The development of a hiPSC-based platform to identify tissue-dependencies of IDH1 R132H.

The application of patient-derived (PD) in vitro tumor models represents the classical strategy for clinical translational oncology research. Using these cellular heterogeneous cultures for the isolation of cancer stem cells (CSCs), suggested to be the main driver for disease malignancy, relies on the use of surrogate biomarkers or is based on CSC-enriching culture conditions. However, the ability of those strategies to exclusively and efficiently enrich for CSC pool has been questioned. Here we present an alternative in vitro CSC model based on the oncogenic transformation of single clone-derived human induced pluripotent stem cells (hiPSC). Hotspot mutations in the DNA encoding for the R132 codon of the enzyme isocitrate dehydrogenase 1 (IDH1) and codon R175 of p53 are commonly occurring molecular features of different tumors and were selected for our transformation strategy. By choosing p53 mutant glial tumors as our model disease, we show that in vitro therapy discovery tests on IDH1-engineered synthetic CSCs (sCSCs) can identify kinases-targeting chemotherapeutics that preferentially target tumor cells expressing corresponding genetic alteration. In contrast, neural stem cells (NSCs) derived from the IDH1R132H overexpressing hiPSCs increase their resistance to the tested interventions indicating glial-to-neural tissue-dependent differences of IDH1R132H. Taken together, we provide proof for the potential of our sCSC technology as a potent addition to biomarker-driven drug development projects or studies on tumor therapy resistance. Moreover, follow-up projects such as comparing in vitro drug sensitivity profiles of hiPSC-derived tissue progenitors of different lineages, might help to understand a variety of tissue-related functions of IDH1 mutations.

Longitudinal stability of molecular alterations and drug response profiles in tumor spheroid cell lines enables reproducible analyses.

The utility of patient-derived tumor cell lines as experimental models for glioblastoma has been challenged by limited representation of the in vivo tumor biology and low clinical translatability. Here, we report on longitudinal epigenetic and transcriptional profiling of seven glioblastoma spheroid cell line models cultured over an extended period. Molecular profiles were associated with drug response data obtained for 231 clinically used drugs. We show that the glioblastoma spheroid models remained molecularly stable and displayed reproducible drug responses over prolonged culture times of 30 in vitro passages. Integration of gene expression and drug response data identified predictive gene signatures linked to sensitivity to specific drugs, indicating the potential of gene expression-based prediction of glioblastoma therapy response. Our data thus empowers glioblastoma spheroid disease modeling as a useful preclinical assay that may uncover novel therapeutic vulnerabilities and associated molecular alterations.

CBF1 is clinically prognostic and serves as a target to block cellular invasion and chemoresistance of EMT-like glioblastoma cells.

BACKGROUND: Glioblastoma is the most common and most lethal primary brain cancer. CBF1 (also known as Recombination signal Binding Protein for immunoglobulin kappa J, RBPJ) is the cardinal transcriptional regulator of the Notch signalling network and has been shown to promote cancer stem-like cells (CSCs) in glioblastoma. Recent studies suggest that some of the malignant properties of CSCs are mediated through the activation of pro-invasive programme of epithelial-to-mesenchymal transition (EMT). Little is known whether CBF1 is involved in the EMT-like phenotype of glioma cells. METHODS: In a collection of GBM neurosphere lines, we genetically inhibited CBF1 and investigated the consequences on EMT-related properties, including in vitro invasiveness by Boyden chambers assay, chemoresistance using a clinical drug library screen and glycolytic metabolism assessing live-cell extracellular acidification rate. We also compared CBF1 expression in cells exposed to low and high oxygen tension. In silico analysis in large-scale Western and Eastern patient cohorts investigated the clinical prognostic value of CBF1 expression in low- and high-grade glioma as well as medulloblastoma. RESULTS: Mean CBF1 expression is significantly increased in isocitrate dehydrogenase 1 (IDH1) R132H mutant glioblastoma and serves as prognostic marker for prolonged overall survival in brain tumours, particularly after therapy with temozolomide. Hypoxic regions of glioblastoma have higher CBF1 activation and exposure to low oxygen can induce its expression in glioma cells in vitro. CBF1 inhibition blocks EMT activators such as zinc finger E-box-binding homeobox 1 (ZEB1) and significantly reduces cellular invasion and resistance to clinically approved anticancer drugs. Moreover, we indicate that CBF1 inhibition can impede cellular glycolysis. CONCLUSIONS: Mean CBF1 activation in bulk tumour samples serves as a clinical predictive biomarker in brain cancers but its intratumoral and intertumoral expression is highly heterogeneous. Microenvironmental changes such as hypoxia can stimulate the activation of CBF1 in glioblastoma. CBF1 blockade can suppress glioblastoma invasion in vitro in particular in cells undergone EMT such as those found in the hypoxic niche. Targeting CBF1 can be an effective anti-EMT therapy to impede invasive properties and chemosensitivity in those cells.

Targeting cancer stem-like cells in glioblastoma and colorectal cancer through metabolic pathways.

Cancer stem-like cells (CSCs) are thought to be the main cause of tumor occurrence, progression and therapeutic resistance. Strong research efforts in the last decade have led to the development of several tailored approaches to target CSCs with some very promising clinical trials underway; however, until now no anti-CSC therapy has been approved for clinical use. Given the recent improvement in our understanding of how onco-proteins can manipulate cellular metabolic networks to promote tumorigenesis, cancer metabolism research may well lead to innovative strategies to identify novel regulators and downstream mediators of CSC maintenance. Interfering with distinct stages of CSC-associated metabolics may elucidate novel, more efficient strategies to target this highly malignant cell population. Here recent discoveries regarding the metabolic properties attributed to CSCs in glioblastoma (GBM) and malignant colorectal cancer (CRC) were summarized. The association between stem cell markers, the response to hypoxia and other environmental stresses including therapeutic insults as well as developmentally conserved signaling pathways with alterations in cellular bioenergetic networks were also discussed. The recent developments in metabolic imaging to identify CSCs were also summarized. This summary should comprehensively update basic and clinical scientists on the metabolic traits of CSCs in GBM and malignant CRC.

Isoflurane enhances the malignant potential of glioblastoma stem cells by promoting their viability, mobility in vitro and migratory capacity in vivo.

BACKGROUND: Isoflurane is one of the most common general anaesthetics used during surgical procedures, including tumour resection. However, the effects of isoflurane on the viability and migration capacity of cancer cells, specifically in the context of brain cancer cells, remain unclear. Therefore, the aim of this study was to evaluate the influence that isoflurane has on the function of glioblastoma stem cells (GCSs) in regards to cell proliferation, survival and migration. METHOD: U251-GSCs were exposed to isoflurane at clinically relevant concentrations and incubation times. The effects on proliferation, survival and migration capacities of the cells were evaluated in vitro. The potential risk was assessed in mice by intracranial injection of U251-GSCs pretreated with isoflurane. Furthermore, the average tumour volume and migration distance of U251-GSCs from the tumour centre were calculated. RESULTS: Exposure of U251-GSCs to 1.2% isoflurane for 6 h resulted in increased proliferation (P<0.05) and decreased apoptosis rate (P<0.05) when compared with the control group. In addition, isoflurane exposure caused increased migration capacity in vitro (P<0.05) and the distance migrated was increased in vivo (P<0.05). CONCLUSION: Clinically relevant concentrations and incubation times of isoflurane could promote the viability and mobility of U251-GSCs, suggesting this general anaesthetic may have detrimental effects in glioblastoma by facilitating its growth and migration.

Epithelial-to-mesenchymal(-like) transition as a relevant molecular event in malignant gliomas.

Tumor dissemination and metastatic behavior account for the vast majority of cancer associated mortality. Epithelial tumors achieve this progressive state via epithelial-to-mesenchymal transition (EMT); however, the importance of this process in the neuroepithelial context is currently very controversially discussed. The review describes the current research status concerning EMT-like changes in malignant gliomas including the role of TWIST1, ZEB1/ZEB2 and SNAIl1/SNAIl2 as inducers for cell-invasiveness in GBMs. Furthermore, WNT/ß-catenin signaling with its key-component FRIZZLED4 activating an EMT-like program in malignant gliomas and its relationship to the stem-like phenotype as well as discoveries on micro-RNA-level regulating the EMT-like process are discussed.