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Supplementary Materials and Methods

Proteomics analysis

Figure S2, related to Figure 2. Comparison of glioma development induced in neonatal and adult G/tv-a and C/tv-a wt mice.

Table S1, related to Table 2. Mean AUC values were calculated from all dose-response curves generated for each group.

Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstream signaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas. Cancer Res; 77(7); 1741–52. ©2017 AACR.

Figure S1, related to Figure 1. Double immunofluorescence stainings for GFP and chicken vimentin of neonatal G/tv-a and C/tv-a mouse brains infected with RCAS-eGFP.

Figure S3, related to Figure 4E. Representative photographs of double immunofluorescence stainings of GCs cultured in presence or absence of serum for 7 days.

Figure S4, related to Figure 4. Characterization of mouse neural stem cell cultures CCG/tv-a and CCC/tv-a.

Background Systemic delivery of anti-tumor therapeutic agents to brain tumors is thwarted by the blood-brain barrier (BBB), an organotypic specialization of brain endothelial cells (ECs). A failure of pharmacological compounds to cross BBB is one culprit for the dismal prognosis of glioblastoma (GBM) patients. Identification of novel vascular targets to overcome the challenges posed by the BBB in tumors for GBM treatment is urgently needed. Methods Temozolomide (TMZ) delivery was investigated in CT2A and PDGFB-driven RCAS/tv-a orthotopic glioma models. Transcriptome analysis was performed on ECs from murine gliomas. Mfsd2a deficient, Cav1 deficient, and Mfsd2a EC-specific inducible mice were developed to study the underlying molecular mechanisms. Results We demonstrated that inhibiting Wnt signaling by LGK974 could increase TMZ delivery and sensitize glioma to chemotherapy in both murine glioma models. Transcriptome analysis of ECs from murine gliomas revealed that Wnt signaling inhibition enhanced vascular transcytosis as indicated by the upregulation of PLVAP and downregulation of MFSD2A. Mfsd2a deficiency in mice enhances TMZ delivery in tumors, whereas constitutive expression of Mfsd2a in ECs suppresses the enhanced TMZ delivery induced by Wnt pathway inhibition in murine glioma. In addition, Wnt signaling inhibition enhanced caveolin-1 (Cav1)-positive caveolae-mediated transcytosis in tumor ECs. Moreover, Wnt signaling inhibitor or Mfsd2a deficiency fails to enhance TMZ penetration in tumors from Cav1-deficient mice. Conclusions These results demonstrated that Wnt signaling regulates MFSD2A-dependent TMZ delivery through a caveolae-mediated EC transcytosis pathway. Our findings identify Wnt signaling as a promising therapeutic target to improve drug delivery for GBM treatment.

Glioblastoma’s (GBM) origin, recurrence and resistance to treatment are driven by GBM cancer stem cells (GSCs). Existing transcriptomic characterisations of GBM classify the tumours to three subtypes: classical, proneural, and mesenchymal. The comprehension of how expression patterns of the GBM subtypes are reflected at global proteome level in GSCs is limited.To characterise protein expression in GSCs, we performed in-depth proteogenomic analysis of patient-derived GSCs by RNA-sequencing and mass-spectrometry proteomics. We identified and quantified over 10,000 proteins in two independent GSCs panels, and propose a GSC-associated proteomic signature (GSAPS) that defines two distinct morphological conditions; one defined by a set of proteins expressed in non-mesenchymal - proneural and classical - GSCs (GPC-like), and another expressed in mesenchymal GSCs (GM-like). The expression of GM-like protein set in GBM tissue was associated with hypoxia, necrosis, recurrence, and worse overall survival in GBM patients.In a proof-of-concept proteogenomic approach, we discovered 252 non-canonical peptides expressed in GSCs, i.e., protein sequences that are variant or derive from genome regions previously considered protein-non-coding. We report new variants of the heterogeneous ribonucleoproteins (HNRNPs), which are implicated in mRNA splicing. Furthermore, we show that per-gene mRNA-protein correlations in GSCs are moderate and vary compared to GBM tissue.

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. Patients usually undergo surgery followed by aggressive radio- and chemotherapy with the alkylating agent temozolomide (TMZ). Still, median survival is only 12–15 months after diagnosis. Many human cancers including GBMs demonstrate addiction to MYC transcription factor signaling and can become susceptible to inhibition of MYC downstream genes. JQ1 is an effective inhibitor of BET Bromodomains, a class of epigenetic readers regulating expression of downstream MYC targets. Here, we show that BET inhibition decreases viability of patient-derived GBM cell lines. We propose a distinct expression signature of MYCN-elevated GBM cells that correlates with significant sensitivity to BET inhibition. In tumors showing JQ1 sensitivity, we found enrichment of pathways regulating cell cycle, DNA damage response and repair. As DNA repair leads to acquired chemoresistance to TMZ, JQ1 treatment in combination with TMZ synergistically inhibited proliferation of MYCN-elevated cells. Bioinformatic analyses further showed that the expression of MYCN correlates with Aurora Kinase A levels and Aurora Kinase inhibitors indeed showed synergistic efficacy in combination with BET inhibition. Collectively, our data suggest that BET inhibitors could potentiate the efficacy of either TMZ or Aurora Kinase inhibitors in GBM treatment.

Glioblastoma is a devastating disease with an overall median survival of 8 months from diagnosis. The majority of patients die of a tumor relapse in close proximity of the resected primary tumor. Glioblastoma is intensely researched but most studies have been performed on tissues and cultures derived from the bulk tumor while it is the remaining edge cells that cause lethality. Investigations of edge cells are rare and few experimental models exist. Here we have established and analyzed a series of matched cell cultures derived from the tumor bulk and outer edge of six IDH wildtype glioblastoma patients with the purpose to understand glioblastoma edge cell biology. Tumor samples were resected guided by 5-ALA fluorescence using neuro-navigation and stringent procedures to not contaminate edge samples with bulk tumor cells. First bulk tumor samples were resected from 5-ALA fluorescent tissue. After removal of all fluorescent areas and careful irrigation of the cavity the edge sample was resected 1-2 cm outside of the fluorescent border in a non-eloquent area. Following dissociation the samples were used in sphere assays and for explantation. There was a significant difference in self-renewal across all patients between matched bulk and edge cultures, in line with results from sphere assays on acute cells, suggesting maintenance of glioblastoma cell properties in established cultures. Invasion analysis showed a reverse significant difference between matched bulk and edge cultures strengthening a general functional distinction between tumor bulk and edge cells across patients. To investigate the molecular basis of our findings we performed whole exome sequencing (WES) and combined single cell RNA- and ATAC-sequencing (10X Multiome). Analyses are ongoing but WES data does not support genetic causes for their differences while the 10X Multiome data indicate that epigenetic regulation may underlie the different properties of bulk and edge glioblastoma cells.

Glioblastoma (GBM) is an aggressive nervous system tumor with a mean survival time of 12-14 months. Cells with neural stem cell-like properties can be derived from GBM tumors. These cells seem to escape conventional irradiation treatment, chemotherapy, and surgery, and may play a crucial role for relapse. It is therefore urgent to develop novel approaches for reliable detection of neural stem cell-like cells in GBM. Here we report a luminescent conjugated oligothiophene (LCO), named GlioStem (p-HTMI), for non-invasive and non-amplified real-time detection of live human patient-derived GBM cells and embryonic neural stem/progenitor cells (NSPCs). Within a maximum of 10 minutes after administration of the molecule in vitro, in the existing media, fluorescence emission was observed without any modulation of the cells or additional vehicle, resulting in efficient detection of cytoplasmic luminescent signal in NSPCs or GBM cells from rodents and humans, detectable at Alexa488/GFP wavelength. GlioStem is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. In vitro, GlioStem was shown to identify fetal cortical NSPCs from rat (FGF2-expanded), embryonic stem cell-derived NSPCs from mouse (FGF2/EGF-expanded), and FGF2-exposed C6 glioma cell cultures from rat, but not any other cell types investigated. Cell sorting experiments of patient-derived, FGF2/EGF-expanded GBM cells demonstrated that GlioStem in addition to NSPC-markers like Nestin and Sox2 labeled the same population (overlap > 90%) of cells as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO GlioStem is a versatile tool for immediate and selective detection of subpopulations of neural stem and glioma cells.

The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide histone modification profiling studies from FFPE samples is either 10–20 tissue sections or whole tissue blocks, which prevents better resolved analyses. But it is desirable to consume a minimal amount of FFPE tissue sections in the analysis as clinical tissues of interest are limited. Here, we present FFPE tissue with antibody-guided chromatin tagmentation with sequencing (FACT-seq), the first highly sensitive method to efficiently profile histone modifications in FFPE tissues by combining a novel fusion protein of hyperactive Tn5 transposase and protein A (T7−pA−Tn5) transposition and T7 in vitro transcription. FACT-seq generates high-quality chromatin profiles from different histone modifications with low number of FFPE nuclei. We proved a very small piece of FFPE tissue section containing ∼4000 nuclei is sufficient to decode H3K27ac modifications with FACT-seq. H3K27ac FACT-seq revealed disease-specific super enhancers in the archived FFPE human colorectal and human glioblastoma cancer tissue. In summary, FACT-seq allows decoding the histone modifications in archival FFPE tissues with high sensitivity and help researchers to better understand epigenetic regulation in cancer and human disease.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor which lacks efficient treatment and predictive biomarkers. Expression of the epithelial stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been described in GBM, but its functional role has not been conclusively elucidated. Here, we have investigated the role of LGR5 in a large repository of patient-derived GBM stem cell (GSC) cultures. The consequences of LGR5 overexpression or depletion have been analyzed using in vitro and in vivo methods, which showed that, among those with highest LGR5 expression (LGR5high ), there were two phenotypically distinct groups: one that was dependent on LGR5 for its malignant properties and another that was unaffected by changes in LGR5 expression. The LGR5-responding cultures could be identified by their significantly higher self-renewal capacity as measured by extreme limiting dilution assay (ELDA), and these LGR5high -ELDAhigh cultures were also significantly more malignant and invasive compared to the LGR5high -ELDAlow cultures. This showed that LGR5 expression alone would not be a strict marker of LGR5 responsiveness. In a search for additional biomarkers, we identified LPAR4, CCND2, and OLIG2 that were significantly upregulated in LGR5-responsive GSC cultures, and we found that OLIG2 together with LGR5 were predictive of GSC radiation and drug response. Overall, we show that LGR5 regulates the malignant phenotype in a subset of patient-derived GSC cultures, which supports its potential as a predictive GBM biomarker. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Summary High-grade gliomas (HGGs) are the most aggressive and invasive primary brain tumors. The platelet-derived growth factor (PDGF) signaling pathway drives HGG progression, and enhanced expression of PDGF receptors (PDGFRs) is a well-established aberration in a subset of glioblastomas (GBMs). PDGFRA is expressed in glioma cells, whereas PDGFRB is mostly restricted to the glioma-associated stroma. Here we show that the spatial location of TAMMs correlates with the expansion of a subset of tumor cells that have acquired expression of PDGFRB in both mouse and human low-grade glioma and HCGs. Furthermore, M2-polarized microglia but not bone marrow (BM)-derived macrophages (BMDMs) induced PDGFRB expression in glioma cells and stimulated their migratory capacity. These findings illustrate a heterotypic cross-talk between microglia and glioma cells that may enhance the migratory and invasive capacity of the latter by inducing PDGFRB., Graphical Abstract, Highlights • PDGFRB+ glioma cells are in physical contact with IBA1+ TAMMs in mouse and human glioma • Aggregation of PDGFRB+ glioma cells correlated with the accumulation of IBA1+ TAMMs • Microglia but not bone marrow-derived macrophages induced PDGFRB expression in vitro • M2-polarized microglia stimulated glioma cell migration dependent on PDGFRB, Pathophysiology; Molecular Mechanism of Behavior; Immunology; Cancer

Glioblastoma is the most aggressive type of brain tumor with poor therapeutic response and prognosis. Passage of systemically delivered pharmacological agents into the brain is largely blocked by the blood-brain-barrier (BBB), an organotypic specialization of brain endothelial cells (EC). Tumor vessels in GBM are abnormal and more permeable, but the heterogeneity of BBB breakdown in different parts of the tumor vasculature and at the tumor's invasive front is largely unknown. Here, through single-cell RNA sequencing (scRNA-seq) of freshly isolated ECs from human glioblastoma and paired tumor peripheral tissues, we have constructed a molecular atlas of human brain ECs providing unprecedented molecular insight into the heterogeneity of the human BBB and its molecular alteration in glioblastoma. We identified 5 distinct EC phenotypes representing different states of EC activation and BBB impairment, and associated with different anatomical locations within and around the tumor. This unique data resource provides key information for designing rational therapeutic regimens and optimizing drug delivery.

There is ample support for developmental regulation of glioblastoma stem cells (GSCs). To examine how cell lineage controls GSC function we have performed a cross-species epigenome analysis of mouse and human GSC cultures. We have analyzed and compared the chromatin-accessibility landscape of nine mouse GSC cultures of defined cell of origin and 60 patient-derived GSC cultures by assay for transposase-accessible chromatin using sequencing (ATAC-seq). This uncovered a variability of both mouse and human GSC cultures that was different from transcriptome analysis and better at predicting functional subgroups. In both species the chromatin accessibility-guided clusters were predominantly determined by distal regulatory element (DRE) regions, displayed contrasting sets of transcription factor binding motifs, and exhibited different functional and drug-response properties. Cross-species analysis of DRE regions in accessible chromatin revealed conserved epigenetic regulation of mouse and human GSCs. Human ATAC-seq data produced three distinct clusters with significant overlap to our previous mouse cell of origin- based stratification, where two of the clusters displayed significantly different patient survival. We conclude that epigenetic regulation of GSCs primarily is dictated by developmental origin which controls key GSC properties and affects therapeutic response.

Glioblastoma (GBM) is a malignant brain tumor with few therapeutic options. The disease presents with a complex spectrum of genomic aberrations, but the pharmacological consequences of these aberrations are partly unknown. Here, we report an integrated pharmacogenomic analysis of 100 patient-derived GBM cell cultures from the human glioma cell culture (HGCC) cohort. Exploring 1,544 drugs, we find that GBM has two main pharmacological subgroups, marked by differential response to proteasome inhibitors and mutually exclusive aberrations in TP53 and CDKN2A/B. We confirm this trend in cell and in xenotransplantation models, and identify both Bcl-2 family inhibitors and p53 activators as potentiators of proteasome inhibitors in GBM cells, We can further predict the responses of individual cell cultures to several existing drug classes, presenting opportunities for drug repurposing and design of stratified trials. Our functionally profiled biobank provides a valuable resource for the discovery of new treatments for GBM. Patrik Johansson, Cecilia Krona and Soumi Kundu share first authorship

There is ample support for developmental control of glioblastoma stem cells (GSCs), and a deeper knowledge of their epigenetic regulation could be central to more efficient glioblastoma (GBM) therapies. For this purpose, we analyzed the chromatin-accessibility landscape of nine mouse GSC cultures of defined cell of origin and 60 patient-derived GSC cultures by assay for transposase-accessible chromatin using sequencing (ATAC-seq). This uncovered an epigenetic variability of both mouse and human GSC cultures that differed from transcriptome clusters. Both mouse and human chromatin accessibility-guided clusters were predominantly determined by distal regulatory elements, displayed unique sets of transcription factor motif enrichment, and exhibited different functional and drug-response properties. Cross-species analysis of distal regulatory element regions in accessible chromatin of mouse and human cultures revealed conserved epigenetic regulation of GSCs.

High-grade glioma (HGG) is a group of primary malignant brain tumors with dismal prognosis. Whereas adult HGG has been studied extensively, childhood HGG, a relatively rare disease, is less well-characterized. Here, we present two novel platelet-derived growth factor (PDGF)-driven mouse models of pediatric supratentorial HGG. Tumors developed from two different cells of origin reminiscent of neural stem cells (NSC) or oligodendrocyte precursor cells (OPC). Cross-species transcriptomics showed that both models are closely related to human pediatric HGG as compared with adult HGG. Furthermore, an NSC-like cell-of-origin enhanced tumor incidence, malignancy, and the ability of mouse glioma cells (GC) to be cultured under stem cell conditions as compared with an OPC-like cell. Functional analyses of cultured GC from these tumors showed that cells of NSC-like origin were more tumorigenic, had a higher rate of self-renewal and proliferation, and were more sensitive to a panel of cancer drugs compared with GC of a more differentiated origin. These two mouse models relevant to human pediatric supratentorial HGG propose an important role of the cell-of-origin for clinicopathologic features of this disease. Cancer Res; 77(3); 802–12. ©2016 AACR.

The identity of the glioblastoma (GBM) cell of origin and its contributions to disease progression and treatment response remain largely unknown. We have analyzed how the phenotypic state of the initially transformed cell affects mouse GBM development and essential GBM cell (GC) properties. We find that GBM induced in neural stem-cell-like glial fibrillary acidic protein (GFAP)-expressing cells in the subventricular zone of adult mice shows accelerated tumor development and produces more malignant GCs (mGC1GFAP) that are less resistant to cancer drugs, compared with those originating from more differentiated nestin- (mGC2NES) or 2,′3′-cyclic nucleotide 3′-phosphodiesterase (mGC3CNP)-expressing cells. Transcriptome analysis of mouse GCs identified a 196 mouse cell origin (MCO) gene signature that was used to partition 61 patient-derived GC lines. Human GC lines that clustered with the mGC1GFAP cells were also significantly more self-renewing, tumorigenic, and sensitive to cancer drugs compared with those that clustered with mouse GCs of more differentiated origin.

Glioblastoma (GBM) is the most frequent and deadly primary malignant brain tumor. Hallmarks are extensive intra-tumor and inter-tumor heterogeneity and highly invasive growth, which provide great challenges for treatment. Efficient therapy is lacking and the majority of patients survive less than 1 year from diagnosis. GBM progression and recurrence is caused by treatment-resistant glioblastoma stem cells (GSCs). GSC cultures are considered important models in target identification and drug screening studies. The current state-of-the-art method, to isolate and maintain GSC cultures that faithfully mimic the primary tumor, is to use serum-free (SF) media conditions developed for neural stem cells (NSCs). Here we have investigated the outcome of explanting 218 consecutively collected GBM patient samples under both SF and standard, serum-containing media conditions. The frequency of maintainable SF cultures (SFCs) was most successful, but for a subgroup of GBM specimens, a viable culture could only be established in serum-containing media, called exclusive serum culture (ESC). ESCs expressed nestin and SOX2, and displayed all functional characteristics of a GSC, that is, extended proliferation, sustained self-renewal and orthotopic tumor initiation. Once adapted to the in vitro milieu they were also sustainable in SF media. Molecular analyses showed that ESCs formed a discrete group that was most related to the mesenchymal GBM subtype. This distinct subgroup of GBM that would have evaded modeling in SF conditions only provide unique cell models of GBM inter-tumor heterogeneity.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor in adults, with a median survival of 14.6 months. Recent efforts have focused on identifying clinically relevant subgroups to improve our understanding of pathogenetic mechanisms and patient stratification. Concurrently, the role of immune cells in the tumor microenvironment has received increasing attention, especially T cells and tumor-associated macrophages (TAM). The latter are a mixed population of activated brain-resident microglia and infiltrating monocytes/monocyte-derived macrophages, both of which express ionized calcium-binding adapter molecule 1 (IBA1). This study investigated differences in immune cell subpopulations among distinct transcriptional subtypes of GBM. Human GBM samples were molecularly characterized and assigned to Proneural, Mesenchymal or Classical subtypes as defined by NanoString nCounter Technology. Subsequently, we performed and analyzed automated immunohistochemical stainings for TAM as well as specific T cell populations. The Mesenchymal subtype of GBM showed the highest presence of TAM, CD8+, CD3+ and FOXP3+ T cells, as compared to Proneural and Classical subtypes. High expression levels of the TAM-related gene AIF1, which encodes the TAM-specific protein IBA1, correlated with a worse prognosis in Proneural GBM, but conferred a survival benefit in Mesenchymal tumors. We used our data to construct a mathematical model that could reliably identify Mesenchymal GBM with high sensitivity using a combination of the aforementioned cell-specific IHC markers. In conclusion, we demonstrated that molecularly distinct GBM subtypes are characterized by profound differences in the composition of their immune microenvironment, which could potentially help to identify tumors amenable to immunotherapy.

Glioblastoma (GBM) is the most common malignant brain tumor in adults with a 15 month median survival after diagnosis. Patients receive surgical resection of the tumor, followed by aggressive radio- and chemotherapy with temozolomide (TMZ). Many human cancers including GBM demonstrate addiction to MYC transcription factor signaling and thus become susceptible to inhibition of MYC downstream genes. JQ1 is an effective inhibitor of BET Bromodomains, a class of epigenetic readers regulating expression of MYC genes and their downstream targets. In a panel of 19 patient-derived GBM cell lines, we showed that BET inhibition alone and in combination with TMZ decreases viability by inducing cell cycle arrest, apoptosis and senescence. We propose a distinct expression signature of GBM cells that correlates with a sensitivity to BET inhibition. Cell lines most sensitive to BET inhibition exhibited ten-fold upregulation of MYCN, as compared to resistant cell lines, while we could not find a correlation between MYC expression and sensitivity to the inhibition. Following BET inhibition, JQ1-sensitive cells downregulated MYCN both on mRNA and protein level, while the inhibition of JQ1-resistant cells had no effect on MYCN expression. In JQ1-sensitive cells, we found enrichment of pathways regulating cell cycle, DNA replication, DNA damage response and repair. As DNA repair leads to an acquired chemoresistance to TMZ, JQ1 treatment in combination with TMZ can further inhibit proliferation of TMZ-resistant cells. Intriguingly, JQ1-sensitive cells expressed high levels of OLIG2 and LGR5, suggesting that BET inhibition targets a subset of proneural GBM progenitors. Taken together, we suggest that BET inhibition can increase the potency of TMZ therapy in particular for GBM patients with a MYCN-positive signature. Ongoing in vivo evaluation of JQ1 and TMZ aims to determine the efficacy of the drug combination in inhibiting tumor growth.

Glioblastoma multiforme (GBM, astrocytoma grade IV) is the most common malignant primary brain tumor in adults. Addressing the shortage of effective treatment options for this cancer, we explored repurposing of existing drugs into combinations with potent activity against GBM cells. We report that the phytoalexin pterostilbene is a potentiator of two drugs with previously reported anti-GBM activity, the EGFR inhibitor gefitinib and the antidepressant sertraline. Combinations of either of these two compounds with pterostilbene suppress cell growth, viability, sphere formation and inhibit migration in tumor GBM cell (GC) cultures. The potentiating effect of pterostilbene was observed to a varying degree across a panel of 41 patient-derived GCs, and correlated in a case specific manner with the presence of missense mutation of EGFR and PIK3CA and a focal deletion of the chromosomal region 1p32. We identify pterostilbene-induced cell cycle arrest, synergistic inhibition of MAPK activity and induction of Thioredoxin interacting protein (TXNIP) as possible mechanisms behind pterostilbene's effect. Our results highlight a nontoxic stilbenoid compound as a modulator of anticancer drug response, and indicate that pterostilbene might be used to modulate two anticancer compounds in well-defined sets of GBM patients.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor which lacks efficient treatment and predictive biomarkers. Expression of the epithelial stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been described in GBM, but its functional role has not been conclusively elucidated. Here, we have investigated the role of LGR5 in a large repository of patient-derived GBM stem cell (GSC) cultures. The consequences of LGR5 overexpression or depletion have been analyzed using in vitro and in vivo methods, which showed that, among those with highest LGR5 expression (LGR5

Glioblastoma (GBM) is the most frequent primary malignant brain tumor. The invasive and heterogeneous nature of GBM stem cells (GSCs) provide the basis for extreme therapy resistance of GBM and the majority of patients survive less than one year from diagnosis. Relevant models of GBM are important for basic research and drug screening and development, and the current consensus in the field is that GSCs are most faithfully maintained under serum-free neural stem cell (NSC) conditions. We have used serum-free NSC media to explant 230 patient-derived GBM samples, and have in parallel also explanted the same samples in regular serum-containing media. We found, accordingly, that serum-free media was most effective in generating sustainable cultures (137/230, 60%), called serum-free (SF) cultures. Interestingly, we also found that that there was a subgroup of GBM specimens that could only be established in serum-containing media (31/230, 13%), called exclusive serum-containing (eSC) cultures. We characterized a number of the eSC cultures and found that they expressed typical GSC markers such as MSI-1, BMI-1, nestin, CD44 and SOX2. They also displayed all functional characteristics of GSCs, i.e. extended proliferation, sustained self-renewal and orthotopic tumor initiation. Molecular analyses showed that the eSC cultures were most closely related to but separated from mesenchymal subtype GBM. In summary we have established GSC cultures that would have evaded modeling under serum-free conditions, representing unique cell models of GBM inter-tumor heterogeneity. Key words: Glioblastoma, serum cultures, stem-like cells, GBM heterogeneity

We have recently identified LGR5 as a marker of an immature, neural stem cell-like cell of origin for glioblastoma (GBM). Here we have further investigated the role of LGR5 in GBM. We have found that in a panel of 61 patient-derived GBM stem cell lines, LGR5 was differentially expressed and significantly enriched in those of the proneural subtype. High LGR5 expression in proneural GBM lines (LGR5high-PN) was associated with increased self-renewal and tumorigenicity compared to low expressing (LGR5low) and high expressing mesenchymal (LGR5high-MS) GBM cell lines. Knock-down of LGR5 gene expression in LGR5high-PN lines resulted in decreased self-renewal, while over-expression in both LGR5high-PN and LGR5low lines produced a higher self-renewal capacity of the cells. Furthermore, by comparing LGR5high-PN cells with and without si/shLGR5 we found that LGR5 also could regulate tumor cell invasion in vitro and in vivo. When analyzing transcriptome changes upon LGR5 depletion we detected a transition from the proneural to the mesenchymal subtype in LGR5high-PN GBM cells treated with shLGR5 that was coupled to an upregulation of genes of the TNFA/NFKB pathway. Taken together our data suggests that LGR5 could be an essential regulator of a subset of GBMs. To stratify patients carrying LGR5-dependent GBMs we have identified a number of additional candidate biomarkers, which together with LGR5 could help defining LGR5-driven GBM cells.

Glioblastoma multiforme (GBM) is a disease with an urgent need for deeper understanding and new therapeutic approaches. As in other solid tumors, the tumor microenvironment (TME) is likely to control growth and sensitivity to treatment. Key components of the TME of GBM include the vasculature, microglia and astrocytes. Whereas GBM vasculature and microglia have been extensively studied, less is known about the potential involvement of astrocytes in GBM biology. In this study, astrocytes were investigated with regard to their effect on glioblastoma growth through in vitro approaches and in an in vivo model. Our studies, using different established GBM cell lines, and patient-derived primary GBM cultures, show that co-cultured astrocytes increase GBM cell number by supporting GBM cell proliferation. Furthermore, orthotopic co-injection of astrocytes and GBM cells in mice leads to reduced survival as compared to GBM cell mono-injection. Gene-expression analyses of mono- or co-cultured astrocytes have identified a GBM-cell-induced astrocyte gene signature. Analyses of two publically available GBM gene expression data set demonstrated that the signature of “GBM-educated” astrocytes was associated with significantly shorter survival. These analyses also indicated differences between molecular subgroups of GBM regarding the signature, such that the mesenchymal subset displayed a higher signature score as compared to the other molecular GBM subtypes. A high-throughput screening of approximately 1200 compounds of the “Prestwick-library” has been performed to identify compounds which specifically block the astrocyte-dependent proliferation of U343MG GBM cells. Following the initial screen, 12 compounds have been validated as specifically acting on GBM growth in co-culture. Two of them have been selected for further studies. In summary, this ongoing study suggests that astrocytes contribute to glioblastoma growth and implies this crosstalk as a candidate target for novel therapies. Clinical relevance of the model system is suggested by the prognostic significance of the signature of GBM-educated astrocytes.

Glioblastoma (GBM) is the most frequent and malignant form of primary brain tumor. GBM is essentially incurable and its resistance to therapy is attributed to a subpopulation of cells called glioma stem cells (GSCs). To meet the present shortage of relevant GBM cell (GC) lines we developed a library of annotated and validated cell lines derived from surgical samples of GBM patients, maintained under conditions to preserve GSC characteristics. This collection, which we call the Human Glioblastoma Cell Culture (HGCC) resource, consists of a biobank of 48 GC lines and an associated database containing high-resolution molecular data. We demonstrate that the HGCC lines are tumorigenic, harbor genomic lesions characteristic of GBMs, and represent all four transcriptional subtypes. The HGCC panel provides an open resource for in vitro and in vivo modeling of a large part of GBM diversity useful to both basic and translational GBM research., Highlights • The HGCC resource contains 48 annotated human GBM cell lines and an interactive database • The GBM cell lines are propagated in stem cell conditions and display GSC characteristics • The HGCC resource provides cell lines of all molecular (TCGA) subtypes • All data connected with the HGCC cell lines can be accessed at hgcc.se