Nils Ole Schmidt

A bioinformatic assay for pluripotency in human cells.

Pluripotent stem cells (PSCs) are defined by their potential to generate all cell types of an organism. The standard assay for pluripotency of mouse PSCs is cell transmission through the germline, but for human PSCs researchers depend on indirect methods such as differentiation into teratomas in immunodeficient mice. Here we report PluriTest, a robust open-access bioinformatic assay of pluripotency in human cells based on their gene expression profiles.

Levels of vascular endothelial growth factor, hepatocyte growth factor/scatter factor and basic fibroblast growth factor in human gliomas and their relation to angiogenesis

Angiogenesis is a possible target in the treatment of human gliomas. To evaluate the role of 3 growth factors, vascular endothelial growth factor (VEGF), hepatocyte growth factor/scatter factor (HGF/SF) and basic fibroblast growth factor (bFGF), in the angiogenic cascade, we determined their levels in extracts of 71 gliomas by enzyme‐linked immunosorbent assay (ELISA). The levels of bFGF were only marginally different between gliomas of World Health Organization (WHO) grade II (low grade) and grades III and IV (high grade). In contrast, the mean concentrations of VEGF were 11‐fold higher in high‐grade tumors and those of HGF/SF 7‐fold, respectively. Both were highly significantly correlated with microvessel density (p < 0.001) as determined by immunostaining for factor VIII‐related antigen. In addition, VEGF and HGF/SF appeared to be independent predictive parameters for glioma microvessel density as determined by multiple regression analysis. We measured the capacity of all 3 factors to induce endothelial tube formation in a collagen gel. In this assay, bFGF was found to be an essential cofactor with which VEGF as well as HGF/SF were able to synergize independently. According to the concentrations of angiogenic factors, extracts from high‐grade tumors were significantly more potent in the tube formation assay than the low‐grade extracts (p = 0.02). Adding neutralizing antibodies to bFGF, VEGF and HGF/SF together with the extracts, tube formation was inhibited by up to 98%, 62% and 54%, respectively. Our findings suggest that bFGF is an essential cofactor for angiogenesis in gliomas, but in itself is insufficient as it is present already in the sparsely vascularized low‐grade tumors. Upon induction of angiogenesis in high‐grade tumors, bFGF may synergize with rising levels of not only VEGF but possibly also with HGF/SF, which appears here to be an independent angiogenic factor. Int. J. Cancer (Pred. Oncol.) 84:10–18, 1999.

Regulatory networks define phenotypic classes of human stem cell lines

Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal and adult sources have been called stem cells, even though they range from pluripotent cells—typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation—to adult stem cell lines, which can generate a far more limited repertoire of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine has highlighted the need for a general, reproducible method for classification of these cells. We report here the creation and analysis of a database of global gene expression profiles (which we call the ‘stem cell matrix’) that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent and differentiated cell types. Using an unsupervised clustering method to categorize a collection of ∼150 cell samples, we discovered that pluripotent stem cell lines group together, whereas other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis we uncovered a protein–protein network (PluriNet) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas and induced pluripotent cells). Analysis of published data showed that the PluriNet seems to be a common characteristic of pluripotent cells, including mouse embryonic stem and induced pluripotent cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotency and self-renewal are under tight control by specific molecular networks.

Evidence for Sequenced Molecular Evolution of IDH1 Mutant Glioblastoma From a Distinct Cell of Origin

PURPOSE Mutation in isocitrate dehydrogenase 1 (IDH1) at R132 (IDH1(R132MUT)) is frequent in low-grade diffuse gliomas and, within glioblastoma (GBM), has been proposed as a marker for GBMs that arise by transformation from lower-grade gliomas, regardless of clinical history. To determine how GBMs arising with IDH1(R132MUT) differ from other GBMs, we undertook a comprehensive comparison of patients presenting clinically with primary GBM as a function of IDH1(R132) mutation status. PATIENTS AND METHODS In all, 618 treatment-naive primary GBMs and 235 lower-grade diffuse gliomas were sequenced for IDH1(R132) and analyzed for demographic, radiographic, anatomic, histologic, genomic, epigenetic, and transcriptional characteristics. RESULTS Investigation revealed a constellation of features that distinguishes IDH1(R132MUT) GBMs from other GBMs (including frontal location and lesser extent of contrast enhancement and necrosis), relates them to lower-grade IDH1(R132MUT) gliomas, and supports the concept that IDH1(R132MUT) gliomas arise from a neural precursor population that is spatially and temporally restricted in the brain. The observed patterns of DNA sequence, methylation, and copy number alterations support a model of ordered molecular evolution of IDH1(R132MUT) GBM in which the appearance of mutant IDH1 protein is an initial event, followed by production of p53 mutant protein, and finally by copy number alterations of PTEN and EGFR. CONCLUSION Although histologically similar, GBMs arising with and without IDH1(R132MUT) appear to represent distinct disease entities that arise from separate cell types of origin as the result of largely nonoverlapping sets of molecular events. Optimal clinical management should account for the distinction between these GBM disease subtypes.

Intravascular Delivery of Neural Stem Cell Lines to Target Intracranial and Extracranial Tumors of Neural and Non-Neural Origin

The remarkable migratory and tumor-tropic capacities of neural stem cells (NSCs and/or neuroprogenitor cells) represent a potentially powerful approach to the treatment of invasive brain tumors, such as malignant gliomas. We have previously shown that whether implanted directly into or at distant sites from an experimental intracranial glioma, NSCs distributed efficiently throughout the main tumor mass and also tracked advancing tumor cells, while stably expressing a reporter transgene. As therapeutic proof-of-concept, NSCs genetically modified to produce the prodrug activating enzyme cytosine deaminase (CD), effected an 80% reduction in the resultant tumor mass, when tumor animals were treated with the systemic prodrug, 5-fluorocytosine. We now extend our findings of the tumor-tropic properties of NSCs (using a well-characterized, clonal NSC line C17.2), by investigating their capacity to target both intracranial and extracranial tumors, when administered into the peripheral vasculature. We furthermore demonstrate their capacity to target extracranial non-neural tumors such as prostate cancer and malignant melanoma. Well-characterized NSC lines (lacZ and/or CD-positive) were injected into the tail vein of adult nude mice with established experimental intracranial and/or subcutaneous flank tumors of neural and non-neural origin. The time course and distribution of NSCs within the tumor and internal organs was assessed in various models. Resulting data suggest that NSCs can localize to various tumor sites when injected via the peripheral vasculature, with little accumulation in normal tissues. Our findings suggest the novel use of intravascularly administered NSCs as an effective delivery vehicle to target and disseminate therapeutic agents to invasive tumors of neural and nonneural origin, both within and outside of the brain.

Vascular endothelial growth factor, hepatocyte growth factor/scatter factor, basic fibroblast growth factor, and placenta growth factor in human meningiomas and their relation to angiogenesis and malignancy.

OBJECTIVE Angiogenesis is mediated by a number of different growth factors and appears vital for tumor growth. The understanding of angiogenic mechanisms could offer new therapeutic perspectives; in this context, the role of four potentially angiogenic growth factors was analyzed in a large series of meningiomas of different grades. METHODS Vascular endothelial growth factor (VEGF), placenta growth factor, hepatocyte growth factor/scatter factor, and basic fibroblast growth factor were quantified in 69 tumors by enzyme-linked immunosorbent assay. Microvessel density and proliferative activity were determined on paraffin sections, and clinical tumor invasiveness was rated. Induction of endothelial chemotaxis and capillary-like tube formation were studied in vitro using modified Boyden chamber assays and three-dimensional collagen gel assays, respectively. RESULTS Tumors included 40 benign (World Health Organization [WHO] Grade I), 21 atypical (WHO Grade II), and 8 anaplastic/malignant (WHO Grade III) meningiomas. We found a correlation between meningioma grade and VEGF content (r = 0.37, P = 0.002), which was 2-fold higher in atypical than in benign meningiomas (P = 0.022) and 10-fold higher in malignant than in benign meningiomas (P = 0.025). Among different subtypes of Grade I meningiomas, VEGF levels were 10-fold higher in meningothelial than in fibrous meningiomas (P = 0.015). None of the other three factors investigated showed any association with tumor grade, microvessel density, or invasiveness, and VEGF also did not correlate with vascularity or invasiveness. Moreover, vascularity did not increase with malignancy grade. Endothelial chemotaxis and capillary-like tube formation in vitro were induced by meningioma extracts and were most effectively blocked by co-addition of antibodies against basic fibroblast growth factor, followed by anti-VEGF, whereas anti-hepatocyte growth factor/scatter factor was not effective. The chemotactic activity of meningioma extracts on endothelial cells correlated with their VEGF content (r = 0.6, P = 0.003). CONCLUSION Meningiomas do not show an angiogenic switch involving VEGF and/or hepatocyte growth factor/scatter factor, as has previously been found in gliomas. Nevertheless, the biological activity of VEGF and basic fibroblast growth factor in meningiomas suggests that both are potential targets for antiangiogenic therapy in meningiomas of all WHO grades.

Targeting of melanoma brain metastases using engineered neural stem/progenitor cells

Brain metastases are an increasingly frequent and serious clinical problem for cancer patients, especially those with advanced melanoma. Given the extensive tropism of neural stem/progenitor cells (NSPCs) for pathological areas in the central nervous system, we expanded investigations to determine whether NSPCs could also target multiple sites of brain metastases in a syngeneic experimental melanoma model. Using cytosine deaminase-expressing NSPCs (CD-NSPCs) and systemic 5-fluorocytosine (5-FC) pro-drug administration, we explored their potential as a cell-based targeted drug delivery system to disseminated brain metastases. Our results indicate a strong tropism of NSPCs for intracerebral melanoma metastases. Furthermore, in our therapeutic paradigm, animals with established melanoma brain metastasis received intracranial implantation of CD-NSPCs followed by systemic 5-FC treatment, resulting in a significant (71%) reduction in tumor burden. These data provide proof of principle for the use of NSPCs for targeted delivery of therapeutic gene products to melanoma brain metastases.

Scatter factor promotes motility of human glioma and neuromicrovascular endothelial cells

Malignant gliomas are characterized by rapid growth, infiltration of normal brain tissue, and high levels of tumor‐associated angiogenesis. The genetic and local environmental tissue factors responsible for the malignant progression from low to high grade gliomas and the highly malignant behavior of glioblastomas are not well understood. In a study of 77 human brain tissue extracts, high grade (III‐IV) tumors had significantly greater scatter factor (SF) content than did low grade tumors or non‐neoplastic tissue. To investigate the potential significance of SF accumulation in gliomas, we measured the effects of SF on DNA synthesis and motility of cultured human glioma cell lines. SF stimulated DNA synthesis in 7/10 glioma cell lines and in 3/3 neuromicrovascular endothelial cell (NMVEC) lines, consistent with our previous report that SF stimulated cell proliferation of a few human glioma cell lines. SF markedly stimulated the chemotactic migration of 10/10 glioma cell lines as well as 3/3 NMVEC lines. In addition, SF stimulated the 2‐dimensional migration of glioma cells on culture surfaces coated with specific extracellular matrix molecules (collagen IV, laminin, and fibronection). As expected based on these biologic responses to SF, 10/10 glioma lines and 4/4 NMVEC lines expressed mRNA for c‐met, the SF receptor. To assess the possible in vivo significance of these migration assays, we compared the chemotactic response of a glioma cell line to human brain cyst fluids and tumor extracts that contained high or low SF concentrations. Fluids and extracts with high SF content tended to induce higher levels of chemotactic migration than did fluids and extracts with low SF content. Addition of anti‐SF monoclonal antibody (MAb) inhibited migration induced by fluids and extracts with high SF content by about 30–50%. Int. J. Cancer 75:19–28, 1998.© 1998 Wiley‐Liss, Inc.

Glioma-produced extracellular matrix influences brain tumor tropism of human neural stem cells

SummaryA major obstacle in the treatment of gliomas is the invasive capacity of the tumor cells. Previous studies have demonstrated the capability of neural stem cells (NSCs) to target these disseminated tumor cells and to serve as therapeutic delivery vehicles. Less is known about the factors involved in brain tumor tropism of NSCs and their interactions within the tumor environment. As gliomas progress and invade, an extensive modulation of the extracellular matrix (ECM) occurs. Tumor-ECM derived from six glioblastoma cell lines, ECM produced by normal human astrocytes and purified ECM compounds known to be upregulated in the glioma environment were analyzed for their effects on NSCs motility in vitro. We found that tumor-produced ECM was highly permissive for NSC migration. Laminin was the most permissive substrate for human NSC migration, and tenascin-C the strongest inducer of a directed human NSC migration (haptotaxis). A positive correlation between the degree of adhesion and migration of NSCs on different ECM compounds exists, as for glioma cells. Our in vitro data suggest that the ECM of malignant gliomas is a modulator of NSC migration. ECM proteins preferentially expressed in areas of glioma cell invasion may provide a permissive environment for NSC tropism to disseminated tumor cells.

Volume Reconstruction Techniques Improve the Correlation Between Histological and in vivo Tumor Volume Measurements in Mouse Models of Human Gliomas

Assessment of therapy efficacy using animal models of tumorigenic cancer requires the ability to accurately measure changes in tumor volume over the duration of disease course. In order to be meaningful, in vivo tumor volume measurements by non-invasive techniques must correlate with tumor volume measurements from endpoint histological analysis. Tumor volume is frequently assessed by endpoint histological analyses approximating the tumor volume with geometric primitives such as spheroids and ellipsoids. In this study we investigated alternative techniques for quantifying histological volume measurements of tumors in a xenograft orthotopic mouse model of human glioblastoma multiforme, and compared these to in vivo tumor volume measurements based on magnetic resonance imaging (MRI) data. Two techniques leveraging three-dimensional (3D) image analysis methods were investigated. The first technique involves the reconstruction of a smoothed polygonal model representing the tumor volume from histological section images and is intended for accuracy and qualitative assessment of tumor burden by visualization, while a second technique which approximates the tumor volume as a series of slabs is presented as an abbreviated process intended to produce quantitatively similar volume measurements with a minimum of effort required on behalf of the investigator. New software (QuickVol) designed for use in the first technique, is also discussed. In cases where tumor growth is asymmetric and invasive, we found that 3D analysis techniques using histological section images produced volume measurements more consistent with in vivo volume measurements based on MRI data, than approximation of tumor volume using geometric primitives. Visualizations of the volumes represented by each of these techniques qualitatively support this finding, and suggest that future research using mouse models of glioblastoma multiforme (genetically engineered or xenograft) will benefit from the use of these or similar alternative tumor volume measurement techniques.