Although gliomas are thought to arise from a neural-glial lineage of ectodermal origin, many gliomas express a mesenchymal signature. Behnan et al. review the origins of this signature in glioma: it may arise via the tumour stroma, via NF1 -mutation in tumour cells and be influenced by the cell of origin, or arise in response to radiotherapy/chemotherapy/anti-angiogenic treatment.

BackgroundGlioblastomas are invasive therapy resistant brain tumors with extremely poor prognosis. The Glioma initiating cell (GIC) population contributes to therapeutic resistance and tumor recurrence. Targeting GIC-associated gene candidates could significantly impact GBM tumorigenicity. Here, we investigate a protein kinase, PBK/TOPK as a candidate for regulating growth, survival and in vivo tumorigenicity of GICs.MethodsPBK is highly upregulated in GICs and GBM tissues as shown by RNA and protein analyses. We knocked down PBK using shRNA vectors and inhibited the function of PBK protein with a pharmacological PBK inhibitor, HITOPK-032. We assessed viability, tumorsphere formation and apoptosis in three patient derived GIC cultures.ResultsGene knockdown of PBK led to decreased viability and sphere formation and in one culture an increase in apoptosis. Treatment of cells with inhibitor HITOPK-032 (5 μM and 10 μM) almost completely abolished growth and elicited a large increase in apoptosis in all three cultures. HI-TOPK-032 treatment (5 mg/kg and 10 mg/kg bodyweight) in vivo resulted in diminished growth of experimentally induced subcutaneous GBM tumors in mice. We also carried out multi-culture assays of cell survival to investigate the relative effects on GICs compared with the normal neural stem cells (NSCs) and their differentiated counterparts. Normal NSCs seemed to withstand treatment slightly better than the GICs.ConclusionOur study of identification and functional validation of PBK suggests that this candidate can be a promising molecular target for GBM treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0398-x) contains supplementary material, which is available to authorized users.

The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These brain tumor-derived mesenchymal stem cells (BT-MSCs) are defined with the phenotype (LinSca-11CD91CD441CD1661/2) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro and that the non-MSC population is nontumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild-type GL261 inoculated into green fluorescent protein (GFP)-transgenic mice and GL261-GFP cells inoculated into wild-type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD441CD91CD1661 both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus, we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients.

Glioblastoma Multiforme (GBM) is characterized by high cancer cell heterogeneity and the presence of a complex tumor microenvironment. Those factors are a key obstacle for the treatment of this tumor type. To model the disease in mice, the current strategy is to grow GBM cells in serum-free non-adherent condition, which maintains their tumor-initiating potential. However, the so-generated tumors are histologically different from the one of origin. In this work, we performed high-throughput marker expression analysis and investigated the tumorigenicity of GBM cells enriched under different culture conditions. We identified a marker panel that distinguished tumorigenic sphere cultures from non-tumorigenic serum cultures (high CD56, SOX2, SOX9, and low CD105, CD248, αSMA). Contrary to previous work, we found that ‘mixed cell cultures' grown in serum conditions are tumorigenic and express cancer stem cell (CSC) markers. As well, 1% serum plus bFGF and TGF-α preserved the tumorigenicity of sphere cultures and induced epithelial-to-mesenchymal transition gene expression. Furthermore, we identified 12 genes that could replace the 840 genes of The Cancer Genome Atlas (TCGA) used for GBM-subtyping. Our data suggest that the tumorigenicity of GBM cultures depend on cell culture strategies that retain CSCs in culture rather than the presence of serum in the cell culture medium.

The biology of glioblastoma invasion and its mechanisms are poorly understood. We demonstrate using time-lapse microscopy that grafting of glioblastoma (GBM) tumorspheres into rodent brain slices results in experimental ex vivo tumors with invasive properties that recapitulate the invasion observed after orthotopic transplantation into the rodent brain. The migratory movements and mitotic patterns were clearly modified by signals extrinsic to the invading cells. The cells migrated away from the tumorspheres, and removal of the spheres reduced the directed invasive movement. The cell cultures contained different populations of invasive cells that had distinct morphology and invasive behavior patterns. Grafts of the most invasive GBM culture contained 91±8% cells with an invasive phenotype, characterized by small soma with a distinct leading process. Conversely, the majority of cells in less invasive GBM grafts were phenotypically heterogeneous: only 6.3±4.1% of the cells had the invasive phenotype. Grafts of highly and moderately invasive cultures had different proportions of cells that advanced into the brain slice parenchyma during the observation period: 89.2±2.2% and 23.1±6.8%, respectively. In grafts with moderately invasive properties, most of the cells (76.8±6.8%) invading the surrounding brain tissue returned to the tumor bulk or stopped centrifugal migration. Our data suggest that the invasion of individual GBM tumors can be conditioned by the prevalence of a cell fraction with particular invasive morphology and by signaling between the tumor core and invasive cells. These findings can be important for the development of new therapeutic strategies that target the invasive GBM cells.

Adult neural progenitor cells (aNPCs) are a potential source for cell based therapy for neurodegenerative diseases and traumatic brain injuries. These cells have been traditionally isolated from hippocampus, subventricular zone and white matter. However, there is still a need for an easily accessible source with better yield to counter the limitations of small surgical samples of previously characterized aNPCs. Here we show that ultrasonic aspirate (UA) samples currently considered as ‘biological waste after surgery,' offer a good source for aNPCs. Furthermore, we show that culture conditions dictated the phenotype of cells across patients. The neurosphere-enriched cells were more similar to freshly isolated brain cells, while cells expanded adherently in serum conditions were similar to mesenchymal stem cells. However, cells expanded in these adherent conditions expressed some NPC and glial markers in addition to active canonical Wnt signaling. This suggests a mesenchymal-neuroectodermal hybrid nature of these cells. Finally, we show that UA-NPCs are comparable to those from neurogenic regions. Our findings suggest that UA samples can be used as a source for fresh and in vitro propagated aNPCs that could have various clinical applications.

Intratumoral heterogeneity is a characteristic of glioblastomas that contain an intermixture of cell populations displaying different glioblastoma subtype gene expression signatures. Proportions of these populations change during tumor evolution, but the occurrence and regulation of glioblastoma subtype transition is not well described. To identify regulators of glioblastoma subtypes we utilized a combination of in vitro experiments and in silico analyses, using experimentally generated as well as publicly available data. Through this combined approach SOX2 was identified to confer a proneural glioblastoma subtype gene expression signature. SFRP2 was subsequently identified as a SOX2-antagonist, able to induce a mesenchymal glioblastoma subtype signature. A subset of patient glioblastoma samples with high SFRP2 and low SOX2 expression was particularly enriched with mesenchymal subtype samples. Phenotypically, SFRP2 decreased tumor sphere formation, stemness as assessed by limiting dilution assay, and overall cell proliferation but increased cell motility, whereas SOX2 induced the opposite effects. Furthermore, an SFRP2/non-canonical-WNT/KLF4/PDGFR/phospho-AKT/SOX2 signaling axis was found to be involved in the mesenchymal transition. Analysis of human tumor tissue spatial gene expression patterns showed distinct expression of SFRP2- and SOX2-correlated genes in vascular and cellular areas, respectively. Finally, conditioned media from SFRP2 overexpressing cells increased CD206 on macrophages. Together, these findings present SFRP2 as a SOX2-antagonist with the capacity to induce a mesenchymal subtype transition in glioma cells located in vascular tumor areas, highlighting its role in glioblastoma tumor evolution and intratumoral heterogeneity.

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