Cecilie Sandberg

Comparison of glioma stem cells to neural stem cells from the adult human brain identifies dysregulated Wnt- signaling and a fingerprint associated with clinical outcome

Glioblastoma is the most common brain tumor. Median survival in unselected patients is <10 months. The tumor harbors stem-like cells that self-renew and propagate upon serial transplantation in mice, although the clinical relevance of these cells has not been well documented. We have performed the first genome-wide analysis that directly relates the gene expression profile of nine enriched populations of glioblastoma stem cells (GSCs) to five identically isolated and cultivated populations of stem cells from the normal adult human brain. Although the two cell types share common stem- and lineage-related markers, GSCs show a more heterogeneous gene expression. We identified a number of pathways that are dysregulated in GSCs. A subset of these pathways has previously been identified in leukemic stem cells, suggesting that cancer stem cells of different origin may have common features. Genes upregulated in GSCs were also highly expressed in embryonic and induced pluripotent stem cells. We found that canonical Wnt-signaling plays an important role in GSCs, but not in adult human neural stem cells. As well we identified a 30-gene signature highly overexpressed in GSCs. The expression of these signature genes correlates with clinical outcome and demonstrates the clinical relevance of GSCs.

A comparison between stem cells from the adult human brain and from brain tumors.

OBJECTIVE To directly compare stem cells from the normal adult human brain (adult human neural stem cells [AHNSC]), Grade II astrocytomas (AC II), and glioblastoma multiforme (GBM), with respect to proliferative and tumor-forming capacity and differentiation potential. METHODS Cells were isolated from tissue obtained during epilepsy surgery (AHNSCs) or tumor surgery (glioma stem cells [GSC]). They were cultured and investigated in vitro or after transplantation in immunodeficient mice. RESULTS Under identical experimental conditions, the following were found: 1) GBM stem cells formed tumors after orthotopic transplantation; AHNSCs showed no sign of tumor formation; 2) GSCs showed a significantly higher growth rate and self-renewal capacity; 3) both the growth rate and telomerase expression were high in GSCs and correlated with malignancy grade (GBM higher than AC II); AHNSCs had low telomerase expression; 4) GSCs invaded normal neurospheres, not vice versa; 5) both AHNSCs and stem cells from AC II and GBM responded to differentiation cues with a dramatic decrease in the proliferation index (Ki-67); 6) GSCs differentiated faster than AHNSCs; 7) upon differentiation, AHNSCs produced normal glia and neurons; GSCs produced morphologically aberrant cells often expressing both glial and neuronal antigens; and 8) differentiation of AHNSCs resulted in 2 typical functional phenotypes: neurons (high electrical membrane resistance, ability to generate action potentials) and glial cells (low membrane resistance, no action potentials). In contrast, GSCs resulted in only 1 functional phenotype: cells with high electrical resistance and active membrane properties capable of generating action potentials. CONCLUSION AHNSCs and stem cells from AC II and GBM differ with respect to proliferation, tumor-forming capacity, and rate and pattern of differentiation.

A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain.

Cells isolated from the ciliary body (CB) of the adult human eye possess properties of retinal stem/progenitor cells and can be propagated as spheres in culture. As these cells are isolated from a non-neural epithelium which has neuroepithelial origin, they may have both epithelial and neural lineages. Since it is the properties of neural progenitor cells that are sought after in a future scenario of autotransplantation, we wanted to directly compare human CB spheres with neurospheres derived from the human subventricular zone (SVZ), which is the best characterized neural stem cell niche in the CNS of adults. The CB epithelium was dissected from donor eyes (n = 8). Biopsies from the ventricular wall were harvested during neurosurgery due to epilepsy (n = 7). CB and SVZ tissue were also isolated from Brown Norwegian rats. Dissociated single cells were cultivated in a sphere-promoting medium and passaged every 10-30 days. Fixed spheres were studied by immunohistochemistry, quantitative RT-PCR and scanning/transmission electron microscopy. We found that both CB and SVZ spheres contained a mixed population of cells embedded in extracellular matrix. CB spheres, in contrast to SVZ neurospheres, contained pigmented cells with epithelial morphology that stained for cytokeratins (3/12 + 19), were connected through desmosomes and tight-junctions and produced PEDF. Markers of neural progenitors (nestin, Sox-2, GFAP) were significantly lower expressed in human CB compared to SVZ spheres, and nestin positive cells in the CB spheres also contained pigment. There was higher expression of EGF and TGF-beta receptors in human CB spheres, and a comparative greater activation of the canonical Wnt pathway. These results indicate that adult human CB spheres contain progenitor cells with epithelial properties and limited expression of neural progenitor markers compared to CNS neurospheres. Further studies mapping the regulation between epithelial and neural properties in the adult human CB spheres are vital to fully utilize them as a clinical source of retinal progenitor cells in the future.

Brain tumor stem cells maintain overall phenotype and tumorigenicity after in vitro culturing in serum-free conditions

Traditional in vitro culturing of tumor cells has been shown to induce changes so that cultures no longer represent the tumor of origin. Serum-free culturing conditions are used in a variety of cancers to propagate stem-like cells in vitro. Limited reports, however, exist on the effects of such propagation. We have compared cells from brain tumor biopsies cultivated under serum-free conditions at passages 2 and 10 to describe the effects of in vitro culturing. We were able to establish cell lines from 7 of 10 biopsies from patients with glioblastoma. The cell lines adapted to conditions and had 2.2 times increased population doubling rate at later passages. Karyotyping and comparative genomic hybridization analysis revealed that all examined cell lines had cytogenetic aberrations commonly found in glioblastomas, and there were only minor differences between tumor and early and late passages in the same culture. Whole-transcriptome analysis shows that tumors had interindividual differences. Changes in the overall expression patterns through passaging were modest, with a significant change in only 14 genes; the variation among cultures was, however, reduced through passages. The ability to differentiate differed among tumors but was maintained throughout passaging. The cells initiated tumors upon transplantation to immunodeficient mice with differing phenotypes, but a given cell culture maintained tumor phenotype after serial cultivation. The cultures established maintained individual characteristics specific to culture identity. Thus, each cell culture reflects an image of the tumor--or a personalized model--from which it was derived and remains representative after moderate expansion.

Combined expressional analysis, bioinformatics and targeted proteomics identify new potential therapeutic targets in glioblastoma stem cells

Ovarian cancer is associated with increased expression of the pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which induces tumor cell proliferation, angiogenesis, and metastasis. Even though bortezomib (BZ) has shown remarkable anti-tumor activity in hematological malignancies, it has been less effective in ovarian cancer; however, the mechanisms are not understood. We have recently shown that BZ unexpectedly induces the expression of IL-8 in ovarian cancer cells in vitro, by IκB kinase (IKK)-dependent mechanism. Here, we tested the hypothesis that IKK inhibition reduces the IL-8 production and increases BZ effectiveness in reducing ovarian tumor growth in vivo. Our results demonstrate that the combination of BZ and the IKK inhibitor Bay 117085 significantly reduces the growth of ovarian tumor xenografts in nude mice when compared to either drug alone. Mice treated with the BZ/Bay 117085 combination exhibit smallest tumors, and lowest levels of IL-8. Furthermore, the reduced tumor growth in the combination group is associated with decreased tumor levels of S536P-p65 NFκB and its decreased recruitment to IL-8 promoter in tumor tissues. These data provide the first in vivo evidence that combining BZ with IKK inhibitor is effective, and suggest that using IKK inhibitors may increase BZ effectiveness in ovarian cancer treatment.Glioblastoma (GBM) is both the most common and the most lethal primary brain tumor. It is thought that GBM stem cells (GSCs) are critically important in resistance to therapy. Therefore, there is a strong rationale to target these cells in order to develop new molecular therapies. To identify molecular targets in GSCs, we compared gene expression in GSCs to that in neural stem cells (NSCs) from the adult human brain, using microarrays. Bioinformatic filtering identified 20 genes (PBK/TOPK, CENPA, KIF15, DEPDC1, CDC6, DLG7/DLGAP5/HURP, KIF18A, EZH2, HMMR/RHAMM/CD168, NOL4, MPP6, MDM1, RAPGEF4, RHBDD1, FNDC3B, FILIP1L, MCC, ATXN7L4/ATXN7L1, P2RY5/LPAR6 and FAM118A) that were consistently expressed in GSC cultures and consistently not expressed in NSC cultures. The expression of these genes was confirmed in clinical samples (TCGA and REMBRANDT). The first nine genes were highly co-expressed in all GBM subtypes and were part of the same protein-protein interaction network. Furthermore, their combined up-regulation correlated negatively with patient survival in the mesenchymal GBM subtype. Using targeted proteomics and the COGNOSCENTE database we linked these genes to GBM signalling pathways. Nine genes: PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2 and HMMR should be further explored as targets for treatment of GBM.

Expansion of multipotent stem cells from the adult human brain.

The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells.

Aqp 9 and brain tumour stem cells.

Several studies have implicated the aquaporins (aqp) 1, 4, and 9 in the pathogenesis of malignant brain tumours, suggesting that they contribute to motility, invasiveness, and oedema formation and facilitate metabolism in tumour cells under hypoxic conditions. We have studied the expression of aqp1, 4, and 9 in biopsies from glioblastomas, isolated tumour stem cells grown in a tumoursphere assay and analyzed the progenitor and differentiated cells from these cultures. We have compared these to the situation in normal rat brain, its stem cells, and differentiated cells derived thereof. In short, qPCR in tumour tissue showed presence of aqp1, 4, and 9. In the tumour progenitor population, aqp9 was markedly more highly expressed, whilst in tumour-derived differentiated cells, aqp4 was downregulated. However, immunostaining did not reveal increased protein expression of aqp9 in the tumourspheres containing progenitor cells; in contrast, its expression (both mRNA and protein) was high in differentiated cultures. We, therefore, propose that aquaporin 9 may have a central role in the tumorigenesis of glioblastoma.

A comparative study of the structural organization of spheres derived from the adult human subventricular zone and glioblastoma biopsies

Sphere forming assays have been useful to enrich for stem like cells in a range of tumors. The robustness of this system contrasts the difficulties in defining a stem cell population based on cell surface markers. We have undertaken a study to describe the cellular and organizational composition of tumorspheres, directly comparing these to neurospheres derived from the adult human subventricular zone (SVZ). Primary cell cultures from brain tumors were found to contain variable fractions of cells positive for tumor stem cell markers (CD133 (2-93%)/SSEA1 (3-15%)/CXCR4 (1-72%)). All cultures produced tumors upon xenografting. Tumorspheres contained a heterogeneous population of cells, but were structurally organized with stem cell markers present at the core of spheres, with markers of more mature glial progenitors and astrocytes at more peripheral location. Ultrastructural studies showed that tumorspheres contained a higher fraction of electron dense cells in the core than the periphery (36% and 19%, respectively). Neurospheres also contained a heterogeneous cell population, but did not have an organization similar to tumorspheres. Although tumorspheres clearly display irregular and neoplastic cells, they establish an organized structure with an outward gradient of differentiation. We suggest that this organization is central in maintaining the tumor stem cell pool.

Wnt inhibition is dysregulated in gliomas and its re-establishment inhibits proliferation and tumor sphere formation.

Evidence indicates that the growth of glioblastoma (GBM), the most common and malignant primary brain cancer, is driven by glioma stem cells (GSCs) resistant to current treatment. As Wnt-signaling is pivotal in stem cell maintenance, we wanted to explore its role in GSCs with the objective of finding distinct signaling mechanisms that could serve as potential therapeutic targets. We compared gene expression in GSCs (n=9) and neural stem cells from the adult human brain (ahNSC; n=3) to identify dysregulated genes in the Wnt signaling pathway. This identified a six-gene Wnt signature present in all nine primary GSC cultures, and the combined expression of three of these genes (SFRP1, SFRP4 and FZD7) reduced median survival of glioma patients from 38 to 17 months. Treatment with recombinant SFRP1 protein in primary cell cultures downregulated nuclear β-catenin and decreased in vitro proliferation and sphere formation in a dose-dependent manner. Furthermore, expressional and functional analysis of SFRP1-treated GSCs revealed that SFRP1 halts cell cycling and induces apoptosis. These observations demonstrate that Wnt signaling is dysregulated in GSC, and that inhibition of the Wnt pathway could serve as a therapeutic strategy in the treatment of GBM.

Inhibition of tumor formation and redirected differentiation of glioblastoma cells in a xenotypic embryonic environment

Background: Tissue microenvironment plays key roles in regulating the progression of aggressive tumors. Tumors are uncommon in the early embryo, suggesting that embryonic tissue microenvironments are nonpermissive for tumors. Yet, the effects of embryonic tissue microenvironments on tumor cells have not been extensively studied. We have, therefore, tested the behavior of human glioblastoma multiforme (GBM) cells transplanted into a central neural tissue microenvironment in the chicken embryo. Results: GBM cells were cultured as spheres to enrich for GBM stem cells (GSCs) and transduced with GFP for identification. Within the proliferative embryonic neural tissue, GSC‐enriched GBM cells exhibited reduced proliferation and survival, altered gene expression, and formed no tumors, in marked contrast to their aggressive behavior in vitro and tumor formation in other tissue microenvironments including the chorioallantoic membrane of the chicken embryo and the brain of adult severe combined immunodeficiency (SCID) mice. Surviving cells in the spinal neural tube exhibited tumor‐atypical expression profiles of neuron‐, glia‐, stem cell‐, and tumor‐related genes. Conclusions: Embryonic neural tissue provides a poor environment for GBM cell survival and tumor formation, and redirects differentiation toward a more benign phenotype. Understanding the anti‐tumorigenic effects of this embryonic tissue microenvironment could provide opportunities to develop novel therapies for GBM treatment. Developmental Dynamics 242:1078‐1093, 2013.