standard regimen, independently of their MACC1 levels (MACC1 low: 5.2 m; MACC1 high: 5.5 m). The inclusion of circulating MACC1 transcript levels into the diagnostic workup might improve the accuracy of outcome prediction and thus help to define more precise risk categories of GBM patients. Whether pharmaceutical inhibition of MACC1 expression might be of general benefit for the treatment response and survival of GBM patients remains to be determined. Citation Format: Carsten Hagemann, Nikolas Neuhaus, Mathias Dahlmann, Almut F. Kessler, Dennis Kobelt, Pia Herrmann, Matthias Eyrich, Benjamin Freitag, Thomas Linsenmann, Camelia Monoranu, Ralf-Ingo Ernestus, Mario Löhr, Ulrike S. Stein. Plasma levels of circulating MACC1 transcripts co-segregate with other prognostic markers and treatment response in glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5583.

Abstract 3195: The molecular mechanism of action and cellular targets of TTFields

Abstract Tumor Treating Fields (TTFields) are AC electric fields of intensity 1-2 V/cm in the frequency range 100-300 kHz, which have been shown to be an effective FDA-approved adjuvant therapy for Glioblastoma Multiforme. However, the mechanism of action for TTFields is not well understood. It is known that microtubules (MTs) and actin filaments re-orient themselves and behave abnormally when subjected to external electric fields indicating that they may be cellular targets of TTFields. The purpose of our study was to determine the molecular mechanism of action of TTFields. Previously, using impedance spectroscopy we observed the effect of unpolymerized tubulin and microtubules on ionic conductivity of buffer solution, and found that unlike tubulin, microtubules increased electrical conductance, which peaked at TTField-like frequencies1. Microtubules have been modeled as conductive cables that attract and guide counterions to increase the solution's conductance. protonic transport through the lumen of the microtubule has also been modeled. To investigate the additional possibility of electronic transport along α, β- tubulin dimers we are performing spectroscopic characterization of MTs. Using Dynamic Light Scattering (DLS) we have characterized the thickness of the solvation layer around the tubulin dimer, which elucidates the mechanism of ionic conduction. We have found that the thickness of the solvation layer increases as the concentration of a highly polar molecule such as DMSO is increased. We have found that increasing the solution temperature leads to a reduction of the solvation layer's thickness and have also characterized response to changes in pH of solution. We have polymerized various morphologies of tubulin assemblies, e.g. planar zinc sheets and (300-500 nm diameter) macrotubes to gain insight into associated conductivity processes. We have imaged these interesting morphologies using Transmission Electron Microscopy (TEM) and epifluorescence microscopy. Impedance spectroscopy of microtubules and actin filaments decorated with ligands such as microtubule associated proteins and drugs and anesthetics is under way. Numerical estimates of the magnitudes of the currents, energy and power generated in a cancer cell using TTFields have been computed. This research provides physical insights into the subcellular mechanisms involved in TTField therapy and can assist in its' optimization. 1.Santelices, Iara B., et al. "Response to Alternating Electric Fields of Tubulin Dimers and Microtubule Ensembles in Electrolytic Solutions." Scientific Reports 7.1 (2017): 9594. Citation Format: Aarat P. Kalra, Jack Xiao, Cameron Hough, Piyush Kar, Vahid Rezania, John D. Lewis, Karthik Shankar, Jack A. Tuszynski. The molecular mechanism of action and cellular targets of TTFields [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3195.

Effects of sequentially applied single and combined temozolomide, hydroxychloroquine and AT101 treatment in a long-term stimulation glioblastoma in vitro model.

Glioblastoma multiforme (GBM) is a poorly curable disease due to its heterogeneity that enables single cells to survive treatment regimen and initiate tumor regrowth. Although some progress in therapy has been achieved in the last years, the efficient treatment of GBMs is still a clinical challenge. Besides the standard therapeutic drug temozolomide (TMZ), quinoline-based antimalarial drugs such as hydroxychloroquine (HCQ) and BH3 mimetics such as AT101 were considered as possible drugs for GBM therapy. We investigated the effects of sequentially applied single and combined TMZ, HCQ and AT101 treatments in a long-term stimulation GBM in vitro model. We performed all investigations in parallel in human astrocytes and two differentially TMZ-responsive human GBM cell lines and adjusted used drug concentrations to known liquor/plasma concentrations in patients. We determined amounts of dead cells and still remaining growth rates and depicted our results in a heatmap-like summary to visualize which sequential long-term treatment schedule seemed to be most promising. We showed that sequential stimulations yielded higher cytotoxicity and better tumor growth control in comparison to single TMZ treatment. This was especially the case for the sequences TMZ/HCQ and TMZ + AT101/AT101 which was as effective as the non-sequential combination TMZ + AT101. Importantly, those affected both less and more TMZ-responsive glioma cell lines, whilst being less harmful for astrocytes in comparison to single TMZ treatment. Sequential treatment with mechanistically different acting drugs might be an option to reduce side effects in long-term treatment, for example in local administration approaches.

A randomized controlled phase II trial of vaccination with lysate-loaded, mature dendritic cells integrated into standard radiochemotherapy of newly diagnosed glioblastoma (GlioVax): study protocol for a randomized controlled trial

BackgroundDespite the combination of surgical resection, radio- and chemotherapy, median survival of glioblastoma multiforme (GBM) patients only slightly increased in the last years. Disease recurrence is definite with no effective therapy existing after tumor removal. Dendritic cell (DC) vaccination is a promising active immunotherapeutic approach. There is clear evidence that it is feasible, results in immunological anti-tumoral responses, and appears to be beneficial for survival and quality of life of GBM patients. Moreover, combining it with the standard therapy of GBM may allow exploiting synergies between the treatment modalities. In this randomized controlled trial, we seek to confirm these promising initial results.MethodsOne hundred and thirty-six newly diagnosed, isocitrate dehydrogenase wildtype GBM patients will be randomly allocated (1:1 ratio, stratified by O6-methylguanine-DNA-methyltransferase promotor methylation status) after near-complete resection in a multicenter, prospective phase II trial into two groups: (1) patients receiving the current therapeutic “gold standard” of radio/temozolomide chemotherapy and (2) patients receiving DC vaccination as an add-on to the standard therapy. A recruitment period of 30 months is anticipated; follow-up will be 2 years. The primary objective of the study is to compare overall survival (OS) between the two groups. Secondary objectives are comparing progression-free survival (PFS) and 6-, 12- and 24-month OS and PFS rates, the safety profile, overall and neurological performance and quality of life.DiscussionUntil now, close to 500 GBM patients have been treated with DC vaccination in clinical trials or on a compassionate-use basis. Results have been encouraging, but cannot provide robust evidence of clinical efficacy because studies have been non-controlled or patient numbers have been low. Therefore, a prospective, randomized phase II trial with a sufficiently large number of patients is now mandatory for clear evidence regarding the impact of DC vaccination on PFS and OS in GBM.Trial registrationProtocol code: GlioVax, date of registration: 17. February 2017.Trial identifier: EudraCT-Number 2017–000304-14.German Registry for Clinical Studies, ID: DRKS00013248 (approved primary register in the WHO network) and at ClinicalTrials.gov, ID: NCT03395587. Registered on 11 March 2017.

A Repurposed Drug for Brain Cancer: Enhanced Atovaquone Amorphous Solid Dispersion by Combining a Spontaneously Emulsifying Component with a Polymer Carrier

Glioblastoma multiforme (GBM) is the most common and lethal central nervous system tumor. Recently, atovaquone has shown inhibition of signal transducer and activator transcription 3, a promising target for GBM therapy. However, it is currently unable to achieve therapeutic drug concentrations in the brain with the currently reported and marketed formulations. The present study sought to explore the efficacy of atovaquone against GBM as well as develop a formulation of atovaquone that would improve oral bioavailability, resulting in higher amounts of drug delivered to the brain. Atovaquone was formulated as an amorphous solid dispersion using an optimized formulation containing a polymer and a spontaneously emulsifying component (SEC) with greatly improved wetting, disintegration, dispersibility, and dissolution properties. Atovaquone demonstrated cytotoxicity against GBM cell lines as well as provided a confirmed target for atovaquone brain concentrations in in vitro cell viability studies. This new formulation approach was then assessed in a proof-of-concept in vivo exposure study. Based on these results, the enhanced amorphous solid dispersion is promising for providing therapeutically effective brain levels of atovaquone for the treatment of GBM.

Personalized regulation of glioblastoma cancer stem cells based on biomedical technologies: From theory to experiment (Review).

Glioblastoma multiforme (GBM) is one of the most aggressive brain tumors. GBM represents >50% of primary tumors of the nervous system and ~20% of intracranial neoplasms. Standard treatment involves surgery, radiation and chemotherapy. However, the prognosis of GBM is usually poor, with a median survival of 15 months. Resistance of GBM to treatment can be explained by the presence of cancer stem cells (CSCs) among the GBM cell population. At present, there are no effective therapeutic strategies for the elimination of CSCs. The present review examined the nature of human GBM therapeutic resistance and attempted to systematize and put forward novel approaches for a personalized therapy of GBM that not only destroys tumor tissue, but also regulates cellular signaling and the morphogenetic properties of CSCs. The CSCs are considered to be an informationally accessible living system, and the CSC proteome should be used as a target for therapy directed at suppressing clonal selection mechanisms and CSC generation, destroying CSC hierarchy, and disrupting the interaction of CSCs with their microenvironment and extracellular matrix. These objectives can be achieved through the use of biomedical cellular products.

FoxM1 drives ADAM17/EGFR activation loop to promote mesenchymal transition in glioblastoma

Mesenchymal transition (MES transition) is a hallmark of glioblastoma multiforme (GBM), however, the mechanism regulating the process remains to be elucidated. Here we report that FoxM1 drives ADAM17/EGFR activation loop to promote MES transition in GBM. Firstly, FoxM1 expression was positively associated with ADAM17 expression, and their expression was correlated with the mesenchymal features and overall patient survival of GBM. Overexpressing FoxM1 or ADAM17 increased the mesenchymal phenotype of glioma cells, which could be reversed by silencing FoxM1 or ADAM17. Importantly, FoxM1 bound to the ADAM17 promoter to transcriptionally upregulate its expression. Using gain- and loss-of-function studies, we showed that FoxM1/ADAM17 axis promoted the MES transition in glioma cells. Moreover, tissue microarray analysis and orthotopic xenograft model further confirmed that FoxM1/ADAM17 axis played key roles in malignancy of GBM. Mechanistically, FoxM1/ADAM17 axis activated the EGFR/AKT/GSK3β signaling pathway and ADAM17/EGFR/GSK3β axis could maintain FoxM1 stability in glioma cells. Taken together, our study demonstrated that FoxM1/ADAM17 feedback loop controlled the MES transition and regulated the progression of GBM, raising the possibility that deregulation of this loop might improve the durability of therapies in GBM.

In vitro evaluation of 225 Ac-DOTA-substance P for targeted alpha therapy of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant form of brain tumors with dismal prognosis despite treatment by surgery combined with radiotherapy and chemotherapy. The neuropeptide Substance P (SP) is the physiological ligand of the neurokinin-1 receptor, which is highly expressed in glioblastoma cells. Thus, SP represents a potential ligand for targeted alpha therapy. In this study, a protocol for the synthesis of SP labeled with the alpha emitter 225 Ac was developed and binding affinity properties were determined. The effects of 225 Ac-DOTA-SP were investigated on human glioblastoma cell lines (T98G, U87MG, U138MG) as well as GBM stem cells. A significant dose-dependent reduction in cell viability was detected up to 6days after treatment. Also, colony-forming capacity was inhibited at the lower doses tested. In comparison, treatment with the conventional agent temozolomide showed higher cell viability and colony-forming capacity. 225 Ac-DOTA-SP treatment caused induction of late apoptosis pathways. Cells were arrested to G2/M-phase upon treatment. Increasing doses and treatment time caused additional S-phase arrest. Similar results were obtained using human glioblastoma stem cells, known to show radioresistance. Our data suggest that 225 Ac-DOTA-SP is a promising compound for treatment of GBM.

Serum amyloid A1 in combination with integrin αVβ3 increases glioblastoma cells mobility and progression.

Glioblastoma multiforme (GBM) is a highly malignant type of brain tumor found in humans. GBM cells reproduce quickly, and the median survival time for patients after therapy is approximately 1 year with a high relapse rate. Current therapies and diagnostic tools for GBM are limited; therefore, we searched for a more favorable therapeutic target or marker protein for both therapy and diagnosis. We used mass spectrometry (MS) analysis to identify GBM‐associated marker proteins from human plasma and GBM cell cultures. Additional plasma and 52 brain tissues obtained from patients with gliomas were used to validate the association rate of serum amyloid A1 (SAA1) in different grades of gliomas and its distribution in tumors. Microarray database analysis further validated the coefficient of SAA1 levels in gliomas. The cellular mechanisms of SAA1 in GBM proliferation and infiltration were investigated in vitro. We analyzed the correlation between SAA1 and patients' medication requirement to demonstrate the clinical effects of SAA1 in GBM. SAA1 was identified from MS analysis, and its level was revealed to be correlated with the disease grade, clinical severity, and survival rate of patients with gliomas. In vitro cultures, including GBM cells and normal astrocytes, revealed that SAA1 promotes cell migration and invasion through integrin αVβ3 to activate the Erk signaling pathway. Magnetic resonance imaging and tumor region‐specific microarray analysis identified a correlation between SAA1 and GBM cell infiltration in patients. In summary, our results demonstrate that SAA1 in combination with integrin αV and β3 can serve as an indicator of high glioblastoma risk. We also identified the cellular mechanisms of SAA1 contributing to GBM progression, which can serve as the basis for future GBM therapy.

Duocarmycin SA, a potent antitumor antibiotic, sensitizes glioblastoma cells to proton radiation

New treatment modalities for glioblastoma multiforme (GBM) are urgently needed. Proton therapy is considered one of the most effective forms of radiation therapy for GBM. DNA alkylating agents such as temozolomide (TMZ) are known to increase the radiosensitivity of GBM to photon radiation. TMZ is a fairly impotent agent, while duocarmycin SA (DSA) is an extremely potent cytotoxic agent capable of inducing a sequence-selective alkylation of duplex DNA. Here, the effects of sub-nM concentrations of DSA on the radiosensitivity of a human GBM cell line (U-138) to proton irradiation were examined. Radiation sensitivity was determined by viability, apoptosis, necrosis and clonogenic assays. DSA concentrations as low as 0.001 nM significantly sensitized U-138 cells to proton irradiation. DSA demonstrates synergistic cytotoxicity against GBM cells treated with proton radiation in vitro, which may represent a novel therapeutic alternative for the treatment of GBM.

Glioblastoma-specific anti-TUFM nanobody for in-vitro immunoimaging and cancer stem cell targeting.

Glioblastoma multiforme (GBM) is the most common and lethal form of brain tumor. The prognosis for patients remains poor, despite the combination of new preoperative and intraoperative neuroimaging, radical surgery, and recent advances in radiotherapy and chemotherapy. To improve GBM therapy and patient outcome, sustained drug delivery to glioma cells is needed, while minimizing toxicity to adjacent neurons and glia cells. This might be achieved through an anti-proteomic approach based on nanobodies, the single-domain antigen-binding fragments of heavy-chain antibodies of the camelid adaptive immune system. We report here on the validation and quantification of a nanobody raised against mitochondrial translation elongation factor (TUFM). Differential expression of TUFM was examined in different GBM cell lines and GBM tissue at the protein and mRNA levels, as compared to their expression in neural stem cells and normal brain tissue. We further used in-silico modelling and immunocytochemistry to define the specificity of anti-TUFM nanobody (Nb206) towards GBM stem cells, as compared to GBM cell lines (U251MG and U87MG cells). Due to its specificity and pronounced inhibitory effect on GBM stem cell growth, we propose the use of this anti-TUFM nanobody for GBM in vitro immunoimaging and potentially also cancer stem cell targeting.

Drug Repositioning in Glioblastoma: A Pathway Perspective.

Glioblastoma multiforme (GBM) is the most malignant primary adult brain tumor. The current standard of care is surgical resection, radiation, and chemotherapy treatment, which extends life in most cases. Unfortunately, tumor recurrence is nearly universal and patients with recurrent glioblastoma typically survive <1 year. Therefore, new therapies and therapeutic combinations need to be developed that can be quickly approved for use in patients. However, in order to gain approval, therapies need to be safe as well as effective. One possible means of attaining rapid approval is repurposing FDA approved compounds for GBM therapy. However, candidate compounds must be able to penetrate the blood-brain barrier (BBB) and therefore a selection process has to be implemented to identify such compounds that can eliminate GBM tumor expansion. We review here psychiatric and non-psychiatric compounds that may be effective in GBM, as well as potential drugs targeting cell death pathways. We also discuss the potential of data-driven computational approaches to identify compounds that induce cell death in GBM cells, enabled by large reference databases such as the Library of Integrated Network Cell Signatures (LINCS). Finally, we argue that identifying pathways dysregulated in GBM in a patient specific manner is essential for effective repurposing in GBM and other gliomas.

Role of Chimeric Antigen Receptor T Cell Therapy in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common primary malignant cancer of brain, which is extremely aggressive and carries a dreadful prognosis. Current treatment protocol runs around radiotherapy, surgical resection, and temozolomide with median overall survival of around 12-15months. Due to its heterogeneity and mutational load, immunotherapy with chimeric antigen receptor (CAR) T cell therapy can be a promising treatment option for recurrent glioblastoma. Initial phase 1 studies have shown that this therapy is safe without dose-limiting side effects and it also has a better clinical outcome. Therefore, CAR T cell therapy can be a great future tool in our armamentarium to treat advanced GBM. In this article, we have explained the structure, mechanism of action, and rationale of CAR T cell therapy in GBM; we also discussed various antigenic targets and clinical outcome of initial studies of this novel therapy.

PD-L1 confers glioblastoma multiforme malignancy via Ras binding and Ras/Erk/EMT activation

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor due to the lack of effective therapeutic drugs. Cancer therapy targeting programmed cell death protein 1 (PD-1) or programmed death ligand-1 (PD-L1) is of revolutionary. However, the role of intrinsic PD-L1, which determines immune-therapy outcomes, remains largely unclear. Here we demonstrated an oncogenic role of PD-L1 via binding and activating Ras in GBM cells. RNA-sequencing transcriptome data revealed that PD-L1 significantly altered gene expression enriched in cell growth/migration/invasion pathways in human GBM cells. PD-L1 overexpression and knockout or knockdown demonstrated that PD-L1 promoted GBM cell proliferation and migration in vitro and in vivo. Mechanistically, PD-L1 prominently activated epithelial mesenchymal transition (EMT) process in a MEK/Erk- but not PI3K/Akt-dependent manner. Further, we identified intracellular interactions of PD-L1 and H-Ras, which led to Ras/Erk/EMT activation. Finally, we demonstrated that PD-L1 overexpression promoted while knockdown abolished GBM development and invasion in orthotopic GBM models of rodents. Taken together, we found that intracellular PD-L1 confers GBM cell malignancy and aggressiveness via binding Ras and activating the downstream Erk-EMT signaling. Thus, these results shed important insights in improving efficacy of immune therapy for GBM as well as other malignant tumors.

Therapeutic approach in glioblastoma multiforme with primitive neuroectodermal tumor components: Case report and review of the literature.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant glioma that is treated with first-line therapy, using surgical resection followed by local radiotherapy and concomitant/adjuvant temozolomide (TMZ) treatment. GBM is characterised by a high local recurrence rate and a low response to therapy. Primitive neuroectodermal tumour (PNET) of the brain revealed a low local recurrence rate; however, it also exhibited a high risk of cerebrospinal fluid (CSF) dissemination. PNET is treated with surgery followed by craniospinal irradiation (CSI) and platinum-based chemotherapy in order to prevent CSF dissemination. GBM with PNET-like components (GBM/PNET) is an emerging variant of GBM, characterised by a PNET-like clinical behaviour with an increased risk of CSF dissemination; it also may benefit from platinum-based chemotherapy upfront or following failure of GBM therapy. The results presented regarding the management of GBM/PNET are based on case reports or case series, so a standard therapeutic approach for GBM/PNET is not defined, constituing a challenging diagnostic and therapeutic dilemma. In this report, a case of a recurrent GBM/PNET treated with surgical resection and radiochemotherapy as Stupp protocol, and successive platinum-based chemotherapy due to the development of leptomeningeal dissemintation and an extracranial metastasis, is discussed. A review of the main papers regarding this rare GBM variant and its therapeutic approach are also reported. In conclusion, GBM/PNET should be treated with a multimodal approach including surgery, chemoradiotherapy, and/or the early introduction of CSI and platinum-based chemotherapy upfront or at recurrence.

The Role of Wnt Signal in Glioblastoma Development and Progression: A Possible New Pharmacological Target for the Therapy of This Tumor.

Wnt is a complex signaling pathway involved in the regulation of crucial biological functions such as development, proliferation, differentiation and migration of cells, mainly stem cells, which are virtually present in all embryonic and adult tissues. Conversely, dysregulation of Wnt signal is implicated in development/progression/invasiveness of different kinds of tumors, wherein a certain number of multipotent cells, namely “cancer stem cells”, are characterized by high self-renewal and aggressiveness. Hence, the pharmacological modulation of Wnt pathway could be of particular interest, especially in tumors for which the current standard therapy results to be unsuccessful. This might be the case of glioblastoma multiforme (GBM), one of the most lethal, aggressive and recurrent brain cancers, probably due to the presence of highly malignant GBM stem cells (GSCs) as well as to a dysregulation of Wnt system. By examining the most recent literature, here we point out several factors in the Wnt pathway that are altered in human GBM and derived GSCs, as well as new molecular strategies or experimental drugs able to modulate/inhibit aberrant Wnt signal. Altogether, these aspects serve to emphasize the existence of alternative pharmacological targets that may be useful to develop novel therapies for GBM.

NKG2D ligands in glioma stem-like cells: expression in situ and in vitro.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor. Tumor stem cells have a major influence on tumor malignancy, and immunological escape mechanisms, involving the Natural Killer Group 2, member D (NKG2D) receptor-ligand-system, are key elements in tumor immuno-surveillance. We analyzed the expression profile and localization of NKG2D ligands (NKG2DL) and embryonic and neural stem cell markers in solid human GBM and stem-like cells isolated from glioma cell lines by qRT-PCR and immunohistochemistry, including quantitative analysis. We also evaluated the effect of Temozolomide (TMZ), the standard chemotherapeutic agent used in GBM therapy, on NKG2DL expression. NKG2DL-positive cells were mostly found scattered and isolated, were detectable in glial fibrillary acidic protein (GFAP)-positive tumor regions and partly in the penumbra of tumor vessels. NKG2DL were found in a distinct tumor stem-like cell subpopulation and were broadly costained with each other. Quantitative analysis revealed, that dependent on the individual NKG2DL investigated, cell portions costained with different stem cell markers varied between small (Musashi-1) and high (KLf-4) amounts. However, a costaining of NKG2DL with CD3γ, typically found in T cells, was also observable, whereas CD11b as a marker for tumor micoglia cells was only rarely costained with NKG2DL. Stem-like cells derived from the glioma cell lines T98G and U251MG showed a distinct expression pattern of NKG2DL and stem cell markers, which seemed to be balanced in a cell line-specific way. With differentiation, T98G displayed less NKG2DL, whereas in U251MG, only expression of most stem cell markers decreased. In addition, stimulation with TMZ led to a significant upregulation of NKG2DL in stem-like cells of both lines. As stem-like glioma cells tend to show a higher expression of NKG2DL than more differentiated tumor cells and TMZ treatment supports upregulation of NKG2DL, the NKG2D system might play an important role in tumor stem cell survival and in GBM therapy.

Kinin-B1 Receptor Stimulation Promotes Invasion and is Involved in Cell-Cell Interaction of Co-Cultured Glioblastoma and Mesenchymal Stem Cells

Glioblastoma multiforme (GBM) represents the most lethal brain tumour, and these tumours have very limited treatment options. Mesenchymal stem cells (MSC) are considered as candidates for advanced cell therapies, due to their tropism towards GBM, possibly affecting their malignancy, thus also representing a potential therapeutic vector. Therefore, we aimed to compare the effects of bone-marrow-derived versus adipose-tissue-derived MSC (BM-/AT-MSC) on heterogeneous populations of tumour cells. This cells’ interplay was addressed by the in-vitro two-dimensional (monolayer) and three-dimensional (spheroid) co-culture models, using U87 and U373 GBM cell lines, expressing genotypically different mesenchymal transcriptome profiles. U87 cell low mesenchymal profile expressed high levels of kinin receptor 1 (B1R) and their invasion was greatly enhanced by the B1R agonist des-Arg9-bradykinin upon BM-MSC co-culturing in 3D co-cultures. This correlated to significantly higher cell-cell interactions in U87/BM-MSC mixed spheroids. This was not observed with the U373 cells and not in AT-MSC co-cultures. Altogether, these data support the on-going exploration of B1R as target for adjuvant approach in GBM therapy. Secondly, the results emphasize the need for further careful exploration of the selectivity regarding the origin of MSC as potential candidates for cell therapies, particular in cancer, where they may adversely affect heterogeneous tumour cell populations.

Apoptotic Effects of Temozolomide and Naturopathic Agents upon Glioblastoma Cells

Temozolomide (TMZ), thymoquinone (TMQ), epigallocatechin gallate (EPIGAL) and staurosporine (STAURO) were used as apoptotic inducing agents acting upon the U87-MG (ATCC, HTB15), LN18 (ATCC, CRL2610) and U118-MG (ATCC, HTB14) glioblastoma multiforme (GBM) cell lines. TMZ is the current drug of choice for primary treatment and adjuvant therapy for recurrent GBM. TMQ and EPIGAL are naturopathic agents, while STAURO is a well-studied apoptotic-inducing agent. The degree and time course of apoptosis were measured by flow cytometry techniques capable of detecting changes in mitochondrial function using the fluorescent dye MitoTracker Deep Red. Phosphatidylserine exposure and plasma membrane permeability were detected simultaneously using violet fluorescent reactive dye (VFRD) in combination with AnnexinV-488. The apoptotic effectiveness of the inducing agents TMZ, TMQ, EPIGAL and STAURO were compared with their ability to inhibit invasiveness and degrade Class I major histocompatibility complex (MHC) determinants. Invasiveness was measured in vitro by the 3D matrigel spheroid invasion assay. The density of the class 1 MHC determinants was measured by flow cytometry. Although TMZ is widely used for the chemotherapeutic treatment of GBM, it was determined that the time course for TMZ induced apoptosis was slower than those of TMQ, EPIGAL and STAURO. Unexpectedly, TMZ was ineffective in its ability to inhibit in vitro invasiveness and did not degrade the class I MHC determinants as effectively as the other apoptotic inducing agents. The findings raise the question of whether in vitro assays of apoptosis and invasiveness are the best measures of the effectiveness of chemotherapeutic agents for the primary treatment and adjuvant therapy of recurrent GBM. The findings also point to the in vivo complexity of the efficacy of chemotherapeutic agents whereby preserving the components of natural and acquired immune mechanisms may be more important than the rapid apoptotic effects of the chemotherapeutic agent.

Abstract B058: Role of IL8-CXCR1/2 axis in glioblastoma cell proliferation, invasion, and vascular mimicry

Abstract Background: Glioblastoma multiforme (GBM) is the most fatal neoplasm of the central nervous system. There is an ongoing quest for improving GBM therapy owing to limited success in current treatment modalities. Chemokines are known to play a critical role in tumor cell proliferation and invasion. Previously we had studied gene expression of complete panel of chemokines and their receptors to identify pro-gliomagenic chemokine axis. Based on our gene expression study and comparison with other publicly available datasets, we identified IL-8 as most frequently upregulated chemokine in GBM as compared to low-grade astrocytoma. In the present study we aimed to elucidate the role of IL-8-CXCR1/2 axes in GBM progression. Methods: The protein expression pattern of IL-8 and its receptors, CXCR1 and CXCR2, in GBM was studied using immunohistochemistry. Furthermore, we checked the impact of targeting IL-8 in vitro in two GBM cell lines to understand its direct role in GBM cell proliferation and invasion. We first checked the expression of IL-8-CXCR1/2 axes by RT-PCR and immunocytochemistry. IL-8-CXCR1/2 axes were targeted using neutralizing antibodies as well as by pharmacologic antagonist reparixin-l-lysine. The impact on cell proliferation and viability was assessed by MTT assay and clonogenic survival assay. Spheroid invasion assay was performed to assess the impact on invasion. Results: The protein localization by immunohistochemistry revealed cytoplasmic expression of IL-8 and CXCR2 in tumor cells while CXCR1 was predominantly present in tumor-associated microvessels, suggesting both autocrine and paracrine modes of signalling. However, the exact biologic action exerted by IL-8-CXCR1/2 axes was unraveled in in vitro experiments where discernible reduction in cell proliferation and survival was observed in U-87MG and LN-18 cell lines on neutralizing IL-8 and its receptors with neutralizing antibodies. Similar effect was also observed on treatment with reparixin-l-lysine. Pharmacologic blocking of CXCR1/2 also resulted in significant reduction in spheroid invasion and clonogenic survival in U-87MG and LN-18 cell lines. Neutralizing CXCR1 alone or in combination with IL-8 resulted in disruption of tubular network in U-87MG cell line, suggesting a possible role in vascular mimicry (VM). Further comparison of CXCR1 and CD34 immunostaining conceded existence of CXCR1+/CD34- microvessels in GBM, confirming the involvement of IL-8-CXCR1 axis in VM structure formation. Conclusion: These results suggest an important role of IL-8-CXCR1/2 axes in GBM biology, which supports their mitogenic as well as angiogenic function, displaying a strong potential as candidate therapeutic target in treatment of GBM. Citation Format: Ira Sharma, Avninder Singh, Fouzia Siraj, Sunita Saxena. Role of IL8-CXCR1/2 axis in glioblastoma cell proliferation, invasion, and vascular mimicry [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B058.