Rolf Bjerkvig

CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells

CD133 is a cell surface marker expressed on progenitors of haematopoietic and endothelial cell lineages. Moreover, several studies have identified CD133 as a marker of brain tumor‐initiating cells. In this study, human glioblastoma multiforme biopsies were engrafted intracerebrally into nude rats. The resulting tumors were serially passaged in vivo, and monitored by magnetic resonance imaging. CD133 expression was analyzed at various passages. Tumors initiated directly from the biopsies expressed little or no CD133, and showed no contrast enhancement suggesting an intact blood‐brain barrier. During passaging, the tumors gradually displayed more contrast enhancement, increased angiogenesis and a shorter survival. Real‐time qPCR and immunoblots showed that this was accompanied by increased CD133 expression. Primary biopsy spheroids and xenograft tumors were subsequently dissociated and flow sorted into CD133 negative and CD133 positive cell populations. Both populations incorporated BrdU in cell culture, and expressed the neural precursor marker nestin. Notably, CD133 negative cells derived from 6 different patients were tumorgenic when implanted into the rat brains. For 3 of these patients, analysis showed that the resulting tumors contained CD133 positive cells. In conclusion, we show that CD133 negative glioma cells are tumorgenic in nude rats, and that CD133 positive cells can be obtained from these tumors. Upon passaging of the tumors in vivo, CD133 expression is upregulated, coinciding with the onset of angiogenesis and a shorter survival. Thus, our findings do not suggest that CD133 expression is required for brain tumor initiation, but that it may be involved during brain tumor progression.

Long-term Cultures of Bone Marrow-Derived Human Mesenchymal Stem Cells Frequently Undergo Spontaneous Malignant Transformation

Human mesenchymal stem cells (hMSC) aid in tissue maintenance and repair by differentiating into specialized cell types. Due to this ability, hMSC are currently being evaluated for cell-based therapies of tissue injury and degenerative diseases. However, extensive expansion ex vivo is a prerequisite to obtain the cell numbers required for human cell-based therapy protocols. Recent studies indicate that hMSC may contribute to cancer development and progression either by acting as cancer-initiating cells or through interactions with stromal elements. If spontaneous transformation ex vivo occurs, this may jeopardize the use of hMSC as therapeutic tools. Whereas murine MSC readily undergo spontaneous transformation, there are conflicting reports about spontaneous transformation of hMSC. We have addressed this controversy in a two-center study by growing bone marrow-derived hMSC in long-term cultures (5-106 weeks). We report for the first time spontaneous malignant transformation to occur in 45.8% (11 of 24) of these cultures. In comparison with hMSC, the transformed mesenchymal cells (TMC) showed a significantly increased proliferation rate and altered morphology and phenotype. In contrast to hMSC, TMC grew well in soft agar assays and were unable to undergo complete differentiation. Importantly, TMC were highly tumorigenic, causing multiple fast-growing lung deposits when injected into immunodeficient mice. We conclude that spontaneous malignant transformation may represent a biohazard in long-term ex vivo expansion of hMSC. On the other hand, this spontaneous transformation process may represent a unique model for studying molecular pathways initiating malignant transformation of hMSC.

Spontaneous Malignant Transformation of Human Mesenchymal Stem Cells Reflects Cross-Contamination: Putting the Research Field on Track - Letter

Several groups, including ours, have published results showing spontaneous transformation of human mesenchymal stem cells (MSC). Recently, we reported in this journal spontaneous transformation of bone marrow-derived human MSC (hMSC), isolated and expanded independently in two laboratories ([1][1

Angiogenesis-independent tumor growth mediated by stem-like cancer cells

In this work, highly infiltrative brain tumors with a stem-like phenotype were established by xenotransplantation of human brain tumors in immunodeficient nude rats. These tumors coopted the host vasculature and presented as an aggressive disease without signs of angiogenesis. The malignant cells expressed neural stem cell markers, showed a migratory behavior similar to normal human neural stem cells, and gave rise to tumors in vivo after regrafting. Serial passages in animals gradually transformed the tumors into an angiogenesis-dependent phenotype. This process was characterized by a reduction in stem cells markers. Gene expression profiling combined with high throughput immunoblotting analyses of the angiogenic and nonangiogenic tumors identified distinct signaling networks in the two phenotypes. Furthermore, proinvasive genes were up-regulated and angiogenesis signaling genes were down-regulated in the stem-like tumors. In contrast, proinvasive genes were down-regulated in the angiogenesis-dependent tumors derived from the stem-like tumors. The described angiogenesis-independent tumor growth and the uncoupling of invasion and angiogenesis, represented by the stem-like cancer cells and the cells derived from them, respectively, point at two completely independent mechanisms that drive tumor progression. This article underlines the need for developing therapies that specifically target the stem-like cell pools in tumors.

The progenitor cell marker NG2/MPG promotes chemoresistance by activation of integrin-dependent PI3K/Akt signaling

Chemoresistance represents a major problem in the treatment of many malignancies. Overcoming this obstacle will require improved understanding of the mechanisms responsible for this phenomenon. The progenitor cell marker NG2/melanoma proteoglycan (MPG) is aberrantly expressed by various tumors, but its role in cell death signaling and its potential as a therapeutic target are largely unexplored. We have assessed cytotoxic drug-induced cell death in glioblastoma spheroids from 15 patients, as well as in five cancer cell lines that differ with respect to NG2/MPG expression. The tumors were treated with doxorubicin, etoposide, carboplatin, temodal, cisplatin and tumor necrosis factor (TNF)α. High NG2/MPG expression correlated with multidrug resistance mediated by increased activation of α3β1 integrin/PI3K signaling and their downstream targets, promoting cell survival. NG2/MPG knockdown with shRNAs incorporated into lentiviral vectors attenuated β1 integrin signaling revealing potent antitumor effects and further sensitized neoplastic cells to cytotoxic treatment in vitro and in vivo. Thus, as a novel regulator of the antiapoptotic response, NG2/MPG may represent an effective therapeutic target in several cancer subtypes.

Expression of the progenitor marker NG2/CSPG4 predicts poor survival and resistance to ionising radiation in glioblastoma

Glioblastoma (GBM) is a highly aggressive brain tumour, where patients respond poorly to radiotherapy and exhibit dismal survival outcomes. The mechanisms of radioresistance are not completely understood. However, cancer cells with an immature stem-like phenotype are hypothesised to play a role in radioresistance. Since the progenitor marker neuron-glial-2 (NG2) has been shown to regulate several aspects of GBM progression in experimental systems, we hypothesised that its expression would influence the survival of GBM patients. Quantification of NG2 expression in 74 GBM biopsies from newly diagnosed and untreated patients revealed that 50% express high NG2 levels on tumour cells and associated vessels, being associated with significantly shorter survival. This effect was independent of age at diagnosis, treatment received and hypermethylation of the O6-methylguanine methyltransferase (MGMT) DNA repair gene promoter. NG2 was frequently co-expressed with nestin and vimentin but rarely with CD133 and the NG2 positive tumour cells harboured genetic aberrations typical for GBM. 2D proteomics of 11 randomly selected biopsies revealed upregulation of an antioxidant, peroxiredoxin-1 (PRDX-1), in the shortest surviving patients. Expression of PRDX-1 was associated with significantly reduced products of oxidative stress. Furthermore, NG2 expressing GBM cells showed resistance to ionising radiation (IR), rapidly recognised DNA damage and effectuated cell cycle checkpoint signalling. PRDX-1 knockdown transiently slowed tumour growth rates and sensitised them to IR in vivo. Our data establish NG2 as an important prognostic factor for GBM patient survival, by mediating resistance to radiotherapy through induction of ROS scavenging enzymes and preferential DNA damage signalling.

Targeting the NG2/CSPG4 Proteoglycan Retards Tumour Growth and Angiogenesis in Preclinical Models of GBM and Melanoma

Aberrant expression of the progenitor marker Neuron-glia 2 (NG2/CSPG4) or melanoma proteoglycan on cancer cells and angiogenic vasculature is associated with an aggressive disease course in several malignancies including glioblastoma multiforme (GBM) and melanoma. Thus, we investigated the mechanism of NG2 mediated malignant progression and its potential as a therapeutic target in clinically relevant GBM and melanoma animal models. Xenografting NG2 overexpressing GBM cell lines resulted in increased growth rate, angiogenesis and vascular permeability compared to control, NG2 negative tumours. The effect of abrogating NG2 function was investigated after intracerebral delivery of lentivirally encoded shRNAs targeting NG2 in patient GBM xenografts as well as in established subcutaneous A375 melanoma tumours. NG2 knockdown reduced melanoma proliferation and increased apoptosis and necrosis. Targeting NG2 in two heterogeneous GBM xenografts significantly reduced tumour growth and oedema levels, angiogenesis and normalised vascular function. Vascular normalisation resulted in increased tumour invasion and decreased apoptosis and necrosis. We conclude that NG2 promotes tumour progression by multiple mechanisms and represents an amenable target for cancer molecular therapy.

Glioma Cell Populations Grouped by Different Cell Type Markers Drive Brain Tumor Growth

Although CD133 has been proposed as a marker for brain tumor-initiating cells, studies show that a tumorigenic potential exists among CD133(-) glioma cells as well. However, it is not established whether the ability of CD133(-) cells to form tumors is a property confined to a small subpopulation, rather than a common trait associated with most glioma cell types. Thus, we used lentiviral vectors expressing green fluorescent protein under lineage-specific promoters to identify CD133(-) glioma cells expressing Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE). Flow cytometry analysis showed the presence of CD133(-) subpopulations expressing these markers in glioma cell lines and in primary cultures from human glioblastoma (GBM) biopsies. Moreover, analysis of cell cycle distribution showed that subgroups expressing Nestin, GFAP, and NSE uniformly contained actively cycling cells, when cultured in serum-containing medium and stem cell medium. These subpopulations were fluorescence-activated cell sorted from CD133(-) U373 glioma cells and implanted intracerebrally in severe combined immunodeficient mice. Moreover, we implanted Nestin-, GFAP-, and NSE-positive glioma cells sorted from a human GBM biopsy, following removal of CD133-positive cells. All the CD133(-) subpopulations produced tumors, with no significant differences in survival or tumor take rates. However, there was a trend toward lower take rates for CD133(-) glioma subpopulations expressing GFAP and NSE. These findings suggest that the ability to form tumors may be a general trait associated with different glioma cell phenotypes, rather than a property limited to an exclusive subpopulation of glioma stem cells.

Invasiveness of primary brain tumors

SummaryPrimary malignant neoplasms of the nervous system differ from other types of malignancy in several ways. Clinical progression is due to local invasive growth, while metastases outside the skull are rare. The tumors show no sharp delimitation from the surrounding normal tissue. At the edge, an ill-defined area of invasive tumor cells, reacting glial cells and inflammatory cells is present. At the same time the primary brain tumors are biologically heterogeneous.In this review, a short survey of markers for malignancy in primary brain tumors is given, and some properties of importance for invasive behavior, are listed. These include different cellular enzymes, phagocytotic property, locomotive and proliferative characteristics.Studies of primary brain tumors in situ show invasive growth into the surrounding brain tissue, often tollowed by hemorrhage and necrosis. In addition spread of tumor cells takes place along preexisting intracranial structures. Recently, several systems for the study of brain tumor invasiveness in culture have been elaborated. Both experimental and human gliomas have been tested. The target tissues include organ culture of embryonic chick heart muscle, chorioallantoic membrane, fetal rat brain tissue and reconstructed vessel walls. It has been shown that glioma cells are able to split junctions between normal cells. They destroy and phagocytose the normal cells and penetrate the normal tissue. The use of brain tissue and reaggregated brain cell cultures as target for glioma cells in culture opens the possibility for an elucidation of invasiveness as one of the most important properties of malignancy in the nervous system.

A novel eGFP-expressing immunodeficient mouse model to study tumor-host interactions.

A NOD/Scid mouse expressing enhanced green fluorescent protein (eGFP) is described, in which human and mouse tumors marked with red fluorescent protein can be established in vivo, both at subcutaneous and orthotopic locations. Using light microscopy as well as multiphoton confocal microscopy techniques, we visualized in detail the intricate colocalization of tumor and host cells in situ. Moreover, using fluorescence‐activated cell sorting (FACS), we were able to completely separate the host cells from the tumor cells, thus providing a system for detailed cellular and molecular analysis of tumor‐host cell interactions. The fact that tumor and host cells can be reliably identified also allowed us to detect double‐positive cells, possibly arising from cell fusion events or horizontal gene transfer. Similarly, the model can be applied for the detection of circulating metastatic cells and for detailed studies on the vascular compartments within tumors, including vasculogenic mimicry. Thus, the model described should provide significant insight into how tumor cells communicate with their microenvironment.—Niclou, S. P., Danzeisen, C., Eikesdal, H. P., Wiig, H., Brons, N. H. C., Poli, A. M. F., Svendsen, A., Torsvik, A., Enger, P. Ø., Terzis, J. A., Bjerkvig, R. A novel eGFP‐expressing immunodeficient mouse model to study tumor‐host interactions. FASEB J. 22, 3120–3128 (2008)