Clonal Variation in Drug and Radiation Response among Glioma-Initiating Cells Is Linked to Proneural-Mesenchymal Transition.

Intratumoral heterogeneity is a hallmark of glioblastoma multiforme and thought to negatively affect treatment efficacy. Here, we establish libraries of glioma-initiating cell (GIC) clones from patient samples and find extensive molecular and phenotypic variability among clones, including a range of responses to radiation and drugs. This widespread variability was observed as a continuum of multitherapy resistance phenotypes linked to a proneural-mesenchymal shift in the transcriptome. Multitherapy resistance was associated with a semi-stable cell state that was characterized by an altered DNA methylation pattern at promoter regions of mesenchymal master regulators and enhancers. The gradient of cell states within the GIC compartment constitutes a distinct form of heterogeneity. Our findings may open an avenue toward the development of new therapeutic rationales designed to reverse resistant cell states.

The Human Glioblastoma Cell Culture Resource: Validated Cell Models Representing All Molecular Subtypes.

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.

Oncogenic signaling is dominant to cell of origin and dictates astrocytic or oligodendroglial tumor development from oligodendrocyte precursor cells.

Stem cells, believed to be the cellular origin of glioma, are able to generate gliomas, according to experimental studies. Here we investigated the potential and circumstances of more differentiated cells to generate glioma development. We and others have shown that oligodendrocyte precursor cells (OPCs) can also be the cell of origin for experimental oligodendroglial tumors. However, the question of whether OPCs have the capacity to initiate astrocytic gliomas remains unanswered. Astrocytic and oligodendroglial tumors represent the two most common groups of glioma and have been considered as distinct disease groups with putatively different origins. Here we show that mouse OPCs can give rise to both types of glioma given the right circumstances. We analyzed tumors induced by K-RAS and AKT and compared them to oligodendroglial platelet-derived growth factor B-induced tumors in Ctv-a mice with targeted deletions of Cdkn2a (p16(Ink4a-/-), p19(Arf-/-), Cdkn2a(-/-)). Our results showed that glioma can originate from OPCs through overexpression of K-RAS and AKT when combined with p19(Arf) loss, and these tumors displayed an astrocytic histology and high expression of astrocytic markers. We argue that OPCs have the potential to develop both astrocytic and oligodendroglial tumors given loss of p19(Arf), and that oncogenic signaling is dominant to cell of origin in determining glioma phenotype. Our mouse data are supported by the fact that human astrocytoma and oligodendroglioma display a high degree of overlap in global gene expression with no clear distinctions between the two diagnoses.

Sox21 inhibits glioma progression in vivo by forming complexes with Sox2 and stimulating aberrant differentiation.

Sox2 is a transcription factor in neural stem cells and keeps the cells immature and proliferative. Sox2 is expressed in primary human glioma such as glioblastoma multiforme (GBM), primary glioma cells and glioma cell lines and is implicated in signaling pathways in glioma connected to malignancy. Sox21, the counteracting partner of Sox2, has the same expression pattern as Sox2 in glioma but in general induces opposite effects. In this study, Sox21 was overexpressed by using a tetracycline-regulated expression system (tet-on) in glioma cells. The glioma cells were injected subcutaneously into immunodeficient mice. The control tumors were highly proliferative, contained microvascular proliferation and large necrotic areas typical of human GBM. Induction of Sox21 in the tumor cells resulted in a significant smaller tumor size, and the effect correlated with the onset of treatment, where earlier treatment gave smaller tumors. Mice injected with glioma cells orthotopically into the brain survived significantly longer when Sox21 expression was induced. Tumors originating from glioma cells with an induced expression of Sox21 exhibited an increased formation of Sox2:Sox21 complexes and an upregulation of S100ß, CNPase and Tuj1. Sox21 appears to decrease the stem-like cell properties of the tumor cells and initiate aberrant differentiation of glioma cells in vivo. Taken together our results indicate that Sox21 can function as a tumor suppressor during gliomagenesis mediated by a shift in the balance between Sox2 and Sox21. The wide distribution of Sox2 and Sox21 in GBM makes the Sox2/Sox21 axis a very interesting target for novel therapy of gliomas.

Mast cell accumulation in glioblastoma with a potential role for stem cell factor and chemokine CXCL12.

Glioblastoma multiforme (GBM) is the most common and malignant form of glioma with high mortality and no cure. Many human cancers maintain a complex inflammatory program triggering rapid recruitment of inflammatory cells, including mast cells (MCs), to the tumor site. However, the potential contribution of MCs in glioma has not been addressed previously. Here we report for the first time that MCs infiltrate KRas+Akt-induced gliomas, using the RCAS/TV-a system, where KRas and Akt are transduced by RCAS into the brains of neonatal Gtv-a- or Ntv-a transgenic mice lacking Ink4a or Arf. The most abundant MC infiltration was observed in high-grade gliomas of Arf-/- mice. MC accumulation could be localized to the vicinity of glioma-associated vessels but also within the tumor mass. Importantly, proliferating MCs were detected, suggesting that the MC accumulation was caused by local expansion of the MC population. In line with these findings, strong expression of stem cell factor (SCF), i.e. the main MC growth factor, was detected, in particular around tumor blood vessels. Further, glioma cells expressed the MC chemotaxin CXCL12 and MCs expressed the corresponding receptor, i.e. CXCR4, suggesting that MCs could be attracted to the tumor through the CXCL12/CXCR4 axis. Supporting a role for MCs in glioma, strong MC infiltration was detected in human glioma, where GBMs contained significantly higher MC numbers than grade II tumors did. Moreover, human GBMs were positive for CXCL12 and the infiltrating MCs were positive for CXCR4. In conclusion, we provide the first evidence for a role for MCs in glioma.

Forced expression of Sox21 inhibits Sox2 and induces apoptosis in human glioma cells.

Numerous studies support a role for Sox2 to keep stem cells and progenitor cells in an immature and proliferative state. Coexpression of Sox2 and GFAP has been found in regions of the adult brain where neural stem cells are present and in human glioma cells. In our study, we have investigated the roles of Sox2 and its counteracting partner Sox21 in human glioma cells. We show for the first time that Sox21 is expressed in both primary glioblastoma and in human glioma cell lines. We found that coexpression of Sox2, GFAP and Sox21 was mutually exclusive with expression of fibronectin. Our result suggests that glioma consists of at least two different cell populations: Sox2(+) /GFAP(+) /Sox21(+) /FN(-) and Sox2(-) /GFAP(-) /Sox21(-) /FN(+) . Reduction of Sox2 expression by using siRNA against Sox2 or by overexpressing Sox21 using a tetracycline-regulated expression system (Tet-on) caused decreased GFAP expression and a reduction in cell number due to induction of apoptosis. We suggest that Sox21 can negatively regulate Sox2 in glioma. Our findings imply that Sox2 and Sox21 may be interesting targets for the development of novel glioma therapy.

Histidine-rich glycoprotein can prevent development of mouse experimental glioblastoma.

Extensive angiogenesis, formation of new capillaries from pre-existing blood vessels, is an important feature of malignant glioma. Several antiangiogenic drugs targeting vascular endothelial growth factor (VEGF) or its receptors are currently in clinical trials as therapy for high-grade glioma and bevacizumab was recently approved by the FDA for treatment of recurrent glioblastoma. However, the modest efficacy of these drugs and emerging problems with anti-VEGF treatment resistance welcome the development of alternative antiangiogenic therapies. One potential candidate is histidine-rich glycoprotein (HRG), a plasma protein with antiangiogenic properties that can inhibit endothelial cell adhesion and migration. We have used the RCAS/TV-A mouse model for gliomas to investigate the effect of HRG on brain tumor development. Tumors were induced with platelet-derived growth factor-B (PDGF-B), in the presence or absence of HRG. We found that HRG had little effect on tumor incidence but could significantly inhibit the development of malignant glioma and completely prevent the occurrence of grade IV tumors (glioblastoma).

Cell type-specific tumor suppression by Ink4a and Arf in Kras-induced mouse gliomagenesis.

Homozygous deletion of the INK4a-ARF locus is one of the most frequent mutations found in human glioblastoma. We have previously shown that combined Ink4a-Arf loss can increase tumor incidence in both glial progenitor cells and astrocytes during mouse gliomagenesis. Here we have investigated the separate contribution of loss of each of the tumor suppressor genes in glial progenitor cells and astrocytes in Akt + Kras-induced gliomagenesis. We show that Arf is the major tumor suppressor gene in both cell types. Arf loss generated glioblastomas from both nestin-expressing glial progenitor cells and glial fibrillary acidic protein-expressing astrocytes, with a significantly higher incidence in astrocytes. Ink4a loss, on the other hand, could only significantly contribute to gliomagenesis from glial progenitor cells and the induced tumors were of lower malignancy than those seen in Arf-deficient mice. Thus, Ink4a and Arf have independent and differential tumor suppressor functions in vivo in the glial cell compartment.