Patient-derived Glioblastoma Stem-like Cells Research Articles

On-chip perivascular niche supporting stemness of patient-derived glioma cells in a serum-free, flowable culture.

Glioblastoma multiforme (GBM) is the most common and the most aggressive type of primary brain malignancy. Glioblastoma stem-like cells (GSCs) can migrate in vascular niches within or away from the tumour mass, increasing tumour resistance to treatments and contributing to relapses. To study individual GSC migration and their interactions with the perivasculature of the tumour microenvironment, there is a need to develop a human organotypic in vitro model. Herein, we demonstrated a perivascular niche-on-a-chip, in a serum-free condition with gravity-driven flow, that supported the stemness of patient-derived GSCs and foetal neural stem cells grown in a three-dimensional environment (3D). Endothelial cells from three organ origins, (i) human brain microvascular endothelial cells (hCMEC/D3), (ii) human umbilical vein endothelial cells (HUVECs) and, (iii) human lung microvascular endothelial cells (HMVEC-L) formed rounded microvessels within the extracellular-matrix integrated microfluidic chip. By optimising cell extraction protocols, systematic studies were performed to evaluate the effects of serum-free media, 3D cell cultures, and the application of gravity-driven flow on the characteristics of endothelial cells and their co-culture with GSCs. Our results showed the maintenance of adherent and tight junction markers of hCMEC/D3 in the serum-free culture and that gravity-driven flow was essential to support adequate viability of both the microvessel and the GSCs in co-culture (>80% viability at day 3). Endpoint biological assays showed upregulation of neovascularization-related genes (e.g., angiopoietins, vascular endothelial growth factor receptors) in endothelial cells co-cultured with GSCs in contrast to the neural stem cell reference that showed insignificant changes. The on-chip platform further permitted live-cell imaging of GSC – microvessel interaction, enabling quantitative analysis of GSC polarization and migration. Overall, our comparative genotypic (i.e. qPCR) and phenotypic (i.e. vessel permeability and GSC migration) studies showed that organotypic (brain cancer cells–brain endothelial microvessel) interactions differed from those within non-tissue specific vascular niches of human origin. The development and optimization of this on-chip perivascular niche, in a serum-free flowable culture, could provide the next level of complexity of an in vitro system to study the influence of glioma stem cells on brain endothelium.

CBIO-19. LOW GLOBAL HISTONE H4 ACETYLATION LEVELS REVEAL CANDIDATE QUIESCENT CELL POPULATIONS IN GLIOMA AND DISTINGUISH TUMORS BY GRADE

Abstract Current standard of care therapy for glioblastoma (GBM) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, the presence of quiescent-like/G0 states, therefore, represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. To gain insight into how glioblastoma cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) harboring a G0-reporter to identify genes that when inhibited trap GSCs in G0-like states. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, which targets both histones (e.g., H4) and non-histone proteins for acetylation. NuA4 functions as a transcriptional activator, whose activities are coordinated with MYC in certain contexts, and also participates in DNA double-strand break repair by facilitating chromatin opening. However, currently little is known about the roles for NuA4 complex in GBM biology. Through modeling KAT5 function in GSC in vitro cultures and in vivo tumors, we find that KAT5 inhibition causes cells to arrest in a G0-like state with high p27 levels, G1-phase DNA content, low protein synthesis rates, low rRNA rates, lower metabolic rate, suppression of cell cycle gene expression, and low histone H4 acetylation. Interestingly, partial inhibition of KAT5 activity slows highly aggressive tumor growth, while increasing p27hi H4-aclow populations. Remarkably, we that low grade gliomas have significantly higher H4-aclow subpopulations and generally lower H4-ac levels than aggressive grade IV tumors. Taken together, our results suggest that NuA4/KAT5 activity may play a key role in quiescence ingress/egress in glioma and that targeting its activity in high grade tumors may effectively “down grade” them, thus, increase patient survival.

Temozolomide-Induced RNA Interactome Uncovers Novel LncRNA Regulatory Loops in Glioblastoma.

Simple SummaryGlioblastoma (GBM) is the most aggressive brain tumor and most resistant to therapy. The identification of novel predictive biomarkers or targets to counteract chemoresistance, requires a better understanding of the GBM primary response to therapy. The aim of our study was to assess the molecular response of GBM to the standard of care chemotherapy by temozolomide (TMZ). We established a comprehensive map of gene expression changes after treatment and discovered that GBM cells elicit a coordinated gene expression program after chemotherapy that differs between sensitive and resistant cells. We found that a novel class of genes expressed as long non-coding RNAs (lncRNAs) is involved in gene regulatory circuits in GBM and could represent novel markers of GBM patient prognosis. By shedding light on the involvement of the non-coding genome in GBM, our results may provide new mechanistic insight on lncRNAs and their importance in chemoresistance. Resistance to chemotherapy by temozolomide (TMZ) is a major cause of glioblastoma (GBM) recurrence. So far, attempts to characterize factors that contribute to TMZ sensitivity have largely focused on protein-coding genes, and failed to provide effective therapeutic targets. Long noncoding RNAs (lncRNAs) are essential regulators of epigenetic-driven cell diversification, yet, their contribution to the transcriptional response to drugs is less understood. Here, we performed RNA-seq and small RNA-seq to provide a comprehensive map of transcriptome regulation upon TMZ in patient-derived GBM stem-like cells displaying different drug sensitivity. In a search for regulatory mechanisms, we integrated thousands of molecular associations stored in public databases to generate a background “RNA interactome”. Our systems-level analysis uncovered a coordinated program of TMZ response reflected by regulatory circuits that involve transcription factors, mRNAs, miRNAs, and lncRNAs. We discovered 22 lncRNAs involved in regulatory loops and/or with functional relevance in drug response and prognostic value in gliomas. Thus, the investigation of TMZ-induced gene networks highlights novel RNA-based predictors of chemosensitivity in GBM. The computational modeling used to identify regulatory circuits underlying drug response and prioritizing gene candidates for functional validation is applicable to other datasets.

Mir-370-3p Impairs Glioblastoma Stem-Like Cell Malignancy Regulating a Complex Interplay between HMGA2/HIF1A and the Oncogenic Long Non-Coding RNA (lncRNA) NEAT1

Glioblastoma (GBM) is the most aggressive and prevalent form of a human brain tumor in adults. Several data have demonstrated the implication of microRNAs (miRNAs) in tumorigenicity of GBM stem-like cells (GSCs). The regulatory functions of miRNAs in GSCs have emerged as potential therapeutic candidates for glioma treatment. The current study aimed at investigating the function of miR-370-3p in glioma progression, as aberrant expression of miR-370-3p, is involved in various human cancers, including glioma. Analyzing our collection of GBM samples and patient-derived GSC lines, we found the expression of miR-370-3p significantly downregulated compared to normal brain tissues and normal neural stem cells. Restoration of miR-370-3p expression in GSCs significantly decreased proliferation, migration, and clonogenic abilities of GSCs, in vitro, and tumor growth in vivo. Gene expression analysis performed on miR-370-3p transduced GSCs, identified several transcripts involved in Epithelial to Mesenchymal Transition (EMT), and Hypoxia signaling pathways. Among the genes downregulated by the restored expression of miR-370-3p, we found the EMT-inducer high-mobility group AT-hook 2 (HMGA2), the master transcriptional regulator of the adaptive response to hypoxia, Hypoxia-inducible factor (HIF)1A, and the long non-coding RNAs (lncRNAs) Nuclear Enriched Abundant Transcript (NEAT)1. NEAT1 acts as an oncogene in a series of human cancers including gliomas, where it is regulated by the Epidermal Growth Factor Receptor (EGFR) pathways, and contributes to tumor growth and invasion. Noteworthy, the expression levels of miR-370-3p and NEAT1 were inversely related in both GBM tumor specimens and GSCs, and a dual-luciferase reporter assay proved the direct binding between miR-370-3p and the lncRNAs NEAT1. Our results identify a critical role of miR-370-3p in the regulation of GBM development, indicating that miR-370-3p acts as a tumor-suppressor factor inhibiting glioma cell growth, migration and invasion by targeting the lncRNAs NEAT1, HMGA2, and HIF1A, thus, providing a potential candidate for GBM patient treatment.

Imidazo[1,2-a]pyridine Derivatives as Aldehyde Dehydrogenase Inhibitors: Novel Chemotypes to Target Glioblastoma Stem Cells.

Glioblastoma multiforme (GBM) is the deadliest form of brain tumor. It is known for its ability to escape the therapeutic options available to date thanks to the presence of a subset of cells endowed with stem-like properties and ability to resist to cytotoxic treatments. As the cytosolic enzyme aldehyde dehydrogenase 1A3 turns out to be overexpressed in these kinds of cells, playing a key role for their vitality, treatments targeting this enzyme may represent a successful strategy to fight GBM. In this work, we describe a novel class of imidazo[1,2-a]pyridine derivatives as aldehyde dehydrogenase 1A3 inhibitors, reporting the evidence of their significance as novel drug candidates for the treatment of GBM. Besides showing an interesting functional profile, in terms of activity against the target enzyme and selectivity toward highly homologous isoenzymes, representative examples of the series also showed a nanomolar to picomolar efficacy against patient-derived GBM stem-like cells, thus proving the concept that targeting aldehyde dehydrogenase might represent a novel and promising way to combat GBM by striking its ability to divide immortally.

COMP-04. MODELING PRECISION ONCOLOGY FOR GLIOBLASTOMA THROUGH INTEGRATION OF DESCRIPTIVE, FUNCTIONAL, AND NETWORK-BASED GENOMICS

Abstract Precision oncology is largely based on the notion that identification and targeting of oncogenic drivers will lead to improved clinical outcomes. However, the promise of precision oncology awaits to be fulfilled for many cancers, including Glioblastoma (GBM), where identification of oncogenic drivers has yet to improve survival rates. Here, we have attempted to systematically identify GBM vulnerabilities by performing genome-wide CRISRP-Cas9 lethality screens in patient-derived GBM stem-like cells (GSCs). In validation studies, we comprehensively retested GSC-specific hits in multiple GSC isolates, which were also genomically profiled (e.g. RNA-seq, exome-seq, CNV), and further integrated these data with CRISPR-Cas9 lethality screens from over 500 human cell lines from the Broad Institute’s CRISPR Avana dataset. As a result, we have begun making GBM dependency predictions and functional associations for top scoring hits, including: tumor developmental subtype; loss of functional redundancy with other genes/proteins; cancer-specific subnetworks of genes involved in mitochondrial protein turnover and membrane trafficking; and genes of unknown function essential for subset of GBMs. A few examples of these categories include the following scenarios. We find ADAR (Adenosine Deaminase RNA Specific) gene dependency is associated with the mesenchymal GBM subtype. The EFR3Agene, which has roles in maintaining active pools of phosphatidylinositol 4-kinase, appears required when the expression of its paralog EFR3Bis low or absent in tumor cells. The F-box protein-encoding gene FBXO42appears non-essential to most human cells lines and neural stem cells, but when knocked out in sensitive GSCs causes mitotic arrest, mitotic catastrophe, and cell death. While still a work in progress, we hope to use these results as a foundation for exploring and illuminating patient-specific molecular vulnerabilities for brain tumors. The results also underscore the need for integration of functional genetic approaches, where gene activities are inhibited, into precision oncology paradigms.

CSIG-04. PAN-RTK INHIBITION OF sLRIG1 MEDIATES AXL DOWNREGULATION IN GLIOBLASTOMA

Abstract INTRODUCTION Aberrant regulation of receptor tyrosine kinase (RTK) activity is characteristic of Glioblastoma (GBM). However, RTK-based targeted therapies have been largely unsuccessful in GBM patients, partially due to the complexity and redundance of RTK signaling. LRIG1 (Leucine-rich Repeats and ImmunoGlobulin-like domains 1) is an important endogenous inhibitor of epidermal growth factor receptor (EGFR) during health and disease, however its mechanism of action is poorly understood. We previously showed that the soluble form of LRIG1 potently inhibits GBM growth in vivo, irrespective of EGFR expression level and status, suggesting the involvement of other RTKs. Here, we aimed to shed light on the molecular mechanisms underlying its anti-cancer activity. MATERIAL AND METHODS We generated a recombinant human soluble LRIG1 protein (rh-sLRIG1) by expressing LRIG1 ectodomain in insect cells via baculovirus infection and subsequent His-tag purification. rh-sLRIG1 was used to treat patient-derived GBM stem-like cells, classical GBM cell lines and patient-derived 3D tumor organoids. Using different cell-based assays, cell proliferation, invasion, cell morphology, as well as protein expression and protein-protein interactions were investigated. RESULTS We find that recombinant sLRIG1 efficiently reduced proliferation, invasion and viability of GBM cells and patient-derived organoids, and modulated cytoskeleton proteins and cell shape. In line with previous data, the effect of recombinant sLRIG1 was independent of EGFR expression. Interestingly sLRIG1 impacted multiple RTKs including AXL, by direct protein downregulation, while EGFR expression was not affected. At the molecular level, we find that sLRIG1 interfereed with AXL dimerization, while no protein interaction with EGFR was detected. CONCLUSION We identify AXL as a novel LRIG1 target and provide evidence that sLRIG1-mediated RTK downregulation requires direct protein-protein interaction. These data pave the way for a potential therapeutic application of recombinant sLRIG1 in the inhibition of growth factor signaling in GBM.

Chemically induced neurite-like outgrowth reveals a multicellular network function in patient-derived glioblastoma cells.

Tumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of glioblastoma multiforme (GBM), the most aggressive type of brain cancer in adults. Here, we show that small-molecule inhibition of RHO-associated serine/threonine kinase proteins (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formed de novo-induced cellular network (iNet) 'webs', which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range Ca2+ signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet webs ex vivo, and may thereby accelerate future studies into the biology of GBM cellular networks.

P11.57 A 3D brain organoid coculture system delineates the invasive cell components in glioblastoma

Abstract BACKGROUND Glioblastoma (GBM) cell infiltration into the surrounding normal brain tissue where the blood brain barrier is intact, represents a major problem for clinical management and therapy. There is a vital need to understand the molecular mechanism that drives tumor cell invasion into the surrounding brain. We have previously developed a 3D coculture model where mature brain organoids are confronted with patient-derived glioblastoma stem-like cells (GSCs). In such a coculture system, single cell invasion into the normal brain tissue can be studied in detail. Here, we first describe in detail, by RNA-seq and proteomics, the differentiation of various neural cell lineages into mature brain organoids as well as their cellular organization. By real-time confocal microscopy and imaging analyses we also determine the speed of tumor cell invasion into the brain. Finally, we used this coculture system to delineate in detail the cellular heterogeneity within the invasive compartment and their gene expression. MATERIAL AND METHODS Immunohistochemistry and immunofluorescence were used to determine the expression and distribution of mature neurons, astrocytes, oligodendrocytes, and microglia within the brain organoids. Proteomics and RNA-seq were used to determine brain development ex-vivo. To assess the clonal composition of the GBM-invasive compartment, we used cellular (RGB) barcoding technology. By advanced imaging, we tracked in real time the invasion of barcoded cells into the brain organoids. Finally, we isolated invasive cells and non-invasive cells from our coculture system and used single cell sequencing to analyze their gene expression profiles and molecular phenotypes. RESULTS Immunohistochemistry and immunofluorescence showed that brain organoids, after 21 days of differentiation, display a highly cellular and structural organization. RNA-seq and proteomics, performed at different time points of organoid differentiation, revealed that the brain organoids develop into mature brain structures after 21 days as verified by a comparative analysis to normal rat brain development in vivo. Imaging analyses showed that multiple clones within the GBMs have the capacity to invade into the brain tissue with an average speed of ~ 20 μm/h. RNA-sec analysis of the invasive compartment revealed a strong up-regulation of genes and pathways associated with anaerobic respiration (glycolysis). CONCLUSION We describe a highly standardized brain organoid coculture system that can be used to delineate GBM invasion ex-vivo. We demonstrate that this platform can be used to unravel the mechanisms that drive GBM invasion into the normal brain.

P11.09 Pan-RTK inhibition of sLRIG1 mediates AXL downregulation in Glioblastoma

Abstract INTRODUCTION Aberrant regulation of receptor tyrosine kinase (RTK) activity is characteristic of Glioblastoma (GBM). However, RTK-based targeted therapies have been largely unsuccessful in GBM patients, partially due to the complexity and redundance of RTK signaling. LRIG1 (Leucine-rich Repeats and ImmunoGlobulindomains protein 1) is known as an endogenous inhibitor of epidermal growth factor receptor (EGFR) during health and disease, however its mechanism of action is poorly understood. We previously showed that the soluble form of LRIG1 potently inhibits of GBM growth in vivo, irrespective of EGFR expression level and status, suggesting the involvement of other RTKs. Here, we aimed to shed light on the molecular mechanisms underlying its anti-cancer activity. MATERIAL AND METHODS We generated a recombinant human soluble LRIG1 protein by expressing LRIG1 ectodomain in insect cells via baculovirus infection and subsequent His-tag purification. rh-sLRIG1 was applied in the medium of classical GBM cell lines, patient-derived GBM stem-like cells and patient-derived 3D tumor organoids. Using different cell-based assays, cell proliferation, invasion, cell morphology, as well as protein expression and protein-protein interactions were investigated. RESULTS We find that recombinant sLRIG1 efficiently reduces proliferation, invasion and viability of GBM cells and patient-derived organoids, and modulates cytoskeleton proteins and cell shape. In line with previous data, the effect of recombinant sLRIG1 is independent of EGFR expression. Interestingly sLRIG1 regulates several RTKs by direct protein downregulation, including AXL, while EGFR expression is not affected. At the molecular level, we find that sLRIG1 interferes with AXL dimerization, while no protein interaction with EGFR is detected. CONCLUSION We identify AXL as a novel LRIG1 target and provide evidence that sLRIG1-mediated RTK downregulation requires direct protein-protein interaction. These data pave the way for a potential therapeutic application of recombinant sLRIG1 in the inhibition of growth factor signaling in GBM.

Novel Semi-Replicative Retroviral Vector Mediated Double Suicide Gene Transfer Enhances Antitumor Effects in Patient-Derived Glioblastoma Models.

As glioblastomas are mostly localized infiltrative lesions, gene therapy based on the retroviral replicating vector (RRV) system is considered an attractive strategy. Combinations of multiple suicide genes can circumvent the limitations associated with each gene, achieving direct and synergistic cytotoxic effects, along with bystander cell killing. In this study, we constructed a semi-and pseudotyped-RRV (sp-RRV) system harboring two suicide genes—herpes simplex virus type 1 thymidine kinase (TK) and yeast cytosine deaminase (CD)—to verify the dissemination and antitumor efficacy of our sp-RRV system (spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD) in seven patient-derived glioblastoma stem-like cells (GSCs). Flow cytometry and high-content analysis revealed a wide range of transduction efficiency and good correlation between the delivery of therapeutic genes and susceptibility to the prodrugs ganciclovir and 5-fluorocytosine in patient-derived GSCs in vitro. Intra-tumoral delivery of spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD, combined with prodrug treatment, synergistically inhibited cell proliferation and angiogenesis while increasing apoptosis and the depletion of tumor-associated macrophages in orthotopic glioblastoma xenografts. Genomic profiling of patient-derived GSCs revealed that the key genes preventing sp-RRV infection and transmission were associated with cell adhesion, migration, development, differentiation, and proliferation. This is the first report demonstrating that a novel sp-RRV-mediated TK/CD double suicide gene transfer system has high oncolytic power against extremely heterogeneous and treatment-refractory glioblastomas.

Myc targeted CDK18 promotes ATR and homologous recombination to mediate PARP inhibitor resistance in glioblastoma

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.

Abstract 413: Emerging principles in synthetic lethality in glioblastoma

Abstract Synthetic lethality occurs when mutations in two otherwise nonessential genes are combined to cause lethality. Because cancer is a disease of genetic alteration, synthetic lethality has been heralded as a method to identity candidate therapeutic targets, e.g., where a target gene could be "synthetic lethal" with a cancer driver mutation. To define synthetic lethal relationships in glioblastoma (GBM), we have performed multiple focused-set and genome-wide CRISPR-Cas9 lethality screens in patient-derived GBM stem-like cells (GSCs) and nontransformed human neural progenitor cells. Because GSCs isolates likely represent a sub-clone of the original tumor and we can determine GSCs' genetic and epigenetic makeup, it is possible to address the concept of synthetic lethality for GBM. To this end, we recently performed comprehensive CRISPR-Cas9 retests of all scoring GBM lethal genes (>900) from screens in three patient isolates with different and overlapping genetic drivers. We then performed secondary retests of high-priority gene targets in 13 GSC harboring various alterations commonly found in GBMs, e.g., EGFRamp, NF1mut, PIK3CAmut, PTENloss/mut, TP53mut, etc. The results were surprising, first in what we did not find. We failed to find synthetic lethal targets for TP53loss/mut, RB1mut, or TERT expression, suggesting that synthetic lethal relationships for these alterations may not exist for GBM. Second, NF1mut interactors defined a broader class of synthetic lethal targets with general overactivity of the RTK/Ras pathway, which can arise from various activating lesions. Third, candidate synthetic lethal relationships can be observed, but, so far, only with EGFRamp, MYC/MYCNamp, and PTEN/PIK3CA alterations. Thus, in general our results suggest that the majority of synthetic lethal relationships in GBM arise from oncogenic activation of the RTK/Ras and PI-3 kinase pathway or amplification of MYC/MYCN. (Synthetic lethal targets will be revealed and discussed at the meeting.) Citation Format: Pia Hoellerbauer, Sonali Arora, Megan Kufeld, Lucas Carter, Emily J. Girard, Heather Feldman, Philip Corrin, James M. Olson, Patrick J. Paddison. Emerging principles in synthetic lethality in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 413.

SPOT-006 Stem cell-associated heterogeneity in glioblastoma is a result of intrinsic tumour plasticity shaped by the microenvironment

IntroductionTo date there is no curative treatment available for patients with Glioblastoma (GBM). The cancer stem cell (CSC) hypothesis posits that GBMs rely on a small subpopulation of cells with stemness properties responsible for tumour progression and recurrence. Recent experimental data from GBM and other cancers however suggest that CSCs may not be a stable entity. The question arises whether such cells are a defined subpopulation of tumour cells or whether they represent a changing entity that any cancer cell can adopt depending on the environmental conditions.Material and methodsTumour cell subpopulations were classified based on their expression of four chosen cell membrane markers (CD133, CD15, A2B5 and CD44). The resulting 16 subpopulations were FACS isolated and analysed for self-renewal and ability to reform the original heterogeneous population in different environments. Mathematical Markov modelling was applied to calculate state transitions between cell states. Intra-tumoral heterogeneity was further interrogated at the single cell transcriptomic level.Results and discussionsSimilar to patient biopsies, we observed markers to be heterogeneously expressed in patient-derived GBM xenografts and stem-like cell cultures. All analysed tumour cell subpopulations carried stem-cell properties and had the capacity to adapt their marker expression profiles to give rise to the original subpopulations. Mathematical modelling revealed a different propensity in reforming the original heterogeneity over time, which was independent of the proliferation index but linked to tumorigenic potential. Although subpopulations varied in their potential to adapt to new environments, all were able to reach a steady state microenvironment-specific equilibrium.ConclusionOur results suggest that GBM CSCs do not represent a clonal entity defined by distinct functional properties and transcriptomic signatures, but rather a cellular state that is determined by environmental conditions. Cellular states are non-hierarchical, reversible and occur via stochastic state transitions of existing populations, striving towards an equilibrium instructed by the microenvironment. Our data provides evidence that stem cell-associated phenotypic heterogeneity in GBM is a result of intrinsic plasticity, which has important implications for the design of treatment strategies targeting cancer stem-like states.

MerTK mediates STAT3–KRAS/SRC-signaling axis for glioma stem cell maintenance

Receptor tyrosine kinase Mer (MerTK) has been shown to be highly expressed in Glioblastoma multiforme (GBM) in comparison to its healthy counterpart and is implicated in brain tumorigenesis. Clarifying the underlying mechanism of MerTK induced invasiveness would result in novel strategies to improve patient’s response to chemotherapeutics. In vitro and in vivo assays were performed to examine the functional role of cancer stem sell (CSC) maintenance in MerTK associated invasiveness. In this article, we demonstrate that apart from GBM cells, MerTK is also upregulated in GBM stem-like cells and associated with an increased infiltrative potential of brain tumors in vivo. Silencing of MerTK suppressed the self-renewal of patient-derived GBM stem-like cells. The signaling mechanisms by which MerTK contributes to CSC maintenance have largely been obscure. Molecular analyses revealed that high expression of the signal transducer and activator of transcription 3 (STAT3)- Kirsten rat sarcoma viral oncogene homolog (KRAS) and proto-oncogene tyrosine-protein kinase SRC axis supports MerTK-induced CSC maintenance in GBM spheroids. Furthermore, a short-hairpin RNA-mediated MerTK knockdown effectively blocked invasiveness and N-cadherin expression in mouse xenografts. Collectively, our results uncover a critical function of MerTK in CSC maintenance. Considering the low basal level of MerTK expression in healthy brain cells, evaluation of MerTK as a therapeutic target should advance the research into better therapeutics for GBM.

Sprouty2 enhances the tumorigenic potential of glioblastoma cells.

BackgroundSprouty2 (SPRY2), a feedback regulator of receptor tyrosine kinase (RTK) signaling, has been shown to be associated with drug resistance and cell proliferation in glioblastoma (GBM), but the underlying mechanisms are still poorly defined.Methods SPRY2 expression and survival patterns of patients with gliomas were analyzed using publicly available databases. Effects of RNA interference targeting SPRY2 on cellular proliferation in established GBM or patient-derived GBM stemlike cells were examined. Loss- or gain-of-function of SPRY2 to regulate the tumorigenic capacity was assessed in both intracranial and subcutaneous xenografts.ResultsSPRY2 was found to be upregulated in GBM, which correlated with reduced survival in GBM patients. SPRY2 knockdown significantly impaired proliferation of GBM cells but not of normal astrocytes. Silencing of SPRY2 increased epidermal growth factor-induced extracellular signal-regulated kinase (ERK) and Akt activation causing premature onset of DNA replication, increased DNA damage, and impaired proliferation, suggesting that SPRY2 suppresses DNA replication stress. Abrogating SPRY2 function strongly inhibited intracranial tumor growth and led to significantly prolonged survival of U87 xenograft-bearing mice. In contrast, SPRY2 overexpression promoted tumor propagation of low-tumorigenic U251 cells.ConclusionsThe present study highlights an antitumoral effect of SPRY2 inhibition that is based on excessive activation of ERK signaling and DNA damage response, resulting in reduced cell proliferation and increased cytotoxicity, proposing SPRY2 as a promising pharmacological target in GBM patients.

Temozolomide affects Extracellular Vesicles Released by Glioblastoma Cells

Glioblastoma multiforme (GBM) is the most aggressive primary tumour within the brain as well as the most common and lethal cerebral cancer, mainly because of treatment failure. Indeed, tumour recurrence is inevitable and fatal in a short period of time. Glioblastoma stem-like cells (GSCs) are thought to participate in tumour initiation, expansion, resistance to treatments, including to the alkylating chemotherapeutic agent temozolomide, and relapse. Here, we assessed whether extracellular vesicles (EVs) released by GSCs could disseminate factors involved in resistance mechanisms. We first characterized EVs either circulating in peripheral blood from newly diagnosed patients or released by patient-derived temozolomide-resistant GSCs. We found that EVs from both sources were mainly composed of particles homogeneous in size (50–100 nm), while they were more abundant in liquid biopsies from GBM patients, as compared to healthy donors. Further, mass spectrometry analysis from GSC-derived EVs unveiled that particles from control and temozolomide-treated cells share core components of EVs, as well as ribosome- and proteasome-associated networks. More strikingly, temozolomide treatment led to the enrichment of EVs with cargoes dedicated to cell adhesion processes. Thus, while relatively inefficient in killing GSCs in vitro, temozolomide could instead increase the release of pro-tumoral information.

Metabolic Heterogeneity Evidenced by MRS among Patient-Derived Glioblastoma Multiforme Stem-Like Cells Accounts for Cell Clustering and Different Responses to Drugs.

Clustering of patient-derived glioma stem-like cells (GSCs) through unsupervised analysis of metabolites detected by magnetic resonance spectroscopy (MRS) evidenced three subgroups, namely clusters 1a and 1b, with high intergroup similarity and neural fingerprints, and cluster 2, with a metabolism typical of commercial tumor lines. In addition, subclones generated by the same GSC line showed different metabolic phenotypes. Aerobic glycolysis prevailed in cluster 2 cells as demonstrated by higher lactate production compared to cluster 1 cells. Oligomycin, a mitochondrial ATPase inhibitor, induced high lactate extrusion only in cluster 1 cells, where it produced neutral lipid accumulation detected as mobile lipid signals by MRS and lipid droplets by confocal microscopy. These results indicate a relevant role of mitochondrial fatty acid oxidation for energy production in GSCs. On the other hand, further metabolic differences, likely accounting for different therapy responsiveness observed after etomoxir treatment, suggest that caution must be used in considering patient treatment with mitochondria FAO blockers. Metabolomics and metabolic profiling may contribute to discover new diagnostic or prognostic biomarkers to be used for personalized therapies.

Glioblastoma stem-like cells secrete the pro-angiogenic VEGF-A factor in extracellular vesicles

ABSTRACTGlioblastoma multiforme (GBM) are mortifying brain tumours that contain a subpopulation of tumour cells with stem-like properties, termed glioblastoma stem-like cells (GSCs). GSCs largely contribute to tumour initiation, propagation and resistance to current anti-cancer therapies. GSCs are situated in perivascular niches, closely associated with brain microvascular endothelial cells, thereby involved in bidirectional molecular and cellular interactions. Moreover, extracellular vesicles are suspected to carry essential information that can adapt the microenvironment to the tumour’s needs, including tumour-induced angiogenesis. In GBM, extracellular vesicles produced by differentiated tumour cells and GSCs were demonstrated to disseminate locally and at distance. Here, we report that the pro-angiogenic pro-permeability factor VEGF-A is carried in extracellular vesicles secreted from ex vivo cultured patient-derived GSCs. Of note, extracellular vesicle-derived VEGF-A contributes to the in vitro elevation of permeability and angiogenic potential in human brain endothelial cells. Indeed, VEGF-A silencing in GSCs compromised in vitro extracellular vesicle-mediated increase in permeability and angiogenesis. From a clinical standpoint, extracellular vesicles isolated from circulating blood of GBM patients present higher levels of VEGF-A, as compared to healthy donors. Overall, our results suggest that extracellular vesicle-harboured VEGF-A targets brain endothelial cells and might impact their ability to form new vessels. Thus, tumour-released EV cargo might emerge as an instrumental part of the tumour-induced angiogenesis and vascular permeability modus operandi in GBM.

Yeast display biopanning identifies human antibodies targeting glioblastoma stem-like cells

Glioblastoma stem-like cells (GSC) are hypothesized to evade current therapies and cause tumor recurrence, contributing to poor patient survival. Existing cell surface markers for GSC are developed from embryonic or neural stem cell systems; however, currently available GSC markers are suboptimal in sensitivity and specificity. We hypothesized that the GSC cell surface proteome could be mined with a yeast display antibody library to reveal novel immunophenotypes. We isolated an extensive collection of antibodies that were differentially selective for GSC. A single domain antibody VH-9.7 showed selectivity for five distinct patient-derived GSC lines and visualized orthotopic GBM xenografts in vivo after conjugation with a near-infrared dye. These findings demonstrate a previously unexplored high-throughput strategy for GSC-selective antibody discovery, to aid in GSC isolation, diagnostic imaging, and therapeutic targeting.