Glioblastoma Cancer Stem Cells Research Articles

Preferential radiosensitization to glioblastoma cancer stem cell-like cells by a Hsp90 inhibitor, N-vinylpyrrolidone-AUY922.

The present study examined the radiosensitization induced by a heat shock protein 90 inhibitor, N-vinylpyrrolidone (NVP)-AUY922, in CD133-positive cells in a hypoxic area of T98G spheroids. CD133-positive cells that are induced in the hypoxic microenvironment of spheroids have previously been reported to exhibit cancer stem cell-like properties. The present study used CD133-positive cells from a glioblastoma cell line (T98G) as cancer stem cell-like cells. CD133-positive and negative cells were sorted from T98G spheroids using fluorescence-activated cell sorting and used for colony formation assay. Colony formation assay results indicated that NVP-AUY922 enhanced radiosensitivity more strongly in CD133-positive cells compared with CD133-negative cells. This result showed that NVP-AUY922 was a preferential radiosensitization candidate targeting glioblastoma cancer stem cells. The mechanisms underlying radiosensitization by NVP-AUY922 are discussed in relation to the properties of cancer stem cells. Overall, HIF-1α inhibition by NVP-AUY922 may induce higher sensitization of cancer stem cells to radiation.

Unexplored Functions of Sex Hormones in Glioblastoma Cancer Stem Cells.

Biological sex impacts a wide array of molecular and cellular functions that impact organismal development and can influence disease trajectory in a variety of pathophysiological states. In nonreproductive cancers, epidemiological sex differences have been observed in a series of tumors, and recent work has identified previously unappreciated sex differences in molecular genetics and immune response. However, the extent of these sex differences in terms of drivers of tumor growth and therapeutic response is less clear. In glioblastoma (GBM), the most common primary malignant brain tumor, there is a male bias in incidence and outcome, and key genetic and epigenetic differences, as well as differences in immune response driven by immune-suppressive myeloid populations, have recently been revealed. GBM is a prototypic tumor in which cellular heterogeneity is driven by populations of therapeutically resistant cancer stem cells (CSCs) that underlie tumor growth and recurrence. There is emerging evidence that GBM CSCs may show a sex difference, with male tumor cells showing enhanced self-renewal, but how sex differences impact CSC function is not clear. In this mini-review, we focus on how sex hormones may impact CSCs in GBM and implications for other cancers with a pronounced CSC population. We also explore opportunities to leverage new models to better understand the contribution of sex hormones vs sex chromosomes to CSC function. With the rising interest in sex differences in cancer, there is an immediate need to understand the extent to which sex differences impact tumor growth, including effects on CSC function.

Quiescent human glioblastoma cancer stem cells drive tumor initiation, expansion, and recurrence following chemotherapy.

We test the hypothesis that glioblastoma harbors quiescent cancer stem cells that evade anti-proliferative therapies. Functional characterization of spontaneous glioblastomas from genetically engineered mice reveals essential quiescent stem-like cells that can be directly isolated from tumors. A derived quiescent cancer-stem-cell-specific gene expression signature is enriched in pre-formed patient GBM xenograft single-cell clusters that lack proliferative gene expression. A refined human 118-gene signature is preserved in quiescent single-cell populations from primary and recurrent human glioblastomas. The F3 cell-surface receptor mRNA, expressed in the conserved signature, identifies quiescent tumor cells by antibody immunohistochemistry. F3-antibody-sorted glioblastoma cells exhibit stem cell gene expression, enhance self-renewal in culture, drive tumor initiation and serial transplantation, and reconstitute tumor heterogeneity. Upon chemotherapy, the spared cancer stem cell pool becomes activated and accelerates transition to proliferation. These results help explain conventional treatment failure and lay a conceptual framework for alternative therapies.

MiR-671-5p Inhibition by MSI1 Promotes Glioblastoma Tumorigenesis via Radioresistance, Tumor Motility and Cancer Stem-like Cell Properties.

MicroRNAs (miRNAs) could be potential biomarkers for glioblastoma multiforme (GBM) prognosis and response to therapeutic agents. We previously demonstrated that the cancer stem cell marker Musashi-1 (MSI1) is an RNA binding protein that promotes radioresistance by increasing downstream RNA stability. To identify that MSI1 interacts with miRNAs and attenuates their function, we also get candidate miRNAs from the mRNA seq by predicting with TargetScan software. miR-671-5p in GBM cells interacts with MSI1 by intersecting the precipitated miRNAs with the predicted miRNAs. Notably, overexpression of MSI1 reversed the inhibitory effect of miR-671-5p. The phenotype of miR-671-5p in GBM cells could affect radiosensitivity by modulating the posttranscriptional activity of STAT3. In addition, miR-671-5p could attenuate tumor migration and cancer stem cell (CSC) characteristics by repressing the posttranscriptional activity of TRAF2. MSI1 may regulate GBM radioresistance, CSCs and tumor motility through miR-671-5p inhibition to increasing STAT3 and TRAF2 presentation. In vivo, the GBM tumor size was inversely correlated with miR-671-5p expression, but tumorigenesis was promoted by STAT3 and TRAF2 activation in the miR-671-5p-positive GBM population. miR-671-5p could be activated as a novel therapeutic target for GBM and has potential application as a predictive biomarker of glioblastoma prognosis.

Modelling the microenvironment of the most aggressive brain tumours for preclinical studies

Preclinical and clinical cancer studies use unrepresentative tumour models that do not properly simulate the intricate pathobiology of the human tumour and its complex microenvironment. This is of critical importance for the brain tumour glioblastoma (GBM), one of the most malignant cancers, which almost always relapses despite treatment. Due to the intertumoral and intratumoral heterogeneity of GBM, it is highly resistant to conventional treatment with radiotherapy and chemotherapy. In this review, we describe the heterogeneity of GBM and the unique microenvironment of brain tumours as well as their importance for GBM progression and to set up clinically relevant GBM models for successful preclinical research and drug screening. A combination of cancer cells, cancer stem cells, and cancer-associated cells create an intricate and diverse tumour microenvironment. GBM is very adept at recruiting healthy cells to support itself. The function of these cancer-associated cells varies in different niches, but they all share the common function of promoting tumour growth and spread. Glioblastoma stem cells are one of the central components of GBM treatment resistance, invasiveness, and cancer stem cell niche relationships. All of these aspects are critical when selecting and designing preclinical GBM models.

STEM-15. SerpinB3 DRIVES CANCER STEM CELL SURVIVAL IN GLIOBLASTOMA

Abstract Despite therapeutic interventions for glioblastoma (GBM), self-renewing, therapy-resistant populations of cells referred to as cancer stem cells (CSCs) drive recurrence. Previously, we identified the unique expression of junctional adhesion molecule-A (JAM-A) on CSCs and demonstrated that JAM-A is both necessary and sufficient for self-renewal and tumor growth. Moreover, we determined that JAM-A signals via Akt in GBM CSCs to sustain pluripotency transcription factor activity; however, the entire signaling network has yet to be fully elucidated. To further delineate this pathway, we immunoprecipitated JAM-A from patient-derived GBM CSCs and performed mass spectrometry to determine JAM-A binding proteins. This led to the identification of the cysteine protease inhibitor SerpinB3 as a putative JAM-A binding partner. Using in vitro CSC functional assays, we show that SerpinB3 is necessary for CSC maintenance and survival. In an in vivo orthotopic xenograft model, knockdown of SerpinB3 extended survival. Mechanistically, knockdown of SerpinB3 led to decreased expression of TGF-β, Myc, WNT, and Notch signaling, known regulators of the CSC state. Additionally, knockdown of SerpinB3 increases susceptibility to radiation therapy. SerpinB3 is essential for buffering cells against cathepsin-mediated cell death, and we found that elevated lysosomal membrane permeability after radiation leads to cathepsin release into the cytoplasm. As a result, SerpinB3 knockdown cells have a diminished capacity to inhibit cathepsin-driven cell death after radiation. The addition of the cathepsin inhibitor E64D partially rescues the SerpinB3 knockdown, however, SerpinB3 mutants that are unable to inhibit cathepsins fail to do the same. Taken together, our findings, identify a novel GBM CSC-specific survival mechanism involving a previously uninvestigated cysteine protease inhibitor, SerpinB3, and provide a potential target to increase the efficacy of standard of care GBM therapies against therapy-resistant CSCs.

Antiproliferative activity of CD44 siRNA-PEI-PEG nanoparticles in glioblastoma: involvement of AKT signaling.

Background and purpose:Glioblastoma multiforme (GBM) is the most invasive type of cancer which starts inside the brain. GBM cells were found to have similar properties to glioblastoma cancer stem cells. CD44 can be used as a marker of the cancer stem cells in a subset of glioblastoma tumor cells. Recent studies showed that CD44 is involved in developing cancer cells via the protein kinase B (PKB or AKT) signaling pathway. Therefore, this study aimed to investigate the CD44 mRNA silencing effects on the glioblastoma cell cycle via AKT signaling pathway.Experimental approach:To determine CD44 expression in the samples of the patients with GBM, we used the analysis of data extracted from TCGA database. qRT-PCR and western blotting were used to evaluate the expression level of genes and proteins. Different cell cycles were evaluated by DAPI staining and flow cytometry.Findings/Results:Bioinformatics results showed that CD44 expression level in GBM tumor samples is higher than in normal samples. Effects of poly (ethylene imine)-polyethylene glycol (PEI-PEG)-loaded CD44 siRNA in cell cycle showed that CD44 silencing could inhibit cell cycle in G0-G1 phase by more than 20% compared to the negative control (P < 0.05). Furthermore, PEI-PEG-loaded CD44 siRNA reduces the expression of cyclin D1 and CKD-4. According to our findings, this structure also prevented AKT phosphorylation at Thr-308 and Ser-473.Conclusion and implications:Our results suggest that PEI-PEG-loaded CD44 siRNA may attenuate the cell cycle by suppressing AKT signaling pathway.

Targeting Folate Metabolism Is Selectively Cytotoxic to Glioma Stem Cells and Effectively Cooperates with Differentiation Therapy to Eliminate Tumor-Initiating Cells in Glioma Xenografts.

Glioblastoma (GBM) is one of the deadliest of all human cancers. Developing therapies targeting GBM cancer stem cells or glioma stem cells (GSCs), which are deemed responsible for the malignancy of GBM due to their therapy resistance and tumor-initiating capacity, is considered key to improving the dismal prognosis of GBM patients. In this study, we found that folate antagonists, such as methotrexate (MTX) and pemetrexed, are selectively cytotoxic to GSCs, but not to their differentiated counterparts, normal fibroblasts, or neural stem cells in vitro, and that the high sensitivity of GCSs to anti-folates may be due to the increased expression of RFC-1/SLC19A1, the reduced folate carrier that transports MTX into cells, in GSCs. Of note, in an in vivo serial transplantation model, MTX alone failed to exhibit anti-GSC effects but promoted the anti-GSC effects of CEP1347, an inducer of GSC differentiation. This suggests that folate metabolism, which plays an essential role specifically in GSCs, is a promising target of anti-GSC therapy, and that the combination of cytotoxic and differentiation therapies may be a novel and promising approach to effectively eliminate cancer stem cells.

A natural protein based platform for the delivery of Temozolomide acid to glioma cells

Glioblastoma is one of the most difficult to treat cancers with poor prognosis and survival of around one year from diagnosis. Effective treatments are desperately needed. This work aims to prepare temozolomide acid (TMZA) loaded albumin nanoparticles, for the first time, to target glioblastoma (GL261) and brain cancer stem cells (BL6). TMZA was loaded into human serum albumin nanoparticles (HSA NPs) using the desolvation method. A response surface 3-level factorial design was used to study the effect of different formulation parameters on the drug loading and particle size of NPs. The optimum conditions were found to be: 4 mg TMZA with 0.05% sodium cholate. This yielded NPs with particle size and drug loading of 111.7 nm and 5.5% respectively. The selected formula was found to have good shelf life and serum stability but with a relatively fast drug release pattern. The optimized NPs showed excellent cellular uptake with ∼ 50 and 100% of cells were positive for NP uptake after 24 h incubation with both GL261 and BL6 glioblastoma cell lines, respectively. The selected formula showed high cytotoxicity with ̴ 20% cell viability at 1 mM TMZA after 72 h incubation time. Finally, the fluorescently labelled NPs showed co-localization with the bioluminescent syngeneic BL6 intra-cranial tumour mouse model after intravenous administration.

Excessive new origin firing underlies selective glioma stem cell cytotoxicity induced by replication stress response inhibition

Abstract Aims Glioblastoma (GBM) is a treatment refractory cancer of extreme unmet need which exhibits treatment resistance due to a subpopulation of GBM cancer stem cells which have constitutive DNA damage response activation driven by elevated replication stress (RS). RS response inhibition is potently cytotoxic to GSC, however mechanistic understanding will be key to biomarker discovery and successful clinical translation. We investigated response to combined ATR and PARP inhibition (CAiPi) to gain mechanistic insight and inform biomarker development. Method A panel of patient-derived GBM cell lines were cultured as stem enriched (GSCs) or stem depleted (bulk), to characterise response to combined ATR inhibition (VE821 5μM) and PARP inhibition (Olaparib 1μM), by CellTiter-Glo viability assay. Mechanistic investigations included immunofluorescence of 53BP1 nuclear bodies and DNA fibre analysis. Studies into the importance of PARP trapping included another PARPi Veliparib (1μM), and investigations into inhibition of origin firing used the CDK inhibitor Roscovitine. Results Responses to CAiPi in a panel of primary paired GBM GSCs vs differentiated progeny were heterogenous. CAiPi is selectively GSC cytotoxic in a subpopulation of tumours. DNA fibre analysis identified increased new origin firing with PARPi, which was correlated with increased PARP trapping. Inhibition of origin firing by exposure to roscovitine rescued the CAiPi cytotoxic phenotype, suggesting origin firing has an important role in selective GSC cytotoxicity. A population of treatment-sensitive GSCs with increased numbers of 53BP1 nuclear bodies in G1 phase with CAiPi were identified, indicative of under-replication of DNA in S phase. Conclusion Selective GSC cytotoxicity is induced by CAiPi via dysregulation of replication, by both DNA under-replication resulting in DNA lesions, and the novel finding of increased new origin firing in GSC due to PARPi.

SYST-05. PHASE 2 STUDY OF VAL-083 AND RADIOTHERAPY IN NEWLY DIAGNOSED MGMT-UNMETHYLATED GBM

Abstract VAL-083 is a novel bi-functional DNA targeting agent that induces inter-strand DNA cross-links at N7-guanine, leading to DNA double-strand breaks and cell death. In vitro and in vivo studies have demonstrated VAL-083 circumvents MGMT-mediated chemo-resistance and differentiates it from other therapies used in the treatment of GBM, including temozolomide (TMZ). VAL-083 also acts as a radiosensitizer against GBM cancer stem cells in vitro. A Phase 2 study was conducted to evaluate the safety and tolerability of VAL-083 when administered concurrently with radiation therapy (RT) in newly diagnosed MGMT unmethylated GBM. Stage 1 was a dose-escalation phase to confirm the dose of VAL-083 in this setting. Patients received VAL-083 at 20, 30, or 40 mg/m2/day x 3 days every 21 days in combination with standard radiation treatment (RT) (2 Gy/day, 5 days/week for 6 weeks). Stage 2 was an expansion phase to enroll up to 20 additional patients at the 30 mg/m2/day of VAL-083 with RT. A total of 29 patients were enrolled in the study and completed treatment, with 25 patients receiving 30 mg/m2/day VAL-083. The median number of cycles completed by all patients was 9 (range 2-13). Consistent with our prior experience, myelosuppression was the most common adverse event. Pharmacokinetics (Cmax and AUC) of VAL-083 were broadly linear with respect to dose, and drug half-life was 0.8 hrs. In a sub-group of patients, levels of VAL-083 in CSF were found to be at least as high as those in plasma. The median progression free survival (PFS) for all patients enrolled was 9.3 (95%CI: 6.4-12.0) months. Eighteen (18/29; 62.1%) patients have died, and median overall survival for all patients enrolled was 19.6 (95%CI: 14.0-22.4) months. These results support the potential benefit of VAL-083 as a treatment alternative against GBM tumors with MGMT-mediated resistance to TMZ. Clinicaltrials.gov: NCT03050736.

Characterization of Glioblastoma Cancer Stem Cells Sorted by Sedimentation Field-Flow Fractionation Using an Ultrahigh-Frequency Range Dielectrophoresis Biosensor.

Cancer stem cells (CSCs) appear to be an essential target for cancer therapies, in particular, in brain tumors such as Glioblastoma. Nevertheless, their isolation is made difficult by their low content in culture or tumors (<5% of the tumor mass) and is essentially based on the use of fluorescent or magnetic labeling techniques, increasing the risk of differentiation induction. The use of label-free separation methods such as sedimentation field-flow fractionation (SdFFF) is promising, but it becomes necessary to consider a coupling with a detection and characterization method for future identification and purification of CSCs from patient-derived tumors. In this study, we demonstrate for the first time the capability of using an ultrahigh-frequency range dielectrophoresis fluidic biosensor as a detector. This implies an important methodological adaptation of SdFFF cell sorting by the use of a new compatible carrier liquid DEP buffer (DEP-B). After SdFFF sorting, subpopulations derived from U87-MG and LN18 cell lines undergo biological characterization, demonstrating that using DEP-B as a carrier liquid, we sorted by SdFFF subpopulations with specific differentiation characteristics: F1 = differentiated cells/F2 = CSCs. These subpopulations presented high-frequency crossover (HFC) values similar to those measured for standard differentiated (around 110 MHz) and CSC (around 80 MHz) populations. This coupling appeared as a promising solution for the development of an online integration of these two complementary label-free separation/detection technologies.

The transcription factor ZEB1 regulates stem cell self-renewal and cell fate in the adult hippocampus.

SummaryRadial glia-like (RGL) stem cells persist in the adult mammalian hippocampus, where they generate new neurons and astrocytes throughout life. The process of adult neurogenesis is well documented, but cell-autonomous factors regulating neuronal and astroglial differentiation are incompletely understood. Here, we evaluate the functions of the transcription factor zinc-finger E-box binding homeobox 1 (ZEB1) in adult hippocampal RGL cells using a conditional-inducible mouse model. We find that ZEB1 is necessary for self-renewal of active RGL cells. Genetic deletion of Zeb1 causes a shift toward symmetric cell division that consumes the RGL cell and generates pro-neuronal progenies, resulting in an increase of newborn neurons and a decrease of newly generated astrocytes. We identify ZEB1 as positive regulator of the ets-domain transcription factor ETV5 that is critical for asymmetric division.

Abstract CT172: Phase 2 clinical trial of dianhydrogalactitol (VAL-083) in patients with newly diagnosed MGMT-unmethylated GBM

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain cancer. The majority of glioblastoma multiforme (GBM) patients have an unmethylated MGMT promoter status, leading to limited response to TMZ and decreased survival. Current standard-of-care includes surgery followed by chemoradiation and TMZ. An unmethylated promoter for O6-methylguanine DNA methyltransferase (MGMT) is a validated biomarker for TMZ-resistance and is strongly correlated with poor outcomes. VAL-083 is a novel bi-functional DNA targeting agent that induces inter-strand cross-links at N7-guanine, leading to DNA double-strand breaks and ultimately cell death. VAL-083 circumvents MGMT-mediated drug-resistance and has demonstrated cytotoxicity in MGMT-unmethylated GBM cell lines, cancer stem cells (CSCs) and in vivo models. Furthermore, VAL-083 acts as a radiosensitizer in GBM CSCs and non-CSCs. A Phase 2, open-label, biomarker-driven, study is being conducted to evaluate the tolerability and efficacy of VAL-083 in combination with radiation therapy (RT) in newly diagnosed MGMT-unmethylated GBM patients. A treatment regimen, consisting of a 6-week induction period of VAL-083 given IV at 20, 30, or 40 mg/m2/day x 3 days every 21 days and concurrent radiation (2 Gy daily, 5 days/week) followed by up to 24 weeks of maintenance therapy with single-agent VAL-083, is being evaluated. The study has 2 stages: Stage 1, dose-escalation, has identified a recommended dose of 30 mg/m2/day of VAL-083 in combination with RT as generally safe and well-tolerated. Stage 2, an expansion stage, has enrolled 20 additional patients at 30 mg/m2/day IV infusion on days 1, 2, and 3 of 21-day cycles. As of January 11, 2021, all 29 subjects in the study have been enrolled and completed the treatment stage of the study. Tumor response are assessed by MRI, according to RANO criteria. Efficacy endpoints include progression-free survival (PFS) and overall survival (OS). Additional endpoints include safety evaluations and pharmacokinetic assessments of plasma and CSF samples. The trial is continuing as planned and an update on primary results on safety and efficacy, and pharmacokinetics will be provided at the meeting. Clinicaltrials.gov identifier: NCT03050736. Citation Format: Zhong-ping Chen, Cheng-cheng Guo, Qun-ying Yang, Jia-wei Li, Shao-xiong Wu, John Langlands, Gregory Johnson, Claire Kwan, Sarath Kanekal, Richard Schwartz, Jeffrey Bacha, Anne Steino, Dennis Brown. Phase 2 clinical trial of dianhydrogalactitol (VAL-083) in patients with newly diagnosed MGMT-unmethylated GBM [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 CT172.

Abstract CT238: Phase 2 study of dianhydrogalactitol (VAL-083) inpatients with MGMT-unmethylated, bevacizumab-naivepatients with MGMT-unmethylated, bevacizumab-naïve glioblastoma in the recurrent and adjuvant setting

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain cancer. Current standard-of-care for glioblastoma (GBM) includes surgery followed by concurrent therapy with radiation and temozolomide (TMZ) followed by adjuvant TMZ (days 1-5 every 28 days). Almost all GBM patients experience recurrent/progressive disease, with a median survival of 3-9 months after recurrence. Second-line treatment for recurrent GBM with bevacizumab (BEV) has not improved survival, and effective therapies for GBM are lacking. Unmethylated promoter status for O6-methylguanine DNA methyltransferase (MGMT) is a validated biomarker for TMZ-resistance and is correlated with poor prognosis. VAL-083 is a bi-functional DNA-targeting agent rapidly inducing interstrand cross-links at N7-guanine, leading to DNA double-strand breaks and cell death. VAL-083's cytotoxicity is independent of MGMT status, and VAL-083 overcomes TMZ-resistance in GBM cell lines, GBM cancer stem cells, and in vivo GBM models. The trial described here is an open-label two-arm biomarker-driven phase 2 clinical trial in MGMT-unmethylated GBM patients with either recurrent disease (Group 1) or newly diagnosed GBM patients requiring adjuvant therapy after chemoradiation with TMZ (Group 2). Patients receive VAL-083 IV at 30 or 40 mg/m2/day on days 1, 2, and 3 of a 21-day cycle. For Group 1 the primary objective of this study is to determine the effect of VAL-083 on median overall survival (mOS) in MGMT-unmethylated recurrent GBM patients compared to historical control. For Group 2, the primary objective of this study is to determine the effect of VAL-083 on progression-free survival (PFS) in newly diagnosed GBM patients requiring adjuvant therapy after chemoradiation with TMZ, compared to historical control. Secondary efficacy endpoints include progression-free survival (PFS) (Group 1), overall response rate (ORR), duration of response (DOR), and quality-of-life (QoL). Tumor response will be assessed by MRI approximately every 42 days, as per RANO criteria. The initial starting dose in this study was 40 mg/m2/day, which was subsequently reduced to 30 mg/m2/day to improve tolerance due to myelosuppression. As of January 11, 2021, 35 evaluable subjects have enrolled at a starting dose of 40 mg/m2/day, and 46 evaluable subjects have enrolled at a starting dose of 30 mg/m2/day in Group 1. In Group 2, 31 evaluable subjects had enrolled at a starting dose of 30 mg/m2/day. As anticipated from prior studies with VAL-083, myelosuppression (thrombocytopenia and neutropenia) has been the most common adverse event observed. The trial is continuing as planned, and an enrollment, efficacy and safety data update will be provided at the meeting. Clinicaltrials.gov identifier: NCT02717962 Citation Format: Barabara J. O'Brien, Carlos Kamiya-Matusoka, Shaio-Pei Weathers, WK Alfred Yung, WK Alfred Yung, Monica Loghin, Rebecca Harrison, Nazanin Majd, Stephanie Knight, Ying Yuan, Jeffrey Bacha, Dennis Brown, Anne Steino, Gregory Johnson, Sarath Kanekal, John Langlands, Lorena Lopez, Richard Schwartz, Marta Penas-Prado, John de Groot. Phase 2 study of dianhydrogalactitol (VAL-083) inpatients with MGMT-unmethylated, bevacizumab-naivepatients with MGMT-unmethylated, bevacizumab-naïve glioblastoma in the recurrent and adjuvant setting [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 CT238.

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.

Development of CD133 Targeting Multi-Drug Polymer Micellar Nanoparticles for Glioblastoma - In Vitro Evaluation in Glioblastoma Stem Cells.

Glioblastoma (GBM) is a malignant brain tumor with a poor long-term prognosis due to recurrence from highly resistant GBM cancer stem cells (CSCs), for which the current standard of treatment with temozolomide (TMZ) alone will unlikely produce a viable cure. In addition, CSCs regenerate rapidly and overexpress methyl transferase which overrides the DNA-alkylating mechanism of TMZ, leading to resistance. The objective of this research was to apply the concepts of nanotechnology to develop a multi-drug therapy, TMZ and idasanutlin (RG7388, a potent mouse double minute 2 (MDM2) antagonist), loaded in functionalized nanoparticles (NPs) that target the GBM CSC subpopulation, reduce the cell viability and provide possibility of in vivo preclinical imaging. Polymer-micellar NPs composed of poly(styrene-b-ethylene oxide) (PS-b-PEO) and poly(lactic-co-glycolic) acid (PLGA) were developed by a double emulsion technique loading TMZ and/or RG7388. The NPs were covalently bound to a 15-nucleotide base-pair CD133 aptamer to target the CD133 antigen expressed on the surfaces of GBM CSCs. For diagnostic functionality, the NPs were labelled with radiotracer Zirconium-89 (89Zr). NPs maintained size range less than 100nm, a low negative charge and exhibited the ability to target and kill the CSC subpopulation when TMZ and RG7388 were used in combination. The targeting function of CD133 aptamer promoted killing in GBMCSCs providing impetus for further development of targeted nanosystems for localized therapy in future in vivo models. This work has provided a potential clinical application for targeting GBM CSCs with simultaneous diagnostic imaging.

Altered lipid metabolism marks glioblastoma stem and non-stem cells in separate tumor niches

Glioblastoma (GBM) displays marked cellular and metabolic heterogeneity that varies among cellular microenvironments within a tumor. Metabolic targeting has long been advocated as a therapy against many tumors including GBM, but how lipid metabolism is altered to suit different microenvironmental conditions and whether cancer stem cells (CSCs) have altered lipid metabolism are outstanding questions in the field. We interrogated gene expression in separate microenvironments of GBM organoid models that mimic the transition between nutrient-rich and nutrient-poor pseudopalisading/perinecrotic tumor zones using spatial-capture RNA-sequencing. We revealed a striking difference in lipid processing gene expression and total lipid content between diverse cell populations from the same patient, with lipid enrichment in hypoxic organoid cores and also in perinecrotic and pseudopalisading regions of primary patient tumors. This was accompanied by regionally restricted upregulation of hypoxia-inducible lipid droplet-associated (HILPDA) gene expression in organoid cores and pseudopalisading regions of clinical GBM specimens, but not lower-grade brain tumors. CSCs have low lipid droplet accumulation compared to non-CSCs in organoid models and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. Targeted lipidomic analysis of multiple patient-derived models revealed a significant shift in lipid metabolism between GBM CSCs and non-CSCs, suggesting that lipid levels may not be simply a product of the microenvironment but also may be a reflection of cellular state. CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs, but had significantly increased polyunsaturated fatty acid production due to high fatty acid desaturase (FADS1/2) expression which was essential to maintain CSC viability and self-renewal. Our data demonstrate spatially and hierarchically distinct lipid metabolism phenotypes occur clinically in the majority of patients, can be recapitulated in laboratory models, and may represent therapeutic targets for GBM.

BMP4 to increase connexin43 expression and interaction with β-catenin in glioblastoma stem cells.

e14045 Background: Glioblastoma multiforme (GBM) is the most commonly occurring and most lethal primary central nervous system tumor. Treatment for GBM involves an aggressive combination of surgical resection, radiotherapy, and temozolomide chemotherapy. While intensive therapy has shown marginal improvement in prognosis, the median survival for GBM patients remains between twelve to fourteen months. The aggressiveness of GBM is attributed to the cellular heterogeneity and the invasive nature of the tumor, as well as the presence of glioblastoma cancer stem cells (GSCs). GSCs are a subpopulation of slowly dividing cells within GBM tumors that can survive chemotherapy and irradiation treatments targeting proliferating cells. The remaining quiescent GSCs can then enter an active state of self-renewal and differentiation through asymmetric division that contributes to tumor recurrence by recapitulating the heterogeneous tumor in relatively short order. Previously, we have identified a non-junctional, tumorigenic function for the gap junction protein connexin43 (Cx43) through its interaction with microtubules in GSCs. However, when GSCs are differentiated, this interaction decreases while Cx43 expression increases, suggesting an alternate function for Cx43. Bone Morphogenic Proteins (BMPs) have been implicated in GSC differentiation and their role in human tumorigenesis has remained largely unknown in GSCs. In this study, we aim to determine the effect of BMP on Cx43 expression and localization in GSCs. Methods: In this study, we used GBM patient-derived GSCs isolated from two GBM tumors following resection at Carilion Clinic, and maintained in stem cell medium. The effect of BMP4 signaling on Cx43 expression and localization in GSCs was determined using RT-qPCR, immunofluorescence, western blot, and co-immunoprecipitation. Results: Preliminary data show that BMP4 treatment increases Cx43 expression at the protein and mRNA levels through nuclear translocation and transcriptional activity of β-catenin. In addition, BMP4 treatment increases the interaction between Cx43 and β-catenin at the plasma membrane. Further studies are underway to determine the implications of disrupting Cx43/β-catenin interaction in GSC differentiation. Conclusions: While additional experiments are needed, our findings may be adapted as a prognostic indicator to determine patients’ susceptibility to traditional GBM therapies, and aid in novel therapeutic development to eradicate GSCs in GBM and prevent tumor recurrence following treatment.

Machine learning revealed stemness features and a novel stemness-based classification with appealing implications in discriminating the prognosis, immunotherapy and temozolomide responses of 906 glioblastoma patients.

Glioblastoma (GBM) is the most malignant and lethal intracranial tumor, with extremely limited treatment options. Immunotherapy has been widely studied in GBM, but none can significantly prolong the overall survival (OS) of patients without selection. Considering that GBM cancer stem cells (CSCs) play a non-negligible role in tumorigenesis and chemoradiotherapy resistance, we proposed a novel stemness-based classification of GBM and screened out certain population more responsive to immunotherapy. The one-class logistic regression algorithm was used to calculate the stemness index (mRNAsi) of 518 GBM patients from The Cancer Genome Atlas (TCGA) database based on transcriptomics of GBM and pluripotent stem cells. Based on their stemness signature, GBM patients were divided into two subtypes via consensus clustering, and patients in Stemness Subtype I presented significantly better OS but poorer progression-free survival than Stemness Subtype II. Genomic variations revealed patients in Stemness Subtype I had higher somatic mutation loads and copy number alteration burdens. Additionally, two stemness subtypes had distinct tumor immune microenvironment patterns. Tumor Immune Dysfunction and Exclusion and subclass mapping analysis further demonstrated patients in Stemness Subtype I were more likely to respond to immunotherapy, especially anti-PD1 treatment. The pRRophetic algorithm also indicated patients in Stemness Subtype I were more resistant to temozolomide therapy. Finally, multiple machine learning algorithms were used to develop a 7-gene Stemness Subtype Predictor, which were further validated in two external independent GBM cohorts. This novel stemness-based classification could provide a promising prognostic predictor for GBM and may guide physicians in selecting potential responders for preferential use of immunotherapy.