Primary Glioblastoma Cell Lines Research Articles

Cucurbitacin B and Evodiamine imparts anti‐neoplastic effects against Glioblastoma Multiforme via PI3‐K/AKT signaling pathway

Glioblastoma Multiforme (GBM) is a highly malignant and aggressive brain tumor. Patients’ survival is around 6 to 12 months even with chemotherapy and surgery. Temozolomide is a first line chemotherapeutic treatment prescribed along with surgery but due to the high resistance to the drug and a low patient survival rate, new drugs and drug combinations must continue to be investigated and developed. Evodiamine, a marketed supplement, and Cucurbitacin B, a compound obtained from natural plants, have been identified as promising candidates to treat various neoplasms but has not been well studied in GBM, and especially in combination. The present study investigated the anti‐proliferating effects of Evodiamine (Evo) and Cucurbitacin‐B (CuB) individually and in combination in U‐87MG cell line (Human Primary Glioblastoma cell line) and elucidated probable mechanisms of action via PI3K/AKT pathway in GBM. Results demonstrated that both CuB and Evo individually decreased the U‐87MG cell viability in a dose dependent manner. The effect was pronounced with drug combination. CuB and Evo imparted their effect via the PI3K/P‐AKT signaling pathway. In addition, Cells treated with CuB and Evo showed a decrease in Bcl2, an anti‐apoptotic factor, and an increase in Bax, a pro‐apoptotic factor, expression; a hallmark of induction of apoptosis. The increased expression of Cleaved Caspase‐3 and p‐c Jun have also been observed. Overall, Cucurbitacin and evodiamine individually and especially in combination proved to be potential anti‐neoplastic agents in U‐87MG cells and may be considered as potential adjuvant therapies for GBM.

Ageritin-The Ribotoxin-like Protein from Poplar Mushroom (Cyclocybe aegerita) Sensitizes Primary Glioblastoma Cells to Conventional Temozolomide Chemotherapy.

Here, we propose Ageritin, the prototype of the ribotoxin-like protein family, as an adjuvant treatment to control the growth of NULU and ZAR, two primary human glioblastoma cell lines, which exhibit a pharmacoresistance phenotype. Ageritin is able to inhibit NULU and ZAR growth with an IC50 of 0.53 ± 0.29 µM and 0.42 ± 0.49 µM, respectively. In this study, Ageritin treatment highlighted a macroscopic genotoxic response through the formation of micronuclei, which represents the morphological manifestation of genomic chaos induced by this toxin. DNA damage was not associated with either the deregulation of DNA repair enzymes (i.e., ATM and DNA-PK), as demonstrated by quantitative PCR, or reactive oxygen species. Indeed, the pretreatment of the most responsive cell line ZAR with the ROS scavenger N-acetylcysteine (NAC) did not follow the reverse cytotoxic effect of Ageritin, suggesting that this protein is not involved in cellular oxidative stress. Vice versa, Ageritin pretreatment strongly enhanced the sensitivity to temozolomide (TMZ) and inhibited MGMT protein expression, restoring the sensitivity to temozolomide. Overall, Ageritin could be considered as a possible innovative glioblastoma treatment, directly damaging DNA and downregulating the MGMT DNA repair protein. Finally, we verified the proteolysis susceptibility of Ageritin using an in vitro digestion system, and considered the future perspective use of this toxin as a bioconjugate in biomedicine.

The chaperone system in glioblastoma multiforme and derived cell lines: diagnostic and mechanistic implications.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults. Novel treatments are needed to counteract the molecular mechanisms of GBM growth and drug resistance. The chaperone system (CS) members are typically cytoprotective but some, termed Hsp, can become pathogenic and participate in carcinogenesis, along with the vascular endothelial growth factor (VEGF), and we investigated them in GBM biopsies and derived cell lines. The objectives were to identify diagnostic-prognostic biomarkers and gather information for developing chaperonotherapy. Cell lines from GBMs were established, characterized (morphology, growth characteristics, and specific markers), and stored. Chaperones and angiogenic factors [Hsp10, Hsp27, Hsp60, Hsp70, Hsp90, FLT-1 (VEGFR-1), FLK1 (KDR, VEGFR-2), and FLT-4 (VEGFR-3)] were observed in cells by immunofluorescence while the chaperones were measured in tumor tissue by immunohistochemistry. Four cell lines were derived from four different GBMs; the cells were spindle shaped or polygonal and grew at high rates as adherent monolayers or clusters without evidence of contact inhibition. The astrocyte-specific glial fibrillary acidic protein (GFAP); and the neuronal NSE, malignancy VIM, and proliferation PCNA, markers were determined. The cells expressed GFAP but no NSE, indicating that they were primary glioblastoma cell lines, with high levels of Hsp10, Hsp27, Hsp60, Hsp90, and Flk1; and low levels of Hsp70, Flt1, and Flt4. Four cell lines were established derived from four out of ten GBM tumors studied. The cell lines showed intense positivity for chaperones studied and factors connected to malignancy and the tumors showed increased levels of chaperones, making them potential diagnostic-prognostic biomarkers and targets for anti-cancer compounds.

Glioblastoma recurrent cells switch between ATM and ATR pathway as an alternative strategy to survive radiation stress.

Primary treatment modality for glioblastoma (GBM) post-surgery is radiation therapy. Due to increased DNA damage repair capacity of resistant residual GBM cells, recurrence is inevitable in glioblastoma and unfortunately the recurrent tumours are resistant to the conventional therapy. Here we used our previously described in vitro radiation survival model generated from primary GBM patient samples and cell lines, which recapitulates the clinical scenario of therapy resistance and relapse. Using the parent and recurrent GBM cells from these models, we show that similar to parent GBM, the recurrent GBM cells also elicit a competent DNA damage response (DDR) post irradiation. However, the use of apical DNA damage repair sensory kinase (ATM and/or ATR) is different in the recurrent cells compared to parent cells. Consistently, we demonstrate that there is a differential clonogenic response of parent and recurrent GBM cells to the ATM and ATR kinase inhibitors with recurrent samples switching between these sensory kinases for survival emphasizing on the underlying heterogeneity within and across GBM samples. Taken together, here we report that recurrent tumours utilize an alternate DDR kinase to overcome radiation induced DNA damage. Since there is no effective treatment specifically for recurred GBM patients, these findings provide a rationale for developing newer treatment option to sensitize recurrent GBM samples by detecting in clinics the ability of cells to activate a DNA damage repair kinase different from their parent counterparts.

Bee Venom Effect on Glioblastoma Cells Viability and Gelatinase Secretion.

BackgroundThe involvement of MMP-2 and MMP-9 in the pathogenesis of various kinds of cancers including glioblastoma is well documented. The evaluation of the anticancer potential of honey bee (Apis mellifera) venom (BV) consisting of the inhibition of MMP-2 and MMP-9 secretion in a glioblastoma cell culture model was the aim of the study.Methods8-MG-BA and GAMG human primary glioblastoma cell lines vs. HT-22 mouse hippocampal neuronal cells were applied for the study. The BV dose (0.5, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, and 5.0 μg/ml) and time-dependent (24, 48, 72 h) cytotoxicity was evaluated with the tetrazolium-based colorimetric assay (MTT test). MMP-2 and MMP-9 activities in the cell culture medium under different BV concentrations were determined by gelatin zymography.ResultsA dose and time-dependent BV effect on cytotoxicity of both glioblastoma cell lines and hippocampus line was observed. The weakest, but statistically important effect was exerted by BV on HT-22 cells. The greatest cytotoxic effect of BV was observed on the 8-MG-BA line, where a statistically significant reduction in viability was observed at the lowest BV dose and the shortest incubation time. The reduction of both gelatinases secretion was observed at 8-MG-BA and GAMG lines without significant effect of HT-22 cell line.ConclusionIn vitro studies indicate that BV has both cytotoxic and inhibitory effects on the secretion of MMP-2 and MMP-9 in selected lines of glioma, suggesting anticancer properties of BV.

PTRF/Cavin-1 as a Novel RNA-Binding Protein Expedites the NF-κB/PD-L1 Axis by Stabilizing lncRNA NEAT1, Contributing to Tumorigenesis and Immune Evasion in Glioblastoma.

BackgroundImmunotherapy, especially checkpoint inhibitors targeting PD-1 or PD-L1, has revolutionized cancer therapy. However, PD-1/PD-L1 inhibitors have not been investigated thoroughly in glioblastoma (GBM). Studies have shown that polymerase 1 and transcript release factor (PTRF/Cavin-1) has an immune-suppressive function in GBM. Thus, the relationship between PTRF and PD-L1 and their role in immune suppression requires further investigation in GBM.MethodsWe used public databases and bioinformatics analysis to investigate the relationship between PTRF and PD-L1. We next confirmed the predicted relationship between PTRF and PD-L1 in primary GBM cell lines by using different experimental approaches. RIP-Seq, RIP, ChIP, and qRT-PCR were conducted to explore the molecular mechanism of PTRF in immunosuppression.ResultsWe found that PTRF stabilizes lncRNA NEAT1 to induce NF-κB and PD-L1 and promotes immune evasion in GBM. PTRF was found to correlate with immunosuppression in the public GBM databases. PTRF increased the level of PD-L1 in primary cell lines from GBM patients. We carried out RIP-Seq of GBM cells and found that PTRF interacts with lncRNA NEAT1 and stabilizes its mRNA. PTRF also promoted the activity of NF-κB by suppressing UBXN1 expression via NEAT1 and enhanced the transcription of PD-L1 through NF-κB activation. Finally, PTRF promoted immune evasion in GBM cells by regulating PD-1 binding and PD-L1 mediated T cell cytotoxicity.ConclusionsIn summary, our study identified the PTRF-NEAT1-PD-L1 axis as a novel immune therapeutic target in GBM.

Chemical synthesis of 4'-thio and 4'-sulfinyl pyrimidine nucleoside analogues.

Analogues of the canonical nucleosides required for nucleic acid synthesis have a longstanding presence and proven capability within antiviral and anticancer research. 4'-Thionucleosides, that incorporate bioisosteric replacement of furanose oxygen with sulfur, represent an important chemotype within this field. Established herein is synthetic capability towards a common 4-thioribose building block that enables access to thio-ribo and thio-arabino pyrimidine nucleosides, alongside their 4'-sulfinyl derivatives. In addition, this building block methodology is templated to deliver 4'-thio and 4'-sulfinyl analogues of the established anticancer drug gemcitabine. Cytotoxic capability of these new analogues is evaluated against human pancreatic cancer and human primary glioblastoma cell lines, with observed activities ranging from low μM to >200 μM; explanation for this reduced activity, compared to established nucleoside analogues, is yet unclear. Access to these chemotypes, with thiohemiaminal linkages, will enable a wider exploration of purine and triphosphate analogues and the application of such materials for potential resistance towards relevant hydrolytic enzymes within nucleic acid biochemistries.

Microglial Cytokines Induce Invasiveness and Proliferation of Human Glioblastoma through Pyk2 and FAK Activation

Simple SummaryMicroglia infiltrate most gliomas and have been demonstrated to promote tumor growth, invasion, and treatment resistance. To develop improved treatment methods, that take into consideration the supporting role of microglia in tumor progression, the functional and mechanistic pathways of glioma–microglia interactions need to be identified and experimentally dissected. Our recent studies and literature reports revealed the overexpression of Pyk2 and FAK in glioblastomas. Pyk2 and FAK signaling pathways have been shown to regulate migration and proliferation in glioma cells, including microglia-promoted glioma cell migration. However, the specific factors released by microglia that modulate Pyk2 and FAK to promote glioma invasiveness and proliferation are poorly understood. The aim of this study was to identify key microglia-derived signaling molecules that induce the activation of Pyk2- and FAK-dependent glioma cell proliferation and invasiveness.Glioblastoma is the most aggressive brain tumor in adults. Multiple lines of evidence suggest that microglia create a microenvironment favoring glioma invasion and proliferation. Our previous studies and literature reports indicated the involvement of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) in glioma cell proliferation and invasion, stimulated by tumor-infiltrating microglia. However, the specific microglia-released factors that modulate Pyk2 and FAK signaling in glioma cells are unknown. In this study, 20 human glioblastoma specimens were evaluated with the use of RT-PCR and western blotting. A Pierson correlation test demonstrated a correlation (0.6–1.0) between the gene expression levels for platelet-derived growth factor β(PDGFβ), stromal-derived factor 1α (SDF-1α), IL-6, IL-8, and epidermal growth factor (EGF) in tumor-purified microglia and levels of p-Pyk2 (Y579/Y580) and p-FAK(Y925) in glioma cells. siRNA knockdown against Pyk2 or FAK in three primary glioblastoma cell lines, developed from the investigated specimens, in combination with the cytokine receptor inhibitors gefitinib (1 μM), DMPQ (200 nM), and burixafor (1 μM) identified EGF, PDGFβ, and SDF-1α as key extracellular factors in the Pyk2- and FAK-dependent activation of invadopodia formation and the migration of glioma cells. EGF and IL-6 were identified as regulators of the Pyk2- and FAK-dependent activation of cell viability and mitosis.

α‐Tocopherol‐Conjugated, Open Chain Sugar‐Mimicking Cationic Lipids: Design, Synthesis and In–Vitro Gene Transfection Properties

AbstractSugar based cationic lipids are promising carriers of nucleic acids into various cells. Particularly, abundant hydroxyl functional groups in the sugar‐like moieties are believed to enhance target oriented uptake as well as biocompatibility. Towards this direction, we have synthesized three new cationic lipid analogues with or without sugar‐like multi‐hydroxyl functional head groups. Liposomal formulations of these lipids were developed and thoroughly characterized by means of DNA binding ability, size and zeta potential. Optimal lipid‐DNA complexes were determined by their transfection efficiency in HEK‐293 (Human embryonic kidney) cells. 1,4 gluconic γ‐lactone derived lipid (TS1) and ascorbic acid derived lipid (TS2), have exhibited 25‐fold and 12‐fold increase in transfected HEPG2 (human liver cancer) cells, respectively, when compared with a control lipid without sugar‐like moiety (TA). However, this property was reversed in the case of U87 (human primary glioblastoma) cell line. The results point out that the transfection efficiencies of sugar alcohol‐based cationic lipids are cell‐dependant. Cytotoxicity of the lipid complexes revealed that the sugar‐mimicking cationic lipids are non‐toxic as ≈80 % viable cells were observed in the treated cell lines. In conclusion, the current paper adds knowledge on the sugar based cationic lipids and reiterates that these lipids could yield potential applications towards liver targeted gene delivery.

TAMI-72. DIVERSITY OF CELLULAR COMMUNICATION IN GLIOBLASTOMA

Abstract OBJECTIVE 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-oncology. In this study, we aim to explore the diversity of transcellular signaling and investigate correlations between transcriptional dynamics and functional signaling. METHODS Electrophysiological characterization of GBM was carried out using planar microelectrodes and Ca2+ imaging, in both 2D cell culture as well as in our novel human cortical GBM model. Exposure to physiologically relevant conditions present within the tumor was carried out to identify specific signaling cells of interest and capture the signaling diversity in response to 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 multiple primary GBM cell lines revealed characteristics of scale-free networks (R2=0.875), confirmed in both 2D culture as well as a human neocortical GBM model. When GBM was cultured in a “in-vivo” like environment, basal activity was significantly higher (50%, p=0.01). Cellular signaling was directly correlated to changes in the environment, like hypoxia or glutamatergic activation, and total inhibition of electrical signaling required the usage of synaptic inhibitors. Using single-cell RNA sequencing and proteomics, several synaptogenesis related genes were identified to play a crucial role in the lineage states present in GBM. CRISPR based perturbation of these genes resulted in alterations in cellular morphology and decreased cellular connectivity (p< 0.01), with loss of scale free features (R2=0.35), and transcriptomic loss of developmental lineages (FDR< 0.01), leading to significant inhibition of GBM stress response. 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.

TAMI-64. IMMUNE ENVIRONMENT SHAPES GLIOBLASTOMA HETEROGENEITY IN TIME AND SPACE

Abstract Glioblastomas are embedded into an immunosuppressive microenvironment resulting in limited success of immunotherapies. Although, we and others observed defined myeloid-tumor crosslinks reducing T cell homing and activation, the spatial context of these interactions remained unexplored. Here, we provide evidence, that local T cell infiltration results in a defined activation of myeloid cells causing transcriptional reprogramming of tumor cells reminiscent of reactive transformation in mature astrocytes. Through integration of spatially resolved transcriptomics and imaging mass cytometry (n=18, 39 protein glioma panel) we mapped defined transcriptional responses in areas of high or low T cell infiltration respectively. Functional analysis revealed that areas of large T cell infiltration are enriched for glial (CHI3L1, GFAP and VIM) and inflammatory genes (HLA-DRA, C3, CCL4, CCL3). We found that marker genes of common reactive states in mature astrocytes significantly overlap with the reactive immune program of glioblastoma cells (f-score 0.76, p=2.2e-10). Increased numbers of CD163+ cells were found in surrounding areas of T cell infiltration and spatially linked to defined immunosuppressive release of IL10, recently reported as a major driver of T cell exhaustion. To support our findings, we injected a primary glioblastoma cell line into cortex slices of three different human and rodent donors. After 7 days of tumor growth, we performed scRNA-sequencing of FACS-sorted tumor cells and baseline cell culture cells. Compared to baseline, we found cells of all reported transcriptional states, confirming the dynamic adaptation of cells within a neural environment. In elderly donors, a significant accumulation of reactive immune programs (ANOVA p< 0.001) was observed. Immunostainings confirmed an increased myeloid cell activation in these donors. Our data suggest that inflammatory stimuli in the glioblastoma microenvironment cause transcriptional reprogramming in glioblastoma similar to inflammatory transformation of reactive astrocytes. The spatial exclusivity of these programs highlights the value of a spatial perspective on heterogeneity.

A novel biphenyl diester derivative, AB38b, inhibits glioblastoma cell growth via the ROS-AKT/mTOR pathway

AB38b is a novel biphenyl diester derivative synthesized in our laboratory, and it has been shown to improve the pathology of nephropathy and encephalopathy in diabetic mice. Glioblastoma (GBM) is the most lethal brain tumor, without effective drugs to date. The present study aims at investigating the role of AB38b in GBM growth and revealing the underlying molecular mechanisms. We found that AB38b administration showed a dose- and time-dependent inhibition on cell proliferation in multiple immortalized and primary GBM cell lines, but it had no significant effects on human astrocyte cell line. More importantly, AB38b blocked cell cycle progression, induced early apoptosis, decreased the activity of AKT/mTOR pathway, and increased the generation of reactive oxygen species (ROS) in GBM cells. Interestingly, antioxidant treatments could reverse the AB38b-mediated abovementioned effects; overexpression of constitutively active AKT could partially rescue the suppressive effects of Ab38b on GBM cell proliferation. In addition, AB38b administration inhibited the tumor growth, decreased the activity of AKT/mTOR pathway, and prolonged the survival time in GBM animal models, without any adverse influences on the important organs. These findings suggest that AB38b exerts anti-glioma activity via elevating the ROS generation followed by inhibiting the activity of AKT/mTOR pathway.

Feasibility of Photodynamic Therapy for Glioblastoma with the Mitochondria-Targeted Photosensitizer Tetramethylrhodamine Methyl Ester (TMRM).

One of the most challenging problems in the treatment of glioblastoma (GBM) is the highly infiltrative nature of the disease. Infiltrating cells that are non-resectable are left behind after debulking surgeries and become a source of regrowth and recurrence. To prevent tumor recurrence and increase patient survival, it is necessary to cleanse the adjacent tissue from GBM infiltrates. This requires an innovative local approach. One such approach is that of photodynamic therapy (PDT) which uses specific light-sensitizing agents called photosensitizers. Here, we show that tetramethylrhodamine methyl ester (TMRM), which has been used to asses mitochondrial potential, can be used as a photosensitizer to target GBM cells. Primary patient-derived GBM cell lines were used, including those specifically isolated from the infiltrative edge. PDT with TMRM using low-intensity green light induced mitochondrial damage, an irreversible drop in mitochondrial membrane potential and led to GBM cell death. Moreover, delayed photoactivation after TMRM loading selectively killed GBM cells but not cultured rat astrocytes. The efficacy of TMRM-PDT in certain GBM cell lines may be potentiated by adenylate cyclase activator NKH477. Together, these findings identify TMRM as a prototypical mitochondrially targeted photosensitizer with beneficial features which may be suitable for preclinical and clinical translation.

Cytotoxicity Effect of Quinoin, Type 1 Ribosome-Inactivating Protein from Quinoa Seeds, on Glioblastoma Cells.

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients’ gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells’ growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs.

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.

349P The amount of cancer stem cells and the sensitivity to anticancer drugs in glioblastoma primary cell culture

It is assumed that aggressiveness and therapy resistance of glioblastoma multiforme (GBM) is determined by its high heterogeneity and presence of cancer stem cells (CSC). The primary glioblastoma cell lines (pGBM lines) could better than the cultured cell lines reflect the tumor heterogeneity and, thus, provide the additional opportunities for personalized medicine. In our study, we compared the response of the pGBM lines and cell glioblastoma lines to chemotherapy drugs with the CSC content in cell cultures.

Green Synthesis of Silver Nanoparticles Using Diospyros malabarica Fruit Extract and Assessments of Their Antimicrobial, Anticancer and Catalytic Reduction of 4-Nitrophenol (4-NP).

The green synthesis of silver nanoparticles (AgNPs) has currently been gaining wide applications in the medical field of nanomedicine. Green synthesis is one of the most effective procedures for the production of AgNPs. The Diospyros malabarica tree grown throughout India has been reported to have antioxidant and various therapeutic applications. In the context of this, we have investigated the fruit of Diospyros malabarica for the potential of forming AgNPs and analyzed its antibacterial and anticancer activity. We have developed a rapid, single-step, cost-effective and eco-friendly method for the synthesis of AgNPs using Diospyros malabarica aqueous fruit extract at room temperature. The AgNPs began to form just after the reaction was initiated. The formation and characterization of AgNPs were confirmed by UV-Vis spectrophotometry, XRD, FTIR, DLS, Zeta potential, FESEM, EDX, TEM and photoluminescence (PL) methods. The average size of AgNPs, in accordance with TEM results, was found to be 17.4 nm. The antibacterial activity of the silver nanoparticles against pathogenic microorganism strains of Staphylococcus aureus and Escherichia coli was confirmed by the well diffusion method and was found to inhibit the growth of the bacteria with an average zone of inhibition size of (8.4 ± 0.3 mm and 12.1 ± 0.5 mm) and (6.1 ± 0.7 mm and 13.1 ± 0.5 mm) at 500 and 1000 µg/mL concentrations of AgNPs, respectively. The anticancer effect of the AgNPs was confirmed by MTT assay using the U87-MG (human primary glioblastoma) cell line. The IC50 value was found to be 58.63 ± 5.74 μg/mL. The results showed that green synthesized AgNPs exhibited significant antimicrobial and anticancer potency. In addition, nitrophenols, which are regarded as priority pollutants by the United States Environmental Protection Agency (USEPA), can also be catalytically reduced to less toxic aminophenols by utilizing synthesized AgNPs. As a model reaction, AgNPs are employed as a catalyst in the reduction of 4-nitrophenol to 4-aminophenol, which is an intermediate for numerous analgesics and antipyretic drugs. Thus, the study is expected to help immensely in the pharmaceutical industries in developing antimicrobial drugs and/or as an anticancer drug, as well as in the cosmetic and food industries.

CD90low glioma-associated mesenchymal stromal/stem cells promote temozolomide resistance by activating FOXS1-mediated epithelial-mesenchymal transition in glioma cells

BackgroundThe tumour microenvironment contributes to chemotherapy resistance in gliomas, and glioma-associated mesenchymal stromal/stem cells (gaMSCs) are important stromal cell components that play multiple roles in tumour progression. However, whether gaMSCs affect chemotherapy resistance to the first-line agent temozolomide (TMZ) remains unclear. Herein, we explored the effect and mechanism of gaMSCs on resistance to TMZ in glioma cells.MethodsHuman glioma cells (cell line U87MG and primary glioblastoma cell line GBM-1) were cultured in conditioned media of gaMSCs and further treated with TMZ. The proliferation, apoptosis and migration of glioma cells were detected by Cell Counting Kit-8 (CCK-8), flow cytometry and wound-healing assays. The expression of FOXS1 in glioma cells was analysed by gene microarray, PCR and Western blotting. Then, FOXS1 expression in glioma cells was up- and downregulated by lentivirus transfection, and markers of the epithelial-mesenchymal transformation (EMT) process were detected. Tumour-bearing nude mice were established with different glioma cells and treated with TMZ to measure tumour size, survival time and Ki-67 expression. Finally, the expression of IL-6 in gaMSC subpopulations and its effects on FOXS1 expression in glioma cells were also investigated.ResultsConditioned media of gaMSCs promoted the proliferation, migration and chemotherapy resistance of glioma cells. The increased expression of FOXS1 and activation of the EMT process in glioma cells under gaMSC-conditioned media were detected. The relationship of FOXS1, EMT and chemotherapy resistance in glioma cells was demonstrated through the regulation of FOXS1 expression in vitro and in vivo. Moreover, FOXS1 expression in glioma cells was increased by secretion of IL-6 mainly from the CD90low gaMSC subpopulation.ConclusionsCD90low gaMSCs could increase FOXS1 expression in glioma cells by IL-6 secretion, thereby activating epithelial-mesenchymal transition and resistance to TMZ in glioma cells. These results indicate a new role of gaMSCs in chemotherapy resistance and provide novel therapeutic targets.

Abstract 2452: Metformin administration in hypoglycemia compromises cancer cell viability in glioblastoma initiating cells

Abstract Cancer cells show higher energy and biomolecules requirements than healthy cells, making the targeting of cancer metabolic dysfunctions an appealing therapeutic approach. Unfortunately, tackling the tumor metabolism is a daunting task, due to the ability of cancer cells to re-organize their metabolism switching between glycolysis and oxidative phosphorylation. Cancer metabolic plasticity and re-programming is partially supported by altered ionic channels expression and activity, our laboratory has described Chloride Intracellular Channel 1 (CLIC1) as a marker of malignancy in tumor cells and a possible therapeutic target. CLIC1 is a metamorphic protein able to switch between cytoplasmic and transmembrane forms (tmCLIC1), the latter characterized by chloride conductance. TmCLIC1 was found to be overexpressed in several solid tumors where supports cancer proliferation and growth, while it is mainly absent in healthy cells. Recently, it has been demonstrated that tmCLIC1 activity is affected by biguanide compounds, in particular metformin. Metformin was recently repurposed as an anticancer drug with unclear mechanisms. We demonstrated that in hypoglycemia, metformin displays increased toxic effect on cancer cells, inducing apoptosis. Metformin inhibits Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A), and hypoglycemia upregulates PP2A subunit B56δ targeting the Glycogen Synthase Kinase 3 Beta (GSK3b). Metformin treatment in combination with hypoglycemia leads to de-phosphorylation of GSK3b protein resulting in degradation of induced myeloid leukemia cell differentiation protein (MCL-1), a pro-survival protein, and cell death (Elgendy et al., Cancer Cell 2019). In primary glioblastoma cell lines enriched in tumor-initiating-cells (GSCs), metformin has proven to be effective in reducing cell growth without affecting viability (Gritti et al., Oncotarget 2014). Here, we are going to show studies trying to establish whether CLIC1 is a component of the established PP2A-mediated signaling network. Our work aims to clarify metformin molecular mechanism of action and to assess a new potential therapeutic approach for solid tumors in which tmCLIC1 is found to be overexpressed. Citation Format: Francesca Cianci, Riccardo Cazzoli, Ivan Verduci, Saverio Minucci, Michele Mazzanti. Metformin administration in hypoglycemia compromises cancer cell viability in glioblastoma initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2452.

Evidence of Reelin Signaling in GBM and Its Derived Cancer Stem Cells.

Glioblastoma (GBM) is the most aggressive and malignant form of primary brain cancer, characterized by an overall survival time ranging from 12 to 18 months. Despite the progress in the clinical treatment and the growing number of experimental data aimed at investigating the molecular bases of GBM development, the disease remains characterized by a poor prognosis. Recent studies have proposed the existence of a population of GBM cancer stem cells (CSCs) endowed with self-renewal capability and a high tumorigenic potential that are believed to be responsible for the resistance against common chemotherapy and radiotherapy treatments. Reelin is a large secreted extracellular matrix glycoprotein, which contributes to positioning, migration, and laminar organization of several central nervous system structures during brain development. Mutations of the reelin gene have been linked to disorganization of brain structures during development and behavioral anomalies. In this study, we explored the expression of reelin in GBM and its related peritumoral tissue and performed the same analysis in CSCs isolated from both GBM (GCSCs) and peritumoral tissue (PCSCs) of human patients. Our findings reveal (i) the higher expression of reelin in GBM compared to the peritumoral tissue by immunohistochemical analysis, (ii) the mRNA expression of both reelin and its adaptor molecule Dab1 in either CSC subtypes, although at a different extent; and (iii) the contribution of CSCs-derived reelin in the migration of human primary GBM cell line U87MG. Taken together, our data indicate that the expression of reelin in GBM may represent a potential contribution to the regulation of GBM cancer stem cells behavior, further stimulating the interest on the reelin pathway as a potential target for GBM treatment.