Glioblastoma Cultures Research Articles

CSIG-05. DISSOCIATION OF THE INTRAMOLECULARLY CLEAVED N- AND C-TERMINAL FRAGMENTS OF ADHESION G PROTEIN-COUPLED RECEPTOR GPR133 (ADGRD1) INCREASES ITS CANONICAL SIGNALING IN GLIOBLASTOMA

Abstract We previously demonstrated that GPR133 (ADGRD1), an adhesion GPCR that signals via cytosolic cAMP increase, is de novo expressed in glioblastoma (GBM) and enriched in patient-derived glioblastoma stem cells. Knockdown of GPR133 reduces GBM cell proliferation and tumorsphere formation, and abolishes orthotopic xenograft initiation in vivo. GPR133’s requirement for GBM growth and its absence in non-malignant brain suggest its therapeutic potential, yet its mechanisms of action and activation remained unclear. Here, we demonstrate in patient-derived GBM cultures and HEK293T cells that GPR133 gets intramolecularly cleaved into N-terminal and C-terminal fragments (NTF and CTF) right after synthesis in the endoplasmic reticulum. The resulting NTF and CTF remain non-covalently bound to each other, until the mature receptor reaches the plasma membrane, where we observe dissociation of the extracellular NTF from the transmembrane-spanning CTF. While cleavage is not required for correct subcellular trafficking, the cleaved wild-type GPR133 generates significantly higher cytosolic cAMP levels than an uncleavable point mutant GPR133 (H543R), suggesting that cleavage and dissociation are involved in receptor activation. To test this hypothesis in a more controllable proxy system, we generated a fusion of the CTF of GPR133 and the N-terminus of human protease-activated receptor 1 (hPAR1). Indeed, acute thrombin-induced cleavage and shedding of the hPAR1 NTF increases intracellular cAMP levels generated by the GPR133 CTF. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. To test whether extracellular binding proteins could influence NTF shedding and/or GPR133 signaling activation, we conducted ligand discovery screens and indeed found a new GPR133 binding protein in GBM cells, which is capable of influencing receptor signaling. Together, these findings provide critical insights into GPR133’s mechanism of activation, that will guide future approaches of therapeutic targeting of GPR133 in GBM.

TAMI-35. DETECTING SINGLE-CELL INTERACTIONS IN ORGANOTYPIC CULTURES OF GLIOBLASTOMA USING BARCODED RABIES VIRUS

Abstract Cell-cell interactions are thought to drive tumor-promoting signals in the microenvironment of glioblastoma, but standard approaches for single cell analysis do not directly identify cell interactions and the mechanisms that mediate them. We recently developed a novel method to analyze cell-cell interactions—rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNAseq). RABID-seq was first implemented in transgenic mice to investigate the interactions of astrocytes with other cells in the CNS enabling the study of astrocyte connectome perturbations and candidate therapeutic targets in multiple sclerosis and its pre-clinical model, experimental autoimmune encephalomyelitis (EAE). Here, we report the first use of RABID-seq in human tissues in organotypic cultures established from three IDH-wildtype glioblastoma (GBM) patients. In organotypic GBM cultures, initial infection by pseudotyped barcoded rabies virus deficient for viral glycoprotein was achieved after previous culture transduction with a lentivirus containing the avian TVA receptor and rabies glycoprotein under the human EF1a promoter. We employed this system to initially infect approximately 1,000 malignant or non malignant cells in the tumor microenvironment. After five days, infected cells were isolated from cultures and processed for single cell analysis using SMART seq. We were able to capture at least 6,000 interacting cells per tumor specimen, from which barcodes were recovered and cDNA was sent for sequencing. Here we present connectomic data from our initial cohort of three glioblastoma patients as an introduction to RABID-seq, with a focus on astrocyte-tumor interactions. Candidate mechanisms of cellular interactions will undergo functional validation in murine models of glioblastoma.

TAMI-10. CHARACTERIZATION OF CANCER-ASSOCIATED FIBROBLASTS IN GBM AND DEFINING THEIR PRO-TUMORAL EFFECTS

Abstract Cancer-associated fibroblasts (CAFs) constitute a key component of the tumor microenvironment. While pro-tumoral CAFs have been identified in some cancers, CAFs had been presumed absent in glioblastoma given the lack of brain fibroblasts. We found that serial trypsinization of primary glioblastoma cultures yields cells that morphologically resemble fibroblasts and transcriptomically resemble CAFs as shown by bulk RNA-seq and single-cell RNA-seq. Moreover, Single-cell RNA-seq from patient GBMs showed a mesenchymal lineage for CAFs. We demonstrate that Glioblastoma CAFs are chemotactically attracted to glioblastoma stem cells (GSCs) and CAFs enriched GSCs. To identify CAF/GSC interaction mediators, we created a resource of inferred crosstalk by mapping the expression of receptors to their cognate ligands/agonists. This analysis suggested PDGF-b and TGF-b as mediators of GSC recruitment and proliferation of CAFs, and osteopontin and hepatocyte growth factor (HGF) as mediators of CAF-induced GSC enrichment, hypothesis confirmed by blocking antibodies. Glioblastoma CAFs also induce hypertrophied vessels and M2 macrophage polarization, the latter through unique CAF production of the EDA fibronectin variant which binds macrophage toll-like receptor 4 (TLR4) in a targetable manner. Glioblastoma CAFs were enriched in the subventricular zone which houses the neural stem cells that produce GSCs. Depleting CAFs in GSC-derived xenografts slowed their in vivo growth. These findings are among the first to identify glioblastoma CAFs and reveal their involvement with GSCs, making them an intriguing target.

Exploring the efficacy of tumor electric field therapy against glioblastoma: An in vivo and in vitro study.

AimsTumor electric fields therapy (TTFields) is emerging as a novel anti‐cancer physiotherapy. Despite recent breakthroughs of TTFields in glioma treatment, the average survival time for glioblastoma patients with TTFields is <2 years, even when used in conjugation with traditional anti‐cancer therapies. To optimize TTFields‐afforded efficacy against glioblastoma, we investigated the cancer cell‐killing effects of various TTFields paradigms using in vitro and in vivo models of glioblastoma.MethodsFor in vitro studies, the U251 glioma cell line or primary cell cultures prepared from 20 glioblastoma patients were treated with the tumor electric field treatment (TEFT) system. Cell number, volume, and proliferation were measured after TEFT at different frequencies (100, 150, 180, 200, or 220 kHz), durations (24, 48, or 72 h), field strengths (1.0, 1.5, or 2.2V/cm), and output modes (fixed or random sequence output). A transwell system was used to evaluate the influence of TEFT on the invasiveness of primary glioblastoma cells. For in vivo studies, the therapeutic effect and safety profiles of random sequence electric field therapy in glioblastoma‐transplanted rats were assessed by calculating tumor size and survival time and evaluating peripheral immunobiological and blood parameters, respectively.ResultsIn the in vitro settings, TEFT was robustly effective in suppressing cell proliferation of both the U251 glioma cell line and primary glioblastoma cell cultures. The anti‐proliferation effects of TEFT were frequency‐ and “dose” (field strength and duration)‐dependent, and contingent on the field sequence output mode, with the random sequence mode (TEFT‐R) being more effective than the fixed sequence mode (TEFT‐F). Genetic tests were performed in 11 of 20 primary glioblastoma cultures, and 6 different genetic traits were identified them. However, TEFT exhibited comparable anti‐proliferation effects in all primary cultures regardless of their genetic traits. TEFT also inhibited the invasiveness of primary glioblastoma cells in transwell experiments. In the in vivo rat model of glioblastoma brain transplantation, treatment with TEFT‐F or TEFT‐R at frequency of 200 kHz and field strength of 2.2V/cm for 14 days significantly reduced tumor volume by 42.63% (TEFT‐F vs. control, p = 0.0002) and 63.60% (TEFT‐R vs. control, p < 0.0001), and prolonged animal survival time by 30.15% (TEFT‐F vs. control, p = 0.0415) and 69.85% (TEFT‐R vs. control, p = 0.0064), respectively. The tumor‐bearing rats appeared to be well tolerable to TEFT therapies, showing only moderate increases in blood levels of creatine and red blood cells. Adverse skin reactions were common for TEFT‐treated rats; however, skin reactions were curable by local treatment.ConclusionTumor electric field treatment at optimal frequency, strength, and output mode markedly inhibits the cell viability, proliferation, and invasiveness of primary glioblastoma cells in vitro independent of different genetic traits of the cells. Moreover, a random sequence electric field output confers considerable anti‐cancer effects against glioblastoma in vivo. Thus, TTFields are a promising physiotherapy for glioblastoma and warrants further investigation.

The polarity protein Par3 coordinates positively self-renewal and negatively invasiveness in glioblastoma

Glioblastoma (GBM) is a brain malignancy characterized by invasiveness to the surrounding brain tissue and by stem-like cells, which propagate the tumor and may also regulate invasiveness. During brain development, polarity proteins, such as Par3, regulate asymmetric cell division of neuro-glial progenitors and neurite motility. We, therefore, studied the role of the Par3 protein (encoded by PARD3) in GBM. GBM patient transcriptomic data and patient-derived culture analysis indicated diverse levels of expression of PARD3 across and independent from subtypes. Multiplex immunolocalization in GBM tumors identified Par3 protein enrichment in SOX2-, CD133-, and NESTIN-positive (stem-like) cells. Analysis of GBM cultures of the three subtypes (proneural, classical, mesenchymal), revealed decreased gliomasphere forming capacity and enhanced invasiveness upon silencing Par3. GBM cultures with suppressed Par3 showed low expression of stemness (SOX2 and NESTIN) but higher expression of differentiation (GFAP) genes. Moreover, Par3 silencing reduced the expression of a set of genes encoding mitochondrial enzymes that generate ATP. Accordingly, silencing Par3 reduced ATP production and concomitantly increased reactive oxygen species. The latter was required for the enhanced migration observed upon silencing of Par3 as anti-oxidants blocked the enhanced migration. These findings support the notion that Par3 exerts homeostatic redox control, which could limit the tumor cell-derived pool of oxygen radicals, and thereby the tumorigenicity of GBM.

RARE INVASIVE FUNGAL SPECIES IN SUSCEPTIBLE HOSTS

TOPIC: Critical Care TYPE: Medical Student/Resident Case Reports INTRODUCTION: We present a case of a 31 year old immunocompromised patient secondary to lupus complicated by renal failure on hemodialysis who presented with fever diagnosed with a rare invasive fungal infection. CASE PRESENTATION: Patient presented due to an ongoing fever of 2 months after recently being treated with empiric antibiotics for presumed pneumonia and septic arthritis of his knee at an outside hospital. Work up for fever of unknown origin was obtained and started on empiric antibiotics. On day 4 of admission, CT head was obtained due to an acute focal neurologic deficit (Image 1) which showed a lesion in the left basal ganglia and subsequently had to be intubated fur acute hypoxic respiratory failure. Due to his immunocompromised state, CNS targeting empiric antibiotics and antifungals were initiated. Biopsy of the lesion and repeat brain imaging was delayed due to thrombocytopenia and hemodynamic instability. All work up for his fever was negative except for a positive fungitell and his subsequent imaging showed rapidly progressing lesion to encompass the left cerebral hemisphere and extending to the right cerebral hemisphere (Image 2). Urgent frontal craniotomy with biopsy initially reported high grade glioblastoma but final culture of the sample grew Scedosporium boydii. Voriconazole was started; however, due to the extensive spread of the fungi the patient passed away shortly after. DISCUSSION: Clinical spectrum of Scedosporiosis in immunocompromised hosts range from keratitis, endophthalmitis, otitis, sinusitis, pulmonary infections and rarely, CNS infections [1-8]. Risk factors include diabetes, transplant recipients, leukemia, lupus, lymphoma and drowning [1,3]. Brain abscess, meningitis and mycotic aneurysms are common CNS manifestations and diagnosis are usually delayed due to difficulty isolating the Scedosporium spp [1-3]. Histopathology showing hyaline hyphae with regular septation and dichotomous branching can be confused with Aspergillus or Fusarium spp [1,2,4,5]. Isolation of the fungus with appropriate culture and media is crucial in accuracy of diagnosis and for subsequent treatment plan. Scedosporium spp are resistance to amphotericin B and has wide variability of susceptibility to itraconazole, voriconazole, posaconazole and micafungin [1-3]. If diagnosed early, surgical resection of the abscess is preferred treatment option with voriconazole depending on susceptibility[1-3]. Therefore, early diagnosis with appropriate mode of isolation is crucial in treating invasive fungal infection due to high morbidity and mortality associated. CONCLUSIONS: Accuracy of sample isolation is difficult for Scedosporium and usually leads to delay in treatment. In our patient, only the biopsy of the brain abscess showed histopathological evidence of Scedosporium spp., reminding us of the importance of early recognition of possible rare causes of infection in susceptible hosts. REFERENCE #1: Ramirez-Garcia A, Pellon A, Rementeria A, Buldain I, Barreto-Bergter E, Rollin-Pinheiro R, de Meirelles JV, Xisto MIDS, Ranque S, Havlicek V, Vandeputte P, Govic YL, Bouchara JP, Giraud S, Chen S, Rainer J, Alastruey-Izquierdo A, Martin-Gomez MT, López-Soria LM, Peman J, Schwarz C, Bernhardt A, Tintelnot K, Capilla J, Martin-Vicente A, Cano-Lira J, Nagl M, Lackner M, Irinyi L, Meyer W, de Hoog S, Hernando FL. Scedosporium and Lomentospora: an updated overview of underrated opportunists. Med Mycol. 2018 Apr 1;56(suppl_1):102-125. doi: 10.1093/mmy/myx113. PMID: 29538735. REFERENCE #2: Tortorano AM, Richardson M, Roilides E, van Diepeningen A, Caira M, Munoz P, Johnson E, Meletiadis J, Pana ZD, Lackner M, Verweij P, Freiberger T, Cornely OA, Arikan-Akdagli S, Dannaoui E, Groll AH, Lagrou K, Chakrabarti A, Lanternier F, Pagano L, Skiada A, Akova M, Arendrup MC, Boekhout T, Chowdhary A, Cuenca-Estrella M, Guinea J, Guarro J, de Hoog S, Hope W, Kathuria S, Lortholary O, Meis JF, Ullmann AJ, Petrikkos G, Lass-Flörl C; European Society of Clinical Microbiology and Infectious Diseases Fungal Infection Study Group; European Confederation of Medical Mycology. ESCMID and ECMM joint guidelines on diagnosis and management of hyalohyphomycosis: Fusarium spp., Scedosporium spp. and others. Clin Microbiol Infect. 2014 Apr;20 Suppl 3:27-46. doi: 10.1111/1469-0691.12465. PMID: 24548001. REFERENCE #3: Seidel D, Meißner A, Lackner M, Piepenbrock E, Salmanton-García J, Stecher M, Mellinghoff S, Hamprecht A, Durán Graeff L, Köhler P, Cheng MP, Denis J, Chedotal I, Chander J, Pakstis DL, Los-Arcos I, Slavin M, Montagna MT, Caggiano G, Mares M, Trauth J, Aurbach U, Vehreschild MJGT, Vehreschild JJ, Duarte RF, Herbrecht R, Wisplinghoff H, Cornely OA. Prognostic factors in 264 adults with invasive Scedosporium spp. and Lomentospora prolificans infection reported in the literature and FungiScope®. Crit Rev Microbiol. 2019 Feb;45(1):1-21. doi: 10.1080/1040841X.2018.1514366. Epub 2019 Jan 10. Erratum in: Crit Rev Microbiol. 2019 Feb 18;:1. PMID: 30628529. DISCLOSURES: No relevant relationships by Chandni Bheeman, source=Web Response No relevant relationships by Sung Hong, source=Web Response No relevant relationships by Karan Omidvari, source=Web Response No relevant relationships by Anuja Pradhan, source=Web Response No relevant relationships by Ronak Shah, source=Web Response

P02.02 Modeling of response to irradiation in recurrence glioblastoma’s cells culture

Abstract BACKGROUND Radiosensitivity of glioblastoma (GB) cells of local relapses may be markedly different from the primary tumor. Optimal doses and regimes of re-irradiation GB recurrence is not determined yet. MATERIAL AND METHODS GO1 primary GB cell culture was obtained during removal of a recurrent tumor after combined treatment, including irradiation of the surgical bed. Сell’s culture was irradiated by photon beams with energy 6 MeV and dose rate 600 MU/min. Irradiation performed in 1, 3 and 5 fractions, by 10 different doses for each regime. The dose range was determined experimentally for one fraction (5–250 Gy); for other regimes it was calculated according to the biological equivalent dose conception (3 fractions: 5–450 Gy, 5 fractions: 5–550 Gy). The proliferative activity of cells was investigate by MTT test. The results were normalized to the control. Dose-effect curves were plotted for each irradiation regime.The experimental data were approximated by calculated curves obtained by selecting the optimal parameters of the LQ-model and it’s modification. RESULTS Irradiation of GO1 by 1 fraction with the dose 5–250 Gy, causes a slow decrease in proliferative activity, which reaches a minimum value of 23% at 150 Gy and then remains constant. After irradiation by 3 fractions, proliferative activity of the GO1 gradually decrease only at a total dose over 120 Gy and reaches 37% after 450 Gy. When GO1 was irradiated in 5 fractions, a similar dose-effect curve was obtained, gradual decrease was observed to a value of 52% in the range of 250–500 Gy. Thus, the experimental dose-effect curves for irradiation of recurrence GB cells for 3 and 5 fractions have the appreciable “shoulder”, which could be explained by increased radioresistance. When approximating the experimental data by fitting the parameters of the LQ-model, the use of α/β = 8 provided the slope of the curve, close to the experimental data. For reflecting the “shoulder” an additional summand was introduced into the mathematical expression for the number of proliferating cells - a 105 Gy for 3 fractions and 255 Gy for 5 fractions. CONCLUSION Modified LQ-model could be used for an adequate mathematical description of the effectiveness of fractionated irradiation in relapsed GB culture cells in vitro. It’s necessary to introduce a summand into the formula that determines the formation of a “shoulder” on the dose-effect curve for this. The research was supported financially by RFBR (Project No. 18-29-01061).

OS06.9A Diversity of cellular communication in glioblastoma

Abstract BACKGROUND Owing to recent advances in understanding of the active functional states exhibited within glioblastoma (GBM), intra-tumoral cellular signaling has moved into focus of neuro-oncological research. In our study, we aim to explore the diversity of transcellular signaling and investigate correlations to transcriptional dynamics and cellular behavior. MATERIAL AND METHODS Electrophysiological mapping of primary GBM cultures was performed by planar microelectrodes, in conjunction with calcium imaging in a human neocortical section based GBM model. Exposure to conditions that are physiologically present within the tumor was carried out to identify specific signaling cells of interest and signaling diversity presented as response to specific environmental conditions. Transcriptional dynamics and plasticity were examined by means of scRNA-sequencing with CRISPR based perturbation, spatial transcriptomics and deep long-read RNA-sequencing. RESULTS Electrophysiological profiles of primary GBM cell lines revealed highly variable network activity. Despite these different characteristics, all profiled primary cell-lines exhibited characteristics of scale-free networks, confirmed in a human neocortical GBM model. When the GBM was allowed to grow in “in-vivo” like environment, basal activity was significantly increased, owing to interactions with elements within the neural environment. Cellular signaling was directly correlated to changes in the environment, like hypoxia or glutamatergic activation, and total inhibition of electrical signaling was achieved only with a combination of both gap junction and synaptic inhibitors. Using single-cell sequencing and proteomics, we identified several genes related to synaptogenesis that plays a crucial role in network formation and consequently transcellular signaling. CRISPR based perturbation of these genes resulted in alterations in cellular morphology and decreased cellular connectivity, with electrical signaling being significantly attenuated. Single-cell sequencing of perturbed tumor cells in the GBM model revealed a loss of developmental lineages and significant reduction of cellular stress response state. CONCLUSION Our findings highlight the role of electrical signaling in glioblastoma. Cellular stressors induce intercellular signaling, leading to transcriptional adaptation suggesting that there exists a highly complex and powerful mechanism for dynamic transcriptional state adaptation.

The protein kinase LKB1 promotes self-renewal and blocks invasiveness in glioblastoma.

The role of liver kinase B1 (LKB1) in glioblastoma (GBM) development remains poorly understood. LKB1 may regulate GBM cell metabolism and has been suggested to promote glioma invasiveness. After analyzing LKB1 expression in GBM patient mRNA databases and in tumor tissue via multiparametric immunohistochemistry, we observed that LKB1 was localized and enriched in GBM tumor cells that co-expressed SOX2 and NESTIN stemness markers. Thus, LKB1-specific immunohistochemistry can potentially reveal subpopulations of stem-like cells, advancing GBM patient molecular pathology. We further analyzed the functions of LKB1 in patient-derived GBM cultures under defined serum-free conditions. Silencing of endogenous LKB1 impaired 3D-gliomasphere frequency and promoted GBM cell invasion in vitro and in the zebrafish collagenous tail after extravasation of circulating GBM cells. Moreover, loss of LKB1 function revealed mitochondrial dysfunction resulting in decreased ATP levels. Treatment with the clinically used drug metformin impaired 3D-gliomasphere formation and enhanced cytotoxicity induced by temozolomide, the primary chemotherapeutic drug against GBM. The IC50 of temozolomide in the GBM cultures was significantly decreased in the presence of metformin. This combinatorial effect was further enhanced after LKB1 silencing, which at least partially, was due to increased apoptosis. The expression of genes involved in the maintenance of tumor stemness, such as growth factors and their receptors, including members of the platelet-derived growth factor (PDGF) family, was suppressed after LKB1 silencing. The defect in gliomasphere growth caused by LKB1 silencing was bypassed after supplementing the cells with exogenous PFDGF-BB. Our data support the parallel roles of LKB1 in maintaining mitochondrial homeostasis, 3D-gliomasphere survival, and hindering migration in GBM. Thus, the natural loss of, or pharmacological interference with LKB1 function, may be associated with benefits in patient survival but could result in tumor spread.

Transcriptional CDK inhibitors, CYC065 and THZ1 promote Bim-dependent apoptosis in primary and recurrent GBM through cell cycle arrest and Mcl-1 downregulation

Activation of cyclin-dependent kinases (CDKs) contributes to the uncontrolled proliferation of tumour cells. Genomic alterations that lead to the constitutive activation or overexpression of CDKs can support tumourigenesis including glioblastoma (GBM), the most common and aggressive primary brain tumour in adults. The incurability of GBM highlights the need to discover novel and more effective treatment options. Since CDKs 2, 7 and 9 were found to be overexpressed in GBM, we tested the therapeutic efficacy of two CDK inhibitors (CKIs) (CYC065 and THZ1) in a heterogeneous panel of GBM patient-derived cell lines (PDCLs) cultured as gliomaspheres, as preclinically relevant models. CYC065 and THZ1 treatments suppressed invasion and induced viability loss in the majority of gliomaspheres, irrespective of the mutational background of the GBM cases, but spared primary cortical neurons. Viability loss arose from G2/M cell cycle arrest following treatment and subsequent induction of apoptotic cell death. Treatment efficacies and treatment durations required to induce cell death were associated with proliferation velocities, and apoptosis induction correlated with complete abolishment of Mcl-1 expression, a cell cycle-regulated antiapoptotic Bcl-2 family member. GBM models generally appeared highly dependent on Mcl-1 expression for cell survival, as demonstrated by pharmacological Mcl-1 inhibition or depletion of Mcl-1 expression. Further analyses identified CKI-induced Mcl-1 loss as a prerequisite to establish conditions at which the BH3-only protein Bim can efficiently induce apoptosis, with cellular Bim amounts strongly correlating with treatment efficacy. CKIs reduced proliferation and promoted apoptosis also in chick embryo xenograft models of primary and recurrent GBM. Collectively, these studies highlight the potential of these novel CKIs to suppress growth and induce cell death of patient-derived GBM cultures in vitro and in vivo, warranting further clinical investigation.

The Leloir Cycle in Glioblastoma: Galactose Scavenging and Metabolic Remodeling.

Simple SummaryProliferation of glioblastoma (GBM) depends on access to extracellular nutrients. Inadequate tumor perfusion creates a short supply of the most common nutrients, such as glucose (Glc) and glutamine. As a result, GBMs undergo metabolic remodeling and scavenge alternative nutrients from the tumor microenvironment for their growth and proliferation. GBM expresses sugar transporters, such as Glut3 and Glut14. Galactose (Gal) is a good substrate for Glut3/Glut14, and GBM cells can scavenge Gal from the circulation/extracellular space. The Leloir pathway allows GBM to transport and metabolize Gal at physiologic Glc concentrations, providing GBM cells with an alternate energy source. GBM cultures proliferated when grown solely on Gal. GBMs metabolize Gal via the Leloir pathway, glycolysis, and pentose phosphate pathway (PPP) to optimize ATP production, while the mitochondrial metabolism of Gal in GBM is limited. The selective targeting of the Leloir/PPP pathway may provide new treatment strategies for GBM.Background: Glioblastoma (GBM) can use metabolic fuels other than glucose (Glc). The ability of GBM to use galactose (Gal) as a fuel via the Leloir pathway is investigated. Methods: Gene transcript data were accessed to determine the association between expression of genes of the Leloir pathway and patient outcomes. Growth studies were performed on five primary patient-derived GBM cultures using Glc-free media supplemented with Gal. The role of Glut3/Glut14 in sugar import was investigated using antibody inhibition of hexose transport. A specific inhibitor of GALK1 (Cpd36) was used to inhibit Gal catabolism. Gal metabolism was examined using proton, carbon and phosphorous NMR spectroscopy, with 13C-labeled Glc and Gal as tracers. Results: Data analysis from published databases revealed that elevated levels of mRNA transcripts of SLC2A3 (Glut3), SLC2A14 (Glut14) and key Leloir pathway enzymes correlate with poor patient outcomes. GBM cultures proliferated when grown solely on Gal in Glc-free media and switching Glc-grown GBM cells into Gal-enriched/Glc-free media produced elevated levels of Glut3 and/or Glut14 enzymes. The 13C NMR-based metabolic flux analysis demonstrated a fully functional Leloir pathway and elevated pentose phosphate pathway activity for efficient Gal metabolism in GBM cells. Conclusion: Expression of Glut3 and/or Glut14 together with the enzymes of the Leloir pathway allows GBM to transport and metabolize Gal at physiological glucose concentrations, providing GBM cells with an alternate energy source. The presence of this pathway in GBM and its selective targeting may provide new treatment strategies.

Nanocell-mediated delivery of miR-34a counteracts temozolomide resistance in glioblastoma

BackgroundGlioblastoma is the most common primary brain tumor and remains uniformly fatal, highlighting the dire need for developing effective therapeutics. Significant intra- and inter-tumor heterogeneity and inadequate delivery of therapeutics across blood–brain barrier continue to be significant impediments towards developing therapies which can significantly enhance survival. We hypothesize that microRNAs have the potential to serve as effective therapeutics for glioblastoma as they modulate the activity of multiple signaling pathways, and hence can counteract heterogeneity if successfully delivered.MethodsUsing a computational approach, we identified microRNA-34a as a microRNA that maximally reduces the activation status of the three core signaling networks (the receptor tyrosine kinase, p53 and Rb networks) that have been found to be deregulated in most glioblastoma tumors. Glioblastoma cultures were transfected with microRNA-34a or control microRNA to assess biological function and therapeutic potential in vitro. Nanocells were derived from genetically modified bacteria and loaded with microRNA-34a for intravenous administration to orthotopic patient-derived glioblastoma xenografts in mice.ResultsOverexpression of microRNA-34a strongly reduced the activation status of the three core signaling networks. microRNA-34a transfection also inhibited the survival of multiple established glioblastoma cell lines, as well as primary patient-derived xenograft cultures representing the proneural, mesenchymal and classical subtypes. Transfection of microRNA-34a enhanced temozolomide (TMZ) response in in vitro cultures of glioblastoma cells with primary TMZ sensitivity, primary TMZ resistance and acquired TMZ resistance. Mechanistically, microRNA-34a downregulated multiple therapeutic resistance genes which are associated with worse survival in glioblastoma patients and are enriched in specific tumor spatial compartments. Importantly, intravenous administration of nanocells carrying miR-34a and targeted to epidermal growth factor receptor (EGFR) strongly enhanced TMZ sensitivity in an orthotopic patient-derived xenograft mouse model of glioblastoma.ConclusionsTargeted bacterially-derived nanocells are an effective vehicle for the delivery of microRNA-34a to glioblastoma tumors. microRNA-34a inhibits survival and strongly sensitizes a wide range of glioblastoma cell cultures to TMZ, suggesting that combination therapy of TMZ with microRNA-34a loaded nanocells may serve as a novel therapeutic approach for the treatment of glioblastoma tumors.

Three-dimensional model of glioblastoma by co-culturing tumor stem cells with human brain organoids.

ABSTRACTEmerging three-dimensional (3D) cultures of glioblastoma are becoming powerful models to study glioblastoma stem cell behavior and the impact of cell–cell and cell–microenvironment interactions on tumor growth and invasion. Here we describe a method for culturing human glioblastoma stem cells (GSCs) in 3D by co-culturing them with pluripotent stem cell-derived brain organoids. This requires multiple coordinated steps, including the generation of cerebral organoids, and the growth and fluorescence tagging of GSCs. We highlight how to recognize optimal organoid generation and how to efficiently mark GSCs, before describing optimized co-culture conditions. We show that GSCs can efficiently integrate into brain organoids and maintain a significant degree of cell fate heterogeneity, paving the way for the analysis of GSC fate behavior and lineage progression. These results establish the 3D culture system as a viable and versatile GBM model for investigating tumor cell biology and GSC heterogeneity.This article has an associated First Person interview with the first author of the paper.

Preparation of primary glial tumor cell lines

Objective. The aim of this work was to obtain the primary cell lines of brain malignant tumors using the explant method.&#x0D; Materials and methods. Thirteen patients of both sexes, aged 22 to 66, were recruited. The tumor material of the patients was fragmented and placed in flasks with complete nutrient medium for glial tumor cells. Subsequently, the material was photographed at various stages of cultivation, the cell morphology was determined, and the rate of monolayer formation at the zero and first passages was assessed.&#x0D; Results. As a result, thirteen primary human cell lines of glial tumors were obtained: six glioblastoma lines, two glioblastoma lines with anaplastic astrocytoma, one anaplastic oligodendroglioma line, one diffuse astrocytoma line, one oligoastrocytoma line and one diffuse protoplasmic astrocytoma line, one anaplastic astrocytoma line. In the culture of diffuse astrocytoma, there were observed the cells forming a network at the bottom of the flask. In the culture of anaplastic astrocytoma at a confluence of 3050 %, fibroblast-like cells were presented, and at a confluence of 100 %, a monolayer was formed with cells intimately adjacent to each other. In the culture of oligoastrocytoma, both fibroblast-like cells and islets of closely intertwined fusiform cells were observed. The same was typical for the cells of diffuse protoplasmic astrocytoma. Anaplastic oligodendroglioma during the first week of cultivation was represented mainly by round cells with a contrast agent, which subsequently attached and actively proliferated. At a confluence of 3080 %, fibroblast-like cells were observed, and at 100 %, spindle-shaped cells closely adjacent to each other. In cultures of glioblastomas, no specific character of cell growth was revealed: spindle-shaped, fibroblast-like cells and cells with long processes forming a network were encountered. Glioblastoma cultures against the background of anaplastic astrocytoma were represented by a network of cells with long processes.&#x0D; At the zero passage, the rate of formation of a 100 % confluence monolayer ranged from 22 to 85 days. At the first passage, the cells reached a full monolayer within 4 to 25 days. At the zero passage, the longest time among all the samples to form the monolayer with a 100 % confluence needed glioblastoma lines on average 59 days. The shortest time to reach a 100 % confluence was required for cells of diffuse astrocytoma, anaplastic oligodendroglioma and glioblastoma against the background of anaplastic astrocytoma 2224 days.&#x0D; Conclusions. In our work, it was shown that the explant method ensures the production of viable cells of glial tumors and the possibility of their further cultivation.

ИССЛЕДОВАНИЕ МИКРОСАТЕЛЛИТНОЙ НЕСТАБИЛЬНОСТИ В ПЕРВИЧНЫХ КЛЕТОЧНЫХ ЛИНИЯХ НИЗКОДИФФЕРЕНЦИРОВАННЫХ ГЛИАЛЬНЫХ ОПУХОЛЕЙ

Glial tumors, in particular those composed of astrocytes, are the most common group of primary brain tumors. The most common glial tumor is astrocytoma. The biology of glial tumors was routinely studied on immortalized cell lines. However, multiple passages result in a loss of cellular heterogeneity. Therefore, more and more scientific laboratories in their research are beginning to use primary cell lines obtained directly from the patient’s native postoperative material. The question of the timing of the genetic stability of primary cell lines remains open. A special type of genetic instability is microsatellite instability, which affects microsatellites found throughout the genome. Microsatellites have several alleles, which are determined by changes in the number of repetitions of a motif unit. Microsatellite instability is of great importance in the oncogenesis of malignant neoplasms. The aim of this work was to study the microsatellite instability of primary cell lines of poorly differentiated glial tumors at different passages in comparison with the patient’s primary tumor material. Tumor cells of primary cultures of anaplastic astrocytoma and glioblastoma at different passages were used as material for the study. Formalin-fixed paraffin wax (FFPE) slides were used as a microsatellite control. Microsatellite analysis was performed on primary cultures of anaplastic astrocytoma and glioblastoma using the following markers: D17S250, D2S123, D5S346, NR21, NR24, NR27, BAT25, BAT26 by PCR. Microsatellite analysis has shown that primary glioblastoma cell lines are genetically more stable than primary anaplastic astrocytoma cell lines.

Functional impact of intramolecular cleavage and dissociation of adhesion G protein–coupled receptor GPR133 (ADGRD1) on canonical signaling

GPR133 (ADGRD1), an adhesion G protein–coupled receptor (GPCR) whose canonical signaling activates GαS-mediated generation of cytosolic cAMP, has been shown to be necessary for the growth of glioblastoma (GBM), a brain malignancy. The extracellular N terminus of GPR133 is thought to be autoproteolytically cleaved into N-terminal and C- terminal fragments (NTF and CTF, respectively). However, the role of this cleavage in receptor activation remains unclear. Here, we used subcellular fractionation and immunoprecipitation approaches to show that the WT GPR133 receptor is cleaved shortly after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant GPR133 (H543R) in patient-derived GBM cultures and HEK293T cells. After cleavage, the resulting NTF and CTF remain noncovalently bound to each other until the receptor is trafficked to the plasma membrane, where we demonstrated NTF–CTF dissociation occurs. Using a fusion of the CTF of GPR133 and the N terminus of thrombin-activated human protease-activated receptor 1 as a controllable proxy system to test the effect of intramolecular cleavage and dissociation, we also showed that thrombin-induced cleavage and shedding of the human protease-activated receptor 1 NTF increased intracellular cAMP levels. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. These findings add depth to our understanding of the molecular life cycle and mechanism of action of GPR133 and provide critical insights that will inform therapeutic targeting of GPR133 in GBM.

EXTH-50. ACTIVATION OF THE MITOCHONDRIAL CLPP PROTEASE IS SYNTHETICALLY LETHAL WITH HDAC1/2 INHIBITION IN GLIOBLASTOMA MODEL SYSTEMS

Abstract Novel therapeutic targets are critical to unravel for recalcitrant malignancies, such as the most common primary brain tumor in adults, glioblastoma (GBM). Heterogeneity remains a hallmark of primary glial brain tumors and therefore targeting several pathways simultaneously is an appropriate approach. Here, we showed that pharmacological activation of the mitochondrial ClpP protease through utilization of the novel imipridone compounds (ONC206 and ONC212) in combination with global (Panobinostat) and selective (romidepsin) HDAC – inhibitors caused synergistic reduction of viability in established and patient-derived xenograft (PDX) cultures of human GBM. This effect occurred independent of TP53 status and was partially mediated by activation of a cell death with apoptotic features accompanied by activation of initiator and effector caspases as well as cleavage of PARP. Consistently, the combination treatment altered the expression of anti-apoptotic and pro-apoptotic Bcl-2 family members, resulting in down-regulation of Bcl-xL and Mcl-1. Knockdown experiments targeting Noxa, BIM, Bcl-xL and Mcl-1 confirmed a functional implication of these proteins in the reduction of cellular viability mediated by the combination treatment. Importantly, knockdown of the ClpP protease significantly rescued the reduction of cellular viability by ClpP activators and the combination treatment, respectively, suggesting critical involvement of this protein. Finally, using a PDX model, we demonstrated that the combination treatment of romidepsin and ONC206 reduced tumor growth more potently than single treatments or vehicle by enhanced reduction of cellular proliferation and pronounced induction of cell death in vivo. Collectively, these observations suggest that the efficacy of HDAC-inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.

Correlation between Kir4.1 expression and barium-sensitive currents in rat and human glioma cell lines

Gliomas are the most common primary brain tumors and often become apparent through symptomatic epileptic seizures. Glial cells express the inwardly rectifying K+ channel Kir4.1 playing a major role in K+ buffering, and are presumably involved in facilitating epileptic hyperexcitability. We therefore aimed to investigate the molecular and functional expression of Kir4.1 channels in cultured rat and human glioma cells. Quantitative PCR showed reduced expression of Kir4.1 in rat C6 and F98 cells as compared to control. In human U-87MG cells and in patient-derived low-passage glioblastoma cultures, Kir4.1 expression was also reduced as compared to autopsy controls. Testing Kir4.1 function using whole-cell patch-clamp experiments on rat C6 and two human low-passage glioblastoma cell lines (HROG38 and HROG05), we found a significantly depolarized resting membrane potential (RMP) in HROG05 (-29 ± 2 mV, n = 11) compared to C6 (-71 ± 1 mV, n = 12, P < 0.05) and HROG38 (-60 ± 2 mV, n = 12, P < 0.05). Sustained K+ inward or outward currents were sensitive to Ba2+ added to the bath solution in HROG38 and C6 cells, but not in HROG05 cells, consistent with RMP depolarization. While immunocytochemistry confirmed Kir4.1 in all three cell lines including HROG05, we found that aquaporin-4 and Kir5.1 were also significantly reduced suggesting that the Ba2+-sensitive K+ current is generally impaired in glioma tissue. In summary, we demonstrated that glioma cells differentially express functional inwardly rectifying K+ channels suggesting that impaired K+ buffering in cells lacking functional Ba2+-sensitive K+ currents may be a risk factor for increased excitability and thereby contribute to the differential epileptogenicity of gliomas.

Mitochondria transfer from tumor-activated stromal cells (TASC) to primary Glioblastoma cells

The tumor microenvironment (TME) controls many aspects of cancer development but little is known about its effect in Glioblastoma (GBM), the main brain tumor in adults. Tumor-activated stromal cell (TASC) population, a component of TME in GBM, was induced in vitro by incubation of MSCs with culture media conditioned by primary cultures of GBM under 3D/organoid conditions. We observed mitochondrial transfer by Tunneling Nanotubes (TNT), extracellular vesicles (EV) and cannibalism from the TASC to GBM and analyzed its effect on both proliferation and survival. We created primary cultures of GBM or TASC in which we have eliminated mitochondrial DNA [Rho 0 (ρ0) cells]. We found that TASC, as described in other cancers, increased GBM proliferation and resistance to standard treatments (radiotherapy and chemotherapy). We analyzed the incorporation of purified mitochondria by ρ0 and ρ+ cells and a derived mathematical model taught us that ρ+ cells incorporate more rapidly pure mitochondria than ρ0 cells.

A co-formulation of interferons type I and II enhances temozolomide response in glioblastoma with unmethylated MGMT promoter status.

Temozolomide (TMZ) is a chemotherapeutic used for the treatment of glioblastoma. The MGMT repair enzyme (O'-(6)-methyl guanine-DNA-methyltransferase) promoter methylation is a predictive biomarker to TMZ response; interferons (IFNs) type I can downregulate MGMT expression improving survival in patients with unmethylated MGMT promoter. HeberFERON is a co-formulation of IFNs type I and II with higher antiproliferative effect over glioblastoma cell lines than individual IFNs. We investigated the proliferative response of patient-derived glioblastoma cultures to HeberFERON and its combination with TMZ in relation to MGMT promoter methylation and the regulation of MGMT transcript after HeberFERON treatment. Eleven glioblastoma-derived cultures, molecularly classified according to TCGA and MGMT promoter methylation, were assayed for proliferation inhibition with HeberFERON at low doses (1-25 IU/mL) [alone or combined with TMZ] or at higher doses (50-200 IU/mL) using CellTiter-Glo Luminescent Cell Viability Assay (Promega). Eight cultures were further treated with 100 IU/mL of HeberFERON for 72 h, total RNA purified (Qiagen) and converted to cDNA (Superscript III kit, Invitrogen) as quantitative PCR templates. Changes of MGMT&P53 transcripts level were monitored. Response of cultures to HeberFERON is variable, dose-dependent and apparently independent from TCGA classification and MGMT methylation status, based on the eight Classical cultures data. When combining HeberFERON with TMZ there was an increase in cell death for cultures, 2/4 with methylated and 5/5 with unmethylated MGMT promoter. In two out five cultures with unmethylated MGMT status, we observed a decrease of MGMT gene levels and an increase in P53 encoding gene levels. HeberFERON and TMZ combination should be further assayed in glioblastoma, mainly for those with unmethylated MGMT promoter.