Mesenchymal Glioblastoma Cells Research Articles

VC-resist glioblastoma cell state: vessel co-option as a key driver of chemoradiation resistance

Glioblastoma (GBM) is a highly lethal type of cancer. GBM recurrence following chemoradiation is typically attributed to the regrowth of invasive and resistant cells. Therefore, there is a pressing need to gain a deeper understanding of the mechanisms underlying GBM resistance to chemoradiation and its ability to infiltrate. Using a combination of transcriptomic, proteomic, and phosphoproteomic analyses, longitudinal imaging, organotypic cultures, functional assays, animal studies, and clinical data analyses, we demonstrate that chemoradiation and brain vasculature induce cell transition to a functional state named VC-Resist (vessel co-opting and resistant cell state). This cell state is midway along the transcriptomic axis between proneural and mesenchymal GBM cells and is closer to the AC/MES1-like state. VC-Resist GBM cells are highly vessel co-opting, allowing significant infiltration into the surrounding brain tissue and homing to the perivascular niche, which in turn induces even more VC-Resist transition. The molecular and functional characteristics of this FGFR1-YAP1-dependent GBM cell state, including resistance to DNA damage, enrichment in the G2M phase, and induction of senescence/stemness pathways, contribute to its enhanced resistance to chemoradiation. These findings demonstrate how vessel co-option, perivascular niche, and GBM cell plasticity jointly drive resistance to therapy during GBM recurrence.

The imitation game: How glioblastoma outmaneuvers immune attack

Immune evasion and resistance to immunotherapy mark major roadblocks in treating glioblastoma, the deadliest form of brain cancer. In this issue of Cell, Gangoso etal. demonstrate that the immune microenvironment drives glioblastoma cells to hijack myeloid-characteristic transcriptional and epigenetic circuits as a mode of immune evasion.

Mesenchymal glioblastoma-induced mature de-novo vessel formation of vascular endothelial cells in a microfluidic device

High vascularization is a biological characteristic of glioblastoma (GBM); however, an in-vitro experimental model to verify the mechanism and physiological role of vasculogenesis in GBM is not well-established. Recently, we established a self-organizing vasculogenic model using human umbilical vein endothelial cells (HUVECs) co-cultivated with human lung fibroblasts (hLFs). Here, we exploited this system to establish a realistic model of vasculogenesis in GBM. We developed two polydimethylsiloxane (PDMS) devices, a doughnut-hole dish and a 5-lane microfluidic device to observe the contact-independent effects of glioblastoma cells on HUVECs. We tested five patient-derived and five widely used GBM cell lines. Confocal fluorescence microscopy was used to observe the morphological changes in Red Fluorescent Protein (RFP)-HUVECs and fluorescein isothiocyanate (FITC)-dextran perfusion. The genetic and expression properties of GBM cell lines were analyzed. The doughnut-hole dish assay revealed KNS1451 as the only cells to induce HUVEC transformation to vessel-like structures, similar to hLFs. The 5-lane device assay demonstrated that KNS1451 promoted the formation of a vascular network that was fully perfused, revealing the functioning luminal construction. Microarray analysis revealed that KNS1451 is a mesenchymal subtype of GBM. Using a patient-derived mesenchymal GBM cell line, mature de-novo vessel formation could be induced in HUVECs by contact-independent co-culture with GBM in a microfluidic device. These results support the development of a novel in vitro research model and provide novel insights in the neovasculogenic mechanism of GBM and may potentially facilitate the future detection of unknown molecular targets.

Plasma IL-8 and ICOSLG as prognostic biomarkers in glioblastoma.

BackgroundCNS immune privilege has been challenged in recent years. Glioblastoma (GBM) immune dysfunction includes complex interactions with the immune system outside the CNS. The aim of this study was to determine diagnostic and prognostic potential of immune-related proteins in plasma in GBM and interrogate biomarker presence in the brain tumor microenvironment (TME).MethodsOne hundred and fifty-eight patients with glioma WHO grade II–IV were included. Plasma collected at surgery was screened for 92 proteins using proximity extension assay technology and related to clinical outcome. Secretion and expression of candidate prognostic biomarkers were subsequently analyzed in 8 GBM cell lines and public RNAseq data.ResultsPlasma levels of 20 out of 92 screened proteins were significantly different in patients with GBM compared to patients with astrocytoma WHO grade II–III. High plasma interleukin-8 (IL-8) (hazard ratio [HR] = 1.52; P = .0077) and low CD244 (HR = 0.36; P = .0004) were associated with short progression-free survival and high plasma IL-8 (HR = 1.40; P = .044) and low ICOS ligand (ICOSLG) (HR = 0.17; P = .0003) were associated with short overall survival (OS) in newly diagnosed patients with GBM. A similar trend was found for ICOSLG (HR = 0.34; P = .053) in recurrent GBM. IL-8 was mostly secreted and expressed by mesenchymal GBM cell lines and expressed by vascular cells and immune cells in the TME. This was also the case for ICOSLG, although less consistent, and with additional expression in tumor-associated oligodendrocytes.ConclusionsHigh plasma IL-8 and low ICOSLG at surgery are associated with short OS in newly diagnosed GBM. Source of plasma ICOSLG may be found outside the TME.

NKG2D Controls Natural Reactivity of Vγ9Vδ2 T Lymphocytes against Mesenchymal Glioblastoma Cells

Cellular immunotherapies are currently being explored to eliminate highly invasive and chemoradioresistant glioblastoma (GBM) cells involved in rapid relapse. We recently showed that concomitant stereotactic injections of nonalloreactive allogeneic Vγ9Vδ2 T lymphocytes eradicate zoledronate-primed human GBM cells. In the present study, we investigated the spontaneous reactivity of allogeneic human Vγ9Vδ2 T lymphocytes toward primary human GBM cells, in vitro and in vivo, in the absence of any prior sensitization. Through functional and transcriptomic analyses, we extensively characterized the immunoreactivity of human Vγ9Vδ2 T lymphocytes against various primary GBM cultures directly derived from patient tumors. We evidenced that GBM cells displaying a mesenchymal signature are spontaneously eliminated by allogeneic human Vγ9Vδ2 T lymphocytes, a reactivity process being mediated by γδ T-cell receptor (TCR) and tightly regulated by cellular stress-associated NKG2D pathway. This led to the identification of highly reactive Vγ9Vδ2 T lymphocyte populations, independently of a specific TCR repertoire signature. Moreover, we finally provide evidence of immunotherapeutic efficacy in vivo, in the absence of any prior tumor cell sensitization. By identifying pathways implicated in the selective natural recognition of mesenchymal GBM cell subtypes, accounting for 30% of primary diagnosed and 60% of recurrent GBM, our results pave the way for novel targeted cellular immunotherapies.

Abstract 5259: Fatty acid oxidation represents a metabolic vulnerability in glioblastoma in nutrient-deprived conditions

Abstract Glioblastoma represents an aggressive, primary brain tumor with limited treatment options. We performed an integrative, cross-platform analysis coupling global metabolomics and gene expression profiling in >100 patient-derived gliomas to begin to understand the diverse metabolic programs driving the aggressive phenotype of this malignancy. Alterations in fatty acid β-oxidation (FAO) emerged as a key metabolic node differentiating glioblastoma from low-grade astrocytoma, as demonstrated by an accumulation of acylcarnitines. Metabolic heterogeneity was observed within glioblastoma that could further define tumors as FAO ‘high’ and ‘low’. Integrative analyses identified these metabolic subtypes to be enriched with mesenchymal (MES) and proneural (PN) glioblastoma subtypes, respectively. These findings were metabolomically and functionally recapitulated in molecular subtype-specific preclinical models. Analysis of gene expression profiles from these lines uncovered an orchestrated transcriptional program designed to promote fatty acid uptake, activation, and mitochondrial oxidation. Studies designed to determine the biologic consequence of enhanced FAO in glioblastoma only identified a role in glucose-deprived conditions, where it served as a vital, alternate source for ATP synthesis. Based on these findings, we tested the hypothesis that dual targeting of glycolysis and FAO would elicit energetic stress-mediated cell death in glioblastoma. Accordingly, the glycolysis inhibitor 2DG and the FAO inhibitor etomoxir only demonstrated anti-proliferative activity in MES glioblastoma cell lines when used as single agents in vitro, while the combination resulted in robust, necroptosis-mediated cell death. Synergistic anti-tumor activity was observed following combined glycolysis and FAO inhibition when extended in vivo in an orthotopic model. Collectively, our findings suggest FAO provides metabolic plasticity in MES glioblastoma, allowing these cells to adapt to nutrient-deprived microenvironments. Combinatorial strategies designed to inhibit glycolysis and FAO represents an attractive therapeutic approach in glioblastoma. Citation Format: Shiva Kant, Antony Dayalan, Pravin Kesarwani, Prakash Chinnaiyan. Fatty acid oxidation represents a metabolic vulnerability in glioblastoma in nutrient-deprived conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5259.

NF-κB, Mesenchymal Differentiation and Glioblastoma.

Although glioblastoma (GBM) has always been recognized as a heterogeneous tumor, the advent of largescale molecular analysis has enabled robust categorization of this malignancy into several specific subgroups. Among the subtypes designated by expression profiling, mesenchymal tumors have been associated with an inflammatory microenvironment, increased angiogenesis, and resistance to therapy. Nuclear factor-κB (NF-κB) is a ubiquitous transcription factor that plays a prominent role in mediating many of the central features associated with mesenchymal differentiation. This review summarizes the mechanisms by which NF-κB proteins and their co-regulating partners induce the transcriptional network that underlies the mesenchymal phenotype. Moreover, both the intrinsic changes within mesenchymal GBM cells and the microenvironmental factors that modify the overall NF-κB response are detailed.

Abstract 2858: Endoplasmic reticulum stress as possible target for therapy in glioblastoma

Abstract Introduction: Glioblastoma (GBM) is the most aggressive form of brain cancer with an overall survival of patients of only 1-2 years after diagnosis. The current standard treatment is largely ineffective due to high resistance and infiltrative nature of GBM. Cellular heterogeneity in the tumor and the presence of cancer stem cells in GBM (GSCs) are considered the main contributors to aggressiveness. Recently, different molecular subtypes have been found in GBM. Proneural (PN) and Mesenchymal (MES) GBM are most distinct and MES tumors are more aggressive and infiltrative. Drugs that induce Endoplasmic Reticulum Stress (ER stress)/Unfolded Protein Response (UPR) activation provide a potential novel therapy for cancer treatment. In this study we aim to characterize and explore the potential of ER stress-inducing drugs to target GBM. Methods: GBM neurospheres PN and MES were tested for sensitivity to the ER stress inducer Thapsigargin (Tg) by MTS. TGFβ was used to induce mesenchymal transition in PN GBMs. Limiting dilution assay was performed to analyze the spheroid formation capacity upon ER stress induction. Activation of UPR was determined by analyzing protein expression of key signals by Western Blotting or XBP1 splicing assay (RT-PCR). Caspase Glo 3/7 assay was performed as read-out for apoptosis. Results: GBM MES neurospheres cells were 2-fold more sensitive to the ER stress inducing drug Tg compared to PN cells. The reduction of cell viability upon ER stress induction coincided with induction of apoptosis (caspase 3/7 activation). Consistently MES GBM cells showed stronger increase in apoptosis activation. Tg-induced ER stress resulted in 2- 4-fold reduction in spheroid formation capacity of MES GBM cells and higher Tg concentration also reduced spheroid formation capacity in one of the PN GBM cell lines. Interestingly, TGFβ-induced mesenchymal transition sensitized particularly PN GBM cells to Tg. The activation of UPR was confirmed by induction of GRP78 and CHOP expression. In MES GBM cells particularly activation of the ATF6-UPR branch by Tg ER stress induction was detected and XBP1 splicing activation appeared to be stronger in PN cells. No significant differences were observed in PERK activation among the different GBM subtypes. Conclusion: ER stress induction appears to be effective for eradicating GBM neurospheres, particularly the aggressive MES subtype. ER stress also targets the GSC compartment in GBM. ATF6 is notably activated in MES GBM cells, while XBP1 seems more activated in PN GBM cells upon Tg-induced ER stress. The relationship between an UPR branch activation and the differential sensitivity for the ER stress induction will be further explored. Overall, targeting the ER stress response appears a promising approach to target GBM. Citation Format: Natalia M. Peñaranda Fajardo, Coby Meijer, Frank A. E. Kruyt. Endoplasmic reticulum stress as possible target for therapy in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2858.

Abstract 1505: Radiogenomics defines key genomic network driving GBM invasion

Abstract PURPOSE Clinical care and outcome in Glioblastoma (GBM) remains challenging due to the tumor's invasive grwoth. To establish personalized treatment options in GBM, discovery of genetic mechanisms essential for the tumor's invasion is needed. We have previously described radiogenomic approaches to diagnose gene networks non invasively by analyzing genomic data from TCGA. The purpose of the current reseach is to identify a genetic network that drives GBM invasion and can be targeted specifically. METHOD AND MATERIALS Using Kaplan-Meier statistics, the data of the two independent databases TCGA and REMBRANDT were used to validate the genetic netowork's impact on clinical outcome. The genes’ staus was assessed in a panel of human glioma stem cells (GSCs) and conventional proneural, classical and mesenchymal GBM cell lines using RT-PCR. Differentiation potential (Tuj1+ve, S100A+ve, and GFAP+ve), self-renewal (limiting dilution assays), invasion (Boyden chamber) and proliferation (BrdU) were assessed. Gain (lentiviral vectors) and loss (SMARTchoice Inducible shRNA) of function experiments were performed. Orthotopic xenograft models (nude mice) were used to characterize the genes impact in vivo. Potential FDA approved therapeutics were identified using connectivity map. RESULTS Texture analysis based on radiogenomics significantly predicted the genes responsible for invasion of GBM in a non-invasive manner. Invasion in both, in vitro and in vivo was significantly decreased upun downregulation of this gene network. Transcriptome micro-array analysis showed that an upregulation of the described genes results in class switching from proneural to mesenchymal sub-types. Cmap derived therapeutics could significantly inhibit the gene network's activitiy and hence invasion. CONCLUSION The describend genes could be essential drivers of molecular subtypes and invasion in GBM. The therapeutics defined with cmap offer a targetted therapy to adress these key features of GBM pathogenesis. Noninvasive radiogenomics-based identification of tumor subgroups and potential treatment approaches can significantly contribute to personalized therapy. CLINICAL RELEVANCE/APPLICATION The described gene network seems to be key for GBM pathogenesis. Noninvasive, radiogenomics-based subgroup identification and specific novel treatment approaches can significanty contribution to personalized GBM therapy. Citation Format: Rivka R. Colen, Markus Luedi, Sanjay K. Singh, Islam Hassan, Joy Gumin, Erik P. Sulman, Frederick F. Lang, Pascal O. Zinn. Radiogenomics defines key genomic network driving GBM invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1505.

Nuclear factor one B (NFIB) encodes a subtype-specific tumour suppressor in glioblastoma.

Glioblastoma (GBM) is an essentially incurable and rapidly fatal cancer, with few markers predicting a favourable prognosis. Here we report that the transcription factor NFIB is associated with significantly improved survival in GBM. NFIB expression correlates inversely with astrocytoma grade and is lowest in mesenchymal GBM. Ectopic expression of NFIB in low-passage, patient-derived classical and mesenchymal subtype GBM cells inhibits tumourigenesis. Ectopic NFIB expression activated phospho-STAT3 signalling only in classical and mesenchymal GBM cells, suggesting a mechanism through which NFIB may exert its context-dependent tumour suppressor activity. Finally, NFIB expression can be induced in GBM cells by drug treatment with beneficial effects.

Activation of hypoxia signaling induces phenotypic transformation of glioma cells: implications for bevacizumab antiangiogenic therapy.

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), can attenuate tumor-associated edema and improve patient symptoms but based on magnetic resonance imaging, is associated with non-enhancing tumor progression and possibly gliosarcoma differentiation. To gain insight into these findings, we investigated the role of hypoxia and epithelial-mesenchymal transition (EMT)-associated proteins in GBM. Tumor markers of hypoxia and EMT were upregulated in bevacizumab-treated tumors from GBM patients compared to untreated counterparts. Exposure of glioma cells to 1% oxygen tension increased cell proliferation, expression of EMT-associated proteins and enhanced cell migration in vitro. These phenotypic changes were significantly attenuated by pharmacologic knockdown of hypoxia-inducible Factor 1α (HIF1α) or HIF2α, indicating that HIFs represent a therapeutic target for mesenchymal GBM cells. These findings provide insights into potential development of novel therapeutic targeting of angiogenesis-specific pathways in GBM.