Glioma-initiating Cells Research Articles

Exhaustive in vitro evaluation of the 9-drug cocktail CUSP9 for treatment of glioblastoma

The CUSP9 protocol is a polypharmaceutical strategy aiming at addressing the complexity of glioblastoma by targeting multiple pathways. Although the rationale for this 9-drug cocktail is well-supported by theoretical and in vitro data, its effectiveness compared to its 511 possible subsets has not been comprehensively evaluated. Such an analysis could reveal if fewer drugs could achieve similar or better outcomes.We conducted an exhaustive in vitro evaluation of the CUSP9 protocol using COMBImageDL, our specialized framework for testing higher-order drug combinations. This study assessed all 511 subsets of the CUSP9v3 protocol, in combination with temozolomide, on two clonal cultures of glioma-initiating cells derived from patient samples. The drugs were used at fixed, clinically relevant concentrations, and the experiment was performed in quadruplicate with endpoint cell viability and live-cell imaging readouts. Our results showed that several lower-order drug combinations produced effects equivalent to the full CUSP9 cocktail, indicating potential for simplified regimens in personalized therapy. Further validation through in vivo and precision medicine testing is required.Notably, a subset of four drugs (auranofin, disulfiram, itraconazole, sertraline) was particularly effective, reducing cell growth, altering cell morphology, increasing apoptotic-like cells within 4–28 h, and significantly decreasing cell viability after 68 h compared to untreated cells.This study underscores the importance and feasibility of comprehensive in vitro evaluations of complex drug combinations on patient-derived tumor cells, serving as a critical step toward (pre-)clinical development.

Divergent transcriptomic signatures from putative mesenchymal stimuli in glioblastoma cells

In glioblastoma, a mesenchymal phenotype is associated with especially poor patient outcomes. Various glioblastoma microenvironmental factors and therapeutic interventions are purported drivers of the mesenchymal transition, but the degree to which these cues promote the same mesenchymal transitions and the uniformity of those transitions, as defined by molecular subtyping systems, is unknown. Here, we investigate this question by analyzing publicly available patient data, surveying commonly measured transcripts for mesenchymal transitions in glioma-initiating cells (GIC), and performing next-generation RNA sequencing of GICs. Analysis of patient tumor data reveals that TGFβ, TNFα, and hypoxia signaling correlate with the mesenchymal subtype more than the proneural subtype. In cultured GICs, the microenvironment-relevant growth factors TGFβ and TNFα and the chemotherapeutic temozolomide promote expression of commonly measured mesenchymal transcripts. However, next-generation RNA sequencing reveals that growth factors and temozolomide broadly promote expression of both mesenchymal and proneural transcripts, in some cases with equal frequency. These results suggest that glioblastoma mesenchymal transitions do not occur as distinctly as in epithelial-derived cancers, at least as determined using common subtyping ontologies and measuring response to growth factors or chemotherapeutics. Further understanding of these issues may identify improved methods for pharmacologically targeting the mesenchymal phenotype in glioblastoma.

EXPLORING THE EFFECT OF EXOSOMES AND RADIATION ON GLIOBLASTOMA CANCER CELLS

Abstract Glioblastoma is the most common and aggressive type of brain cancer, with higher incidences in aging adults over 75 and little to no long-term survival or cure. The median survival time under the current standard of care, surgical resection and chemoradiation, is 15-18 months. Glioblastoma tumors consist of multiple cell types, including mature brain cells in cell cycle arrest and cancer stem cells (CSCs) that replenish cancer cell populations. However, the cancerous non-stem cells are induced into a cell fate called glioma-initiating cells in response to radiation, where they begin to express stem-like phenotypes. Glioma-initiating cells proliferate at an aggressive rate and are the reason for cancer recurrence, making them a necessary target for new and more effective therapeutics. This research is studying the effect of exposure to exosomes, extracellular vesicles that carry proteins and RNA, on the stem-like phenotype expression of irradiated non-stem cells. Irradiated glioblastoma cells were exposed to varying concentrations of exosomes isolated from glioblastoma CSCs in vitro. The results showed a decrease in ZsGreen expression, a marker for stem-like phenotypes, as exosome concentration increased. Exposure of glioblastoma cancer cells to exosomes post-irradiation decreased the rate of transition to glioma-initiating cells. This suggests that CSC exosomes may signal that stem cells are present, decreasing the need for the formation of glioma-initiating cells. Further research is necessary to better understand the role of exosomes in glioblastoma tumors, however, this result may suggest exosomes as a potential way to delay or reduce the aggressive nature of glioblastoma cancer recurrence.

CTNI-68. PHASE I/II STUDY OF MAINTENANCE THERAPY WITH METFORMIN AND TEMOZOLOMIDE FOR TARGETING GLIOBLASTOMA CANCER STEM CELLS

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive primary brain tumor with poor prognosis. One strategy for overcoming resistance is developing a new therapy targeting the cancer stem/initiating cells. We have shown that the antidiabetic drug metformin (MF) can induce differentiation of stem-like glioma-initiating cells and suppress tumor formation through AMPK-FOXO3 activation (Stem Cells Transl Med. 2012;1(11)). We conducted a phase I/II study to examine the clinical effect of MF combined with standard maintenance temozolomide (TMZ). Patients and METHODS Patients between 20 and 74 years of age with supratentorial GBM were eligible. MF dose-escalation study was performed using a 3 + 3 design as phase I part to determine recommended phase II dose. Dose-limiting toxicities (DLTs) were assessed during the first 6 weeks after MF initiation. After completing phase I study, we proceeded to phase II part. RESULTS The first three patients were treated with 1,500 mg/day MF from 2021 and the next four patients were treated with 2,250 mg/day MF, which is the maximum dose approved in Japan. No DLTs were observed. The most common adverse effects were appetite loss, nausea, and diarrhea, which were observed in three patients. All of them were manageable, with grade 1 or 2 (Cancers (Basel). 2022;14(17)). Based on the phase I study, we proceeded to phase II study with 2,250 mg/day MF from April 2022 and finished recruiting patients for phase II part in May 2023. Primary endpoint of phase II trial is progression-free survival rate at 12 months compared to historical data. CONCLUSION MF dose of up to 2,250 mg/day combined with maintenance TMZ was well tolerated. We report the safety and efficacy of MF with TMZ for GBM.

Therapeutic potential of pentamidine for glioma-initiating cells and glioma cells through multimodal antitumor effects.

Glioma-initiating cells, which comprise a heterogeneous population of glioblastomas, contribute to resistance against aggressive chemoradiotherapy. Using drug reposition, we investigated a therapeutic drug for glioma-initiating cells. Drug screening was undertaken to select candidate agents that inhibit proliferation of two different glioma-initiating cells lines. The alteration of proliferation and stemness of the two glioma-initiating cell lines, and proliferation, migration, cell cycle, and survival of these two differentiated glioma-initiating cell lines and three different glioblastoma cell lines treated with the candidate agent were evaluated. We also used a xenograft glioma mouse model to evaluate anticancer effects of treated glioma cell lines. Among the 1301 agents, pentamidine-an antibiotic for Pneumocystis jirovecii-emerged as a successful antiglioma agent. Pentamidine treatment suppressed proliferation and stemness in glioma-initiating cell lines. Proliferation and migration were inhibited in all differentiated glioma-initiating cells and glioblastoma cell lines, with cell cycle arrest and caspase-dependent apoptosis induction. The in vivo study reproduced the same findings as the in vitro studies. Pentamidine showed a stronger antiproliferative effect on glioma-initiating cells than on differentiated cells. Western blot analysis revealed pentamidine inhibited phosphorylation of signal transducer and activator of transcription 3 in all cell lines, whereas Akt expression was suppressed in glioma-initiating cells but not in differentiated lines. In the present study, we identified pentamidine as a potential therapeutic drug for glioma. Pentamidine could be promising for the treatment of glioblastomas by targeting both glioma-initiating cells and differentiated cells through its multifaceted antiglioma effects.

ACT-19 A REPORT OF PHASE I PART OF PHASE I/II STUDY OF MAINTENANCE THERAPY WITH METFORMIN AND TEMOZOLOMIDE FOR GLIOBLASTOMA

Abstract Background Glioblastoma (GBM) is an aggressive primary brain tumor with poor prognosis. One strategy for overcoming resistance is developing a new therapy targeting the cancer stem/initiating cells. We have shown that the antidiabetic drug metformin (MF) can induce differentiation of stem-like glioma-initiating cells and suppress tumor formation through AMPK-FOXO3 activation. We conducted a phase I/II study to examine the clinical effect of MF combined with standard maintenance temozolomide (TMZ). Here, we report the result of phase I part and the current status of phase II part. Patients and Methods Patients between 20 and 74 years of age with supratentorial GBM histologically diagnosed according to the World Health Organization 2016 classification were eligible. MF dose-escalation was planned using a 3 + 3 design. Dose-limiting toxicities (DLTs) were assessed during the first 6 weeks after MF initiation. Results Between February 2021 and January 2022, the first three patients were treated with 1,500 mg/day MF and the next four patients were treated with 2,250 mg/day MF, which is the maximum dose approved in Japan. The median age of the patients was 41 years. Three tumors (42.9 %) were IDH1/2 mutants and 4 (57.1 %) were IDH1/2 wild-types. No DLTs were observed. The most common adverse effects were appetite loss, nausea, and diarrhea, which were observed in three patients. All of them were manageable, with grade 1 or 2. Only one grade 3 seizure was reported, which was likely related to the tumor. Two patients experienced tumor progression at 6.0 and 6.1 months, and one died 12.2 months after initial surgery. The other five patients remained stable at the last follow-up. Conclusion MF dose of up to 2,250 mg/day combined with maintenance TMZ appeared to be well tolerated, and we proceeded to phase II study with 2,250 mg/day MF.

Metformin with Temozolomide for Newly Diagnosed Glioblastoma: Results of Phase I Study and a Brief Review of Relevant Studies.

Simple SummaryGlioblastoma is an incurable disease, demanding new therapeutic approaches. Our preclinical studies proved that the antidiabetic drug metformin could induce the differentiation of stem-like glioma-initiating cells and suppress tumor formation through AMPK-FOXO3 activation, suggesting the potential of metformin for treating glioblastoma. Taking into consideration that the anti-cancer effects of metformin are known to be dose-dependent, we conducted a dose-escalation phase I study to evaluate the safety and determine the recommended phase II dose of metformin in combination with maintenance temozolomide in patients with newly diagnosed glioblastoma. We show that the 2250 mg/day metformin appeared to be well tolerated with acceptable toxicity. Therefore, we proceed to a phase II study with 2250 mg/day metformin to evaluate its clinical benefits. Cancer stem/initiating cells are resistant to existing radiotherapy or chemotherapy; thus, our strategy targeting glioma-initiating cells using metformin is a novel therapeutic strategy which could possibly improve the outcome of glioblastoma.Glioblastoma (GBM) inevitably recurs due to a resistance to current standard therapy. We showed that the antidiabetic drug metformin (MF) can induce the differentiation of stem-like glioma-initiating cells and suppress tumor formation through AMPK-FOXO3 activation. In this study, we design a phase I/II study to examine the clinical effect of MF. We aim to determine a recommended phase II MF dose with maintenance temozolomide (TMZ) in patients with newly diagnosed GBM who completed standard concomitant radiotherapy and TMZ. MF dose-escalation was planned using a 3 + 3 design. Dose-limiting toxicities (DLTs) were assessed during the first six weeks after MF initiation. Three patients were treated with 1500 mg/day MF and four patients were treated with 2250 mg/day MF between February 2021 and January 2022. No DLTs were observed. The most common adverse effects were appetite loss, nausea, and diarrhea, all of which were manageable. Two patients experienced tumor progression at 6.0 and 6.1 months, and one died 12.2 months after initial surgery. The other five patients remained stable at the last follow-up session. The MF dose of up to 2250 mg/day combined with maintenance TMZ appeared to be well tolerated, and we proceeded to a phase II study with 2250 mg/day MF.

CD95 gene deletion may reduce clonogenic growth and invasiveness of human glioblastoma cells in a CD95 ligand-independent manner

CD95 (Fas/APO-1) is a multifunctional cell surface receptor with antithetic roles. First described to mediate cell death, interactions of CD95 with its natural ligand, CD95L, have also been described to induce tumor-promoting signaling leading to proliferation, invasion and stem cell maintenance, mainly in cancer cells that are resistant to CD95-mediated apoptosis. While activation of CD95-mediated apoptosis in cancer cells may not be clinically practicable due to toxicity, inhibition of tumor-promoting CD95 signaling holds therapeutic potential. In the present study, we characterized CD95 and CD95L expression in human glioma-initiating cells (GIC), a glioblastoma cell population with stem cell features, and investigated the consequences of CRISPR-Cas9-mediated CD95 or CD95L gene deletion. In vitro, GIC expressed CD95 but not CD95L and were sensitive to CD95-mediated apoptosis. Upon genetic deletion of CD95, GIC acquired resistance to CD95L-induced apoptosis but exhibited inferior clonogenic growth, sphere-forming capacity, and invasiveness compared with control cells, suggesting the existence of CD95L-independent constitutive CD95 signaling with tumor-promoting properties in GIC. In vivo, GIC expressed CD95 and a non-canonical form of CD95L lacking the CD95-binding region. CD95 genetic deletion did not prolong survival in immunocompromised GIC-bearing mice. Altogether, these data indicate that canonical CD95L may not be expressed in human GIC and suggest the existence of a CD95L-independent CD95-signaling pathway that maintains some malignancy traits of GIC. The lack of altered survival of tumor-bearing mice after genetic deletion of CD95 suggests that CD95 signaling is not essential to maintain the growth of human GIC xenografted into the brains of nude mice. The ligand-independent tumor-promoting role of constitutive CD95 in our GIC models in vitro highlights the complexity and challenges associated with targeting CD95 with therapeutic intent.

Targeting CXCR4 to suppress glioma-initiating cells and chemoresistance in glioma.

Glioma initiating cells (GICs), also known as glioma stem cells, display the capacity to recapitulate the functional diversity within the tumor. Despite the great progress achieved over the last decades, defining the key molecular regulators of GICs has represented a major obstacle in this field. In our study, data from The Cancer Genome Atlasdatabase illustrated a relationship between C-X-C motif chemokine receptor 4 (CXCR4) expression and the survival of glioma patients. Mechanistically, we further indicated that CXCR4 mediated the upregulation of Kruppel like factor 5(KLF5), a zinc-finger-containing transcription factor, to facilitate the proliferation of GICs. What's more, CXCR4 also enhanced the chemoresistance through KLF5/Bcl2-like 12 (BCl2L12) in glioma. The elevated expression of KLF5 and BCL2L12 induced by CXCR4 was dependent on phosphoinositide 3-kinases (PI3K)/serine/threonine kinase (AKT) signaling. Importantly, combined application of temozolomideand a CXCR4 inhibitor efficiently reversed CXCR4 mediated drugs resistance and improved anticancer effects in vivo. Collectively, our findings confirmed that CXCR4 promoted GICs proliferation via the KLF5/BCL2L12 dependent pathway, which may enrich the understanding of GICs and help drive the design of efficacious therapeutic strategies.

Abstract 3170: Human retroviral sequences transcription in glioma/glioblastoma sample

Abstract In glioblastoma patients, expansion of glioma initiating cells (GICs), a discrete population of cells demonstrating stem-like phenotype, is often associated with drug resistance development and ultimately cancer recurrence. Therefore a better understanding of the molecular and cellular mechanisms underlying the origin of GICs is critical to develop new therapies better targeting glioblastoma. Human endogenous retroviral sequences (HERVs), present in approximately 500,000 copies and comprising nearly 8% of the human genome, may serve as an abundant genetic material equipping GIC with phenotypic plasticity needed to develop stem-like phenotype. Recent epigenomic and transcriptomic data show HERV activation during early human development. Moreover, increased HERV transcription is observed in OCT4/SOX2/KLF4/c-MYC induced pluripotent cells. Finally, activation of HERV loci transcription is also commonly observed in different tumor types. Using publicly available The Cancer Genome Atlas Glioblastoma Multiforme (TCGA-GBM) RNA-seq data collection, we have quantified transcripts for all 13,208 HERV loci annotated in the RepBase database. To improve the accuracy of quantifying the HERV transcriptomic landscape, we reassigned already mapped fragments to the most probable transcript using the Telescope software. Our comprehensive approach allowed us to identify the set of HERV loci uniquely dysregulated in the glioblastoma compared to normal brain tissue. In addition, we compared 540 low-grade glial tumors from TCGA-LGG project (classified as grade II and III gliomas) with 165 glioblastoma samples from TCGA-GBM (classified as grade IV gliomas) using the same bioinformatics pipeline. Comprehensive analysis of transcript levels allowed us to identify HERV assemblage differentially expressed between these two patient cohorts. The correlation between HERV and ORF transcript levels show strong coregulation of HERVs expression with genes involved in establishing/maintaining embryonic/neural/glial stem cells phenotype. To confirm our observations we have up- or downregulated HERV loci combining guide RNA array targeting robustly approximately 90% of HERV LTR5Hs elements with a vector expressing CRISPR-based transcriptional activator or repressor. Downregulation of HERV transcription with this system was associated with the inhibition of clonogenic growth in cell culture. In summary, this study sets the stage for identifying HERV loci that could serve as potential GIC biomarkers, as well as the molecular targets for GIC elimination. Citation Format: Adam J. Lassak, Monika Rak, Melody C. Baddoo, Hui-Yi Lin, Krzysztof Reiss. Human retroviral sequences transcription in glioma/glioblastoma sample [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3170.

Phenotyping clonal populations of glioma stem cell reveals a high degree of plasticity in response to changes of microenvironment

AbstractThe phenotype of glioma-initiating cells (GIC) is modulated by cell-intrinsic and cell-extrinsic factors. Phenotypic heterogeneity and plasticity of GIC is an important limitation to therapeutic approaches targeting cancer stem cells. Plasticity also presents a challenge to the identification, isolation, and propagation of purified cancer stem cells. Here we use a barcode labelling approach of GIC to generate clonal populations over a number of passages, in combination with phenotyping using the established stem cell markers CD133, CD15, CD44, and A2B5. Using two cell lines derived from isocitrate dehydrogenase (IDH)-wildtype glioblastoma, we identify a remarkable heterogeneity of the phenotypes between the cell lines. During passaging, clonal expansion manifests as the emergence of a limited number of barcoded clones and a decrease in the overall number of clones. Dual-labelled GIC are capable of forming traceable clonal populations which emerge after as few as two passages from mixed cultures and through analyses of similarity of relative proportions of 16 surface markers we were able to pinpoint the fate of such populations. By generating tumour organoids we observed a remarkable persistence of dominant clones but also a significant plasticity of stemness marker expression. Our study presents an experimental approach to simultaneously barcode and phenotype glioma-initiating cells to assess their functional properties, for example to screen newly established GIC for tumour-specific therapeutic vulnerabilities.The authors barcoded glioma-initiating cells (GIC) using combinations of virally encoded fluorophores. GIC show a strong tendency to form clonal populations over as few as two passages. Combined with stem cell marker phenotyping and computational analysis, they could trace the fate of such populations. This model presents an approach for rapid assessment of newly established GIC to assess tumour-specific vulnerabilities.

Spatial Heterogeneity of Invading Glioblastoma Cells Regulated by Paracrine Factors.

Glioblastoma (GBM) is the most common and lethal type of malignant primary brain tumor in adults. GBM displays heterogeneous tumor cell population comprising glioma-initiating cells (GICs) with stem cell-like characteristics and differentiated glioma cells. During GBM cell invasion into normal brain tissues, which is the hallmark characteristic of GBM, GICs at the invasion front retain stemness, while cells at the tumor core display cellular differentiation. However, the mechanism of cellular differentiation underlying the formation of spatial cellular heterogeneity in GBM remains unknown. In the present study, we first observed spatially heterogeneous GBM cell populations emerged from an isogenic clonal population of GICs during invasion into a 3D collagen hydrogel in a microfluidic device. Specifically, GICs at the invasion front maintained stemness, while trailing cells displayed astrocytic differentiation. The spatial cellular heterogeneity resulted from the difference in cell density between GICs at the invasion front and trailing cells. Trailing GICs at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they secreted, while cells at the invasion front of low cell density retained stemness due to the lack of paracrine factors. In addition, we demonstrated that interstitial flow suppressed astrocytic differentiation of trailing GICs by the clearance of paracrine factors. Our findings suggest that intercellular crosstalk between tumor cells is an essential factor in developing the spatial cellular heterogeneity of GBM cells with various differentiation statuses. It also provides insights into the development of novel therapeutic strategies targeting GBM cells with stem cell characteristics at the invasion front. Impact Statement We elucidated the mechanism of cellular differentiation underlying the spatial cellular heterogeneity of glioblastoma composed of glioma-initiating cells (GICs) and differentiated glioma cells during invasion in a microfluidic device. Trailing cells at high cell density exhibited astrocytic differentiation through local accumulation of paracrine factors they produced, while cells at the invasion front of low cell density were shown to retain stemness due to the lack of paracrine factors. Our findings provide valuable knowledge for the development of effective therapeutic strategies targeting GICs at the invasion front.

ATX-101, a Peptide Targeting PCNA, Has Antitumor Efficacy Alone or in Combination with Radiotherapy in Murine Models of Human Glioblastoma.

Simple SummaryPCNA is an interesting target for cancertreatment due to its essential activities in DNA replication and repair and its recently discovered regulatory roles in cellular signaling. Here, we demonstrate that ATX-101, a peptide targeting PCNA, has antitumor effects as a single agent and radiosensitizing properties in glioblastoma multiforme models.Cell proliferation requires the orchestrated actions of a myriad of proteins regulating DNA replication, DNA repair and damage tolerance, and cell cycle. Proliferating cell nuclear antigen (PCNA) is a master regulator which interacts with multiple proteins functioning in these processes, and this makes PCNA an attractive target in anticancer therapies. Here, we show that a cell-penetrating peptide containing the AlkB homolog 2 PCNA-interacting motif (APIM), ATX-101, has antitumor activity in a panel of human glioblastoma multiforme (GBM) cell lines and patient-derived glioma-initiating cells (GICs). Their sensitivity to ATX-101 was not related to cellular levels of PCNA, or p53, PTEN, or MGMT status. However, ATX-101 reduced Akt/mTOR and DNA-PKcs signaling, and a correlation between high Akt activation and sensitivity for ATX-101 was found. ATX-101 increased the levels of γH2AX, DNA fragmentation, and apoptosis when combined with radiotherapy (RT). In line with the in vitro results, ATX-101 strongly reduced tumor growth in two subcutaneous xenografts and two orthotopic GBM models, both as a single agent and in combination with RT. The ability of ATX-101 to sensitize cells to RT is promising for further development of this compound for use in GBM.

CSIG-17. IMMUNE MODULATORY ROLE OF TYPE I INTERFERON IN SYNGENEIC MOUSE GLIOMA MODELS

Abstract Glioblastoma is characterized by a poor prognosis and a challenging phenotype for drug development. Although multimodal treatment, including surgery, radio- and chemotherapy is applied, the overall survival remains just above one year. Numerous clinical trials have studied targeted therapies against commonly deregulated pathways, but an efficient targeted drug is yet to be discovered. Likewise, immunotherapy has not been shown to be active. A subset of glioma tumor cells demonstrates stem-like properties; these cells are commonly referred to as glioma initiating cells (GIC). These types of cells are pluripotent and can by definition initiate and recapitulate glioma growth in experimental animals in vivo. Furthermore, these cells are often resistant to conventional therapies. Interferon β (IFN-β) is an immunomodulatory molecule with anti-cancer properties. We have previously shown that IFN-β greatly reduces sphere-formation capability of GIC. It was also confirmed that IFN-β sensitized resistant GIC to irradiation or the chemotherapeutic agent, temozolomide (TMZ). IFN-β treatment significantly prolonged survival in a xenograft model with GIC cells. In the current project, we want to use syngeneic mouse models to study the immunomodulatory effects of type I IFNs. Preliminary results indicate that abrogation of IFN signalling in tumor cells by CRISPR/Cas9 technology prolonged survival in mice only in cell lines which have substantial baseline autocrine IFN signalling. On the contrary, we did not observe a difference in survival when wild-type tumor cells were implanted in either IFNAR1 deficient or proficient hosts. Flow cytometry analysis will elucidate changes in immune cell recruitment and infiltration upon IFN signalling disruption. Moreover, we explore different treatments in combination with IFN-β as there are indications that TMZ or radiotherapy can have synergistic effects with stimulation of interferon type I signalling.

Ultra-hyper-fractionated radiotherapy for high-grade gliomas.

High-grade gliomas (HGGs; WHO grades III and IV) are invariably lethal brain tumors. Low-dose hyper-radiosensitivity (HRS) of HGG is a well-established phenomenon in vitro. However, possibly linked to the unavailability of accurate animal models of the diseases, this therapeutic effect could not be consistently translated to the animal setting, thus impairing its subsequent clinical development. The purpose of this study was to develop radiotherapeutic (RT) schedules permitting to significantly improve the overall survival of faithful animal models of HGG that have been recently made available. We used primary glioma initiating cell (GIC)-driven orthotopic animal models that accurately recapitulate the heterogeneity and growth patterns of the patients' tumors, to investigate the therapeutic effects of low radiation doses toward HGG. With the same total dose, RT fractions ≤0.5Gy twice per week [ultra-hyper-fractionation (ultra-hyper-FRT)] started at early stages of tumor progression (a condition that in the clinical setting often occurs at the end of the guidelines treatment) improved the effectiveness of RT and the animal survival in comparison to standard fractions. For the same cumulative dose, the use of fractions ≤0.5Gy may permit to escape one or more tumor resistance mechanisms thus increasing the effectiveness of RT and the overall animal survival. These findings suggest investigating in the clinical setting the therapeutic effect of an ultra-hyper-FRT schedule promptly extending the conventional RT component of the current guideline ("Stupp") therapeutic protocol.

Multiple Antitumor Molecular Mechanisms Are Activated by a Fully Synthetic and Stabilized Pharmaceutical Product Delivering the Active Compound Sulforaphane (SFX-01) in Preclinical Model of Human Glioblastoma

Frequent relapses and therapeutic resistance make the management of glioblastoma (GBM, grade IV glioma), extremely difficult. Therefore, it is necessary to develop new pharmacological compounds to be used as a single treatment or in combination with current therapies in order to improve their effectiveness and reduce cytotoxicity for non-tumor cells. SFX-01 is a fully synthetic and stabilized pharmaceutical product containing the α-cyclodextrin that delivers the active compound 1-isothiocyanato-4-methyl-sulfinylbutane (SFN) and maintains biological activities of SFN. In this study, we verified whether SFX-01 was active in GBM preclinical models. Our data demonstrate that SFX-01 reduced cell proliferation and increased cell death in GBM cell lines and patient-derived glioma initiating cells (GICs) with a stem cell phenotype. The antiproliferative effects of SFX-01 were associated with a reduction in the stemness of GICs and reversion of neural-to-mesenchymal trans-differentiation (PMT) closely related to epithelial-to-mesenchymal trans-differentiation (EMT) of epithelial tumors. Commonly, PMT reversion decreases the invasive capacity of tumor cells and increases the sensitivity to pharmacological and instrumental therapies. SFX-01 induced caspase-dependent apoptosis, through both mitochondrion-mediated intrinsic and death-receptor-associated extrinsic pathways. Here, we demonstrate the involvement of reactive oxygen species (ROS) through mediating the reduction in the activity of essential molecular pathways, such as PI3K/Akt/mTOR, ERK, and STAT-3. SFX-01 also reduced the in vivo tumor growth of subcutaneous xenografts and increased the disease-free survival (DFS) and overall survival (OS), when tested in orthotopic intracranial GBM models. These effects were associated with reduced expression of HIF1α which, in turn, down-regulates neo-angiogenesis. So, SFX-01 may have potent anti-glioma effects, regulating important aspects of the biology of this neoplasia, such as hypoxia, stemness, and EMT reversion, which are commonly activated in this neoplasia and are responsible for therapeutic resistance and glioma recurrence. SFX-01 deserves to be considered as an emerging anticancer agent for the treatment of GBM. The possible radio- and chemo sensitization potential of SFX-01 should also be evaluated in further preclinical and clinical studies.

Synchrotron-Based Fourier-Transform Infrared Micro-Spectroscopy (SR-FTIRM) Fingerprint of the Small Anionic Molecule Cobaltabis(dicarbollide) Uptake in Glioma Stem Cells.

The anionic cobaltabis (dicarbollide) [3,3′-Co(1,2-C2B9H11)2]−, [o-COSAN]−, is the most studied icosahedral metallacarborane. The sodium salts of [o-COSAN]− could be an ideal candidate for the anti-cancer treatment Boron Neutron Capture Therapy (BNCT) as it possesses the ability to readily cross biological membranes thereby producing cell cycle arrest in cancer cells. BNCT is a cancer therapy based on the potential of 10B atoms to produce α particles that cross tissues in which the 10B is accumulated without damaging the surrounding healthy tissues, after being irradiated with low energy thermal neutrons. Since Na[o-COSAN] displays a strong and characteristic ν(B-H) frequency in the infrared range 2.600–2.500 cm−1, we studied the uptake of Na[o-COSAN] followed by its interaction with biomolecules and its cellular biodistribution in two different glioma initiating cells (GICs), mesenchymal and proneural respectively, by using Synchrotron Radiation-Fourier Transform Infrared (FTIR) micro-spectroscopy (SR-FTIRM) facilities at the MIRAS Beamline of ALBA synchrotron light source. The spectroscopic data analysis from the bands in the regions of DNA, proteins, and lipids permitted to suggest that after its cellular uptake, Na[o-COSAN] strongly interacts with DNA strings, modifies proteins secondary structure and also leads to lipid saturation. The mapping suggests the nuclear localization of [o-COSAN]−, which according to reported Monte Carlo simulations may result in a more efficient cell-killing effect compared to that in a uniform distribution within the entire cell. In conclusion, we show pieces of evidence that at low doses, [o-COSAN]− translocates GIC cells’ membranes and it alters the physiology of the cells, suggesting that Na[o-COSAN] is a promising agent to BNCT for glioblastoma cells.

P13.17 CD95 gene silencing affects growth and invasiveness of glioma-initiating cells in a CD95L-independent manner

Abstract BACKGROUND CD95 (Fas/APO-1) holds a dual role of potential relevance in tumor development. CD95-CD95 ligand (CD95L) signaling regulates apoptotic cell death in CD95-expressing cells, but non-apoptotic, tumor-promoting CD95-CD95L signaling has been likewise described. Therapeutic stimulation of apoptotic CD95 signaling is associated with major clinical side effects. However, inhibition of tumor-promoting CD95 signaling may represent a promising treatment strategy for human cancers where potential tumor-promoting CD95 functions include invasiveness and cancer cell stemness, including glioblastoma. MATERIAL AND METHODS In this study, CD95 and CD95L expression was characterized in human glioma-initiating cells (GIC) in vitro and in vivo. CD95 and CD95L gene knockout (KO) GIC were generated by means of CRISPR-Cas9 and the effects of gene silencing were evaluated by assessing growth, clonogenicity, invasiveness and tumorigenicity in nude mice. RESULTS CD95 expression and sensitivity to exogenous CD95L-induced apoptosis were confirmed in selected GIC in vitro. CD95L expression was not detected. Upon CD95 KO, all GIC acquired resistance to CD95L-induced apoptosis. Furthermore, despite the confirmed absence of CD95L expression in vitro, CD95 KO S-24 GIC revealed decreased cell growth, inferior sphere forming capacity and decreased invasiveness. These data suggested a CD95L-independent tumor-promoting role of CD95 in S-24 GIC. In vivo, however, CD95 KO did not prolong the survival of glioma-bearing mice. Analyses of further GIC models are ongoing. CONCLUSION These data demonstrate that, unlike CD95, CD95L is not expressed in cultured human GIC and that CD95-CD95L interactions are not required for tumor-promoting CD95 signaling. Although CD95 KO is detrimental for S-24 GIC in vitro, CD95 KO alone does not affect survival in S-24 human GIC xenograft-bearing mice.

PRRX1 induced by BMP signaling decreases tumorigenesis by epigenetically regulating glioma-initiating cell properties via DNA methyltransferase 3A.

Glioma‐initiating cells (GICs), a major source of glioblastoma recurrence, are characterized by the expression of neural stem cell markers and the ability to grow by forming nonadherent spheres under serum‐free conditions. Bone morphogenetic proteins (BMPs), members of the transforming growth factor‐β family, induce differentiation of GICs and suppress their tumorigenicity. However, the mechanisms underlying the BMP‐induced loss of GIC stemness have not been fully elucidated. Here, we show that paired related homeobox 1 (PRRX1) induced by BMPs decreases the CD133‐positive GIC population and inhibits tumorigenic activity of GICs in vivo. Of the two splice isoforms of PRRX1, the longer isoform, pmx‐1b, but not the shorter isoform, pmx‐1a, induces GIC differentiation. Upon BMP stimulation, pmx‐1b interacts with the DNA methyltransferase DNMT3A and induces promoter methylation of the PROM1 gene encoding CD133. Silencing DNMT3A maintains PROM1 expression and increases the CD133‐positive GIC population. Thus, pmx‐1b promotes loss of stem cell‐like properties of GICs through region‐specific epigenetic regulation of CD133 expression by recruiting DNMT3A, which is associated with decreased tumorigenicity of GICs.

Effects of the DRD2/3 antagonist ONC201 and radiation in glioblastoma

BackgroundGlioblastoma (GBM) is the deadliest of all brain cancers in adults. The current standard-of-care is surgery followed by radiotherapy and temozolomide, leading to a median survival time of only 15 months. GBM are organized hierarchically with a small number of glioma-initiating cells (GICs), responsible for therapy resistance and tumor recurrence, suggesting that targeting GICs could improve treatment response. ONC201 is a first-in-class anti-tumor agent with clinical efficacy in some forms of high-grade gliomas. Here we test its efficacy against GBM in combination with radiation. MethodsUsing patient-derived GBM lines and mouse models of GBM we test the effects of radiation and ONC201 on GBM self-renewalin vitro and survivalin vivo.A possible resistance mechanism is investigated using RNA-Sequencing. ResultsTreatment of GBM cells with ONC201 reduced self-renewal, clonogenicity and cell viabilityin vitro. ONC201 exhibited anti-tumor effects on radioresistant GBM cells indicated by reduced self-renewal in secondary and tertiary glioma spheres. Combined treatment of ONC201 and radiation prolonged survival in syngeneic and patient-derived orthotopic xenograft mouse models of GBM. Subsequent transcriptome analyses after combined treatment revealed shifts in gene expression signatures related to quiescent GBM populations, GBM plasticity, and GBM stem cells. ConclusionsOur findings suggest that combined treatment with the DRD2/3 antagonist ONC201 and radiation improves the efficacy of radiation against GBMin vitroandin vivothrough suppression of GICs without increasing toxicity in mouse models of GBM. A clinical assessment of this novel combination therapy against GBM is further warranted.