Faculty Bibliography

Gliomas are malignant primary tumors of the central nervous system. Their cell-of-origin is thought to be a neural progenitor or stem cell that acquires mutations leading to oncogenic transformation. Thanks to advances in human stem cell biology, it has become possible to derive human cell types that represent putative cells-of-origin in vitro and model the gliomagenesis process by systematically introducing genetic alterations in these human cells. Here, we present methods to derive human neural stem cells (NSCs) from human embryonic stem cells (hESCs). Because these NSCs are genetically unmodified at baseline, they can be used as a cellular platform to study the effects of individual oncogenic hits in a well-controlled manner in the backdrop of a human genetic background. We also present some key applications of these NSCs, which include their transduction with lentiviral vectors, their neuroglial differentiation and xenografting methods into immunocompromised mice to assess in vivo behavior.

In vitro propagation of patient-derived glioblastoma (GBM) cells can be achieved either by adherent monolayer culture, already described in Chapter 3 , or by tumorsphere culture in suspension. Here, we provide a detailed protocol for establishing patient-derived tumorsphere cultures. Such cultures are enriched for GBM stem cells (GSCs) and can be used to generate orthotopic tumor xenografts in the brain of immunocompromised mice. We also point out nuances in the protocol that can increase the yield of successful cultures from operative specimens.

This chapter describes a straightforward method for isolating glioblastoma stem cells (GSCs) from in vitro tissue cultures via fluorescence-activated cell sorting (FACS) using CD133 as a surface marker. The use of a directly conjugated antibody to an APC fluorophore against the CD133 molecule provides sufficient and clear detection of positive cells from the rest of the population. This strategy avoids an unnecessary secondary antibody incubation step thereby minimizing loss and increasing yield. The same protocol can be applied to other GSC surface markers. The described method allows for quick and efficient purification of GSCs, which can then be used in several downstream applications.

This chapter provides detailed step-by-step instructions for the production of lentiviral particles and the transduction of primary human glioblastoma cultures. Lentiviruses stably transduce both dividing and non-dividing cells, such as quiescent cancer stem cell populations. The viral envelope is pseudotyped with the vesicular stomatitis virus envelope glycoprotein G (VSV-G), which renders the lentiviral particles pantropic, so that they can infect theoretically all cell types. The third generation packaging system used in this protocol produces lentiviruses with important safety features, including replication incompetence and self-inactivation (SIN). The protocol we describe here leads to transduction of primary human glioblastoma cultures with efficiencies of up to 90%.

Several lines of evidence suggest a cellular hierarchy in glioblastoma (GBM). In this hierarchy, GBM stem-like cells (GSCs) play critical roles in tumor progression and recurrence, by virtue of their robust tumor-propagating potential and resistance to conventional chemoradiotherapy. Therefore, targeting GSCs holds significant therapeutic promise. Expression of CD133 (PROM1), a cell surface glycoprotein, has been associated with the GSC phenotype and used as a GSC marker. Here, we describe a protocol that allows the selective lentiviral transduction of CD133-expressing GBM cells. This selectivity is conferred by pseudotyping the lentiviral envelope with a single-chain antibody against an extracellular epitope on CD133. We previously demonstrated the efficacy and specificity of this lentiviral vector using patient-derived GBM cultures. This chapter outlines the preparation of the vector and the transduction of human GBM cells.

The regulation of pH in glioblastoma (GBM) has received significant attention, because it has been linked to tumor metabolism and the stem cell phenotype. The variability in blood perfusion and oxygen tension within tumors suggests that ambient pH values fluctuate across different tumor territories. This chapter describes a detailed protocol for measuring intracellular pH in patient-derived GBM cells in vitro, using the fluorescent pH sensitive dye BCECF.

Histologic heterogeneity in glioblastoma (GBM) is highlighted by regional variability in vascular density. Areas of vascular hyperplasia are interspersed with avascular territories, in which necrosis is surrounded by a zone of hypoxic tumor cells expressing stem cell markers, a phenomenon known as pseudopalisading necrosis. This vascular heterogeneity suggests intratumoral oxygen gradients, which regulate cellular and metabolic adaptations in tumor cells. Quantification of tumor vascularity, blood perfusion and oxygenation is therefore critical. In this chapter, we describe three different methods, all of which involve microscopy to analyze these parameters in tumor xenografts. We present detailed protocols for analysis of tumor endothelium using endothelial markers, blood perfusion by systemic infusion of Evans Blue and oxygen tension by pimonidazole injection, followed by immunostaining.

Orthotopic rodent xenografts are an essential tool for studying glioblastoma in vivo. Xenograft growth as a function of time can only be monitored by noninvasive imaging. This chapter describes in detail how to assess xenograft size and growth using bioluminescent imaging with IVIS (in vivo imaging system). This form of imaging (a) can be performed without the help of a trained technician, (b) is a very quick procedure, allowing simultaneous imaging of up to five animals at a total experimental duration of 15 min, and (c) is cheaper than the alternatives (small animal MRI or CT). This technique relies on the stable expression of luciferase by the xenografted GBM cells. Luciferin, the substrate of luciferase, which is injected into host mice intraperitoneally, distributes throughout the mouse body and crosses the blood brain barrier. Luciferase expressed by the xenografted cells uses this substrate in a catalytic reaction, leading to the emission of visible light, which is detected by the CCD camera of the IVIS imaging system. The intensity of this emitted light correlates to the size of a given xenograft and allows comparisons of xenograft size across different animals, as well as within the same animal across different time points.

Patient-derived xenografts (PDX) provide in vivo glioblastoma (GBM) models that recapitulate actual tumors. Orthotopic tumor xenografts within the mouse brain are obtained by injection of GBM stem-like cells derived from fresh surgical specimens. These xenografts reproduce GBM's histologic complexity and hallmark biological behaviors, such as brain invasion, angiogenesis, and resistance to therapy. This method has become essential for analyzing mechanisms of tumorigenesis and testing the therapeutic effect of candidate agents in the preclinical setting. Here, we describe a protocol for establishing orthotopic tumor xenografts in the mouse brain with human GBM cells.

Purpose: Prior studies have shown correlation between relative cerebral blood volume (rCBV) and patient survival and tumor genomics. The purpose of this study was to determine whether rCBV values correlate with isocitrate dehydrogenase (IDH) mutation status, genome-wide CNV (copy number variation) and patient overall survival in diffuse gliomas. Materials & Methods: 107 treatment naive gliomas (62 patients from TCGA/TCIA dataset and 45 patients from our institute) (44 glioblastoma and 63 lower grade gliomas) with DSC T2* perfusion data were included. IDH mutation and survival data were assayed by the TCGA, and pre-surgical imaging collected by The Cancer Imaging Archive. CNVabundance plots obtained with Illumina 850k EPIC DNA methylation arrays were reviewed in 19 patients. The association of rCBV with tumor genomics, CNV and overall survival were analyzed. Results: IDH-wildtype gliomas (44.8%) demonstrated higher rCBV values (rCBV = 6.87 +/- 3.09) than IDH-mutated gliomas (55.2%, rCBV =2.21 +/- 1.71 for 1p/19q codeleted gliomas and 2.09 +/- 2.00 for non-codeleted gliomas, ANOVA, p<0.0001). rCBV is a significant predictor of overall survival (HR 1.23, p<0.0001). Gliomas with rCBV < 3.80 showed better survival (n = 54, median survival time unobserved) than gliomas with rCBV > 3.8 (n = 53, median 18 months; log-rank p<0.0001). IDHwt gliomas with high rCBV had the worst survival (10.6% surviving at 3 years, 95% CI (4%, 30%)). CNV-S IDHmut 1p19q noncodeleted gliomas demonstrated significantly lower mean rCBV (1.4 +/- 0.4) than CNV-U gliomas (4.0 +/- 1.1, p = 0.009). Conclusion: IDHwt gliomas show higher rCBV than IDHmut gliomas irrespective of the glioma grade. Higher rCBV measurements are associated with poorer survival in the entire cohort and also within IDHmut and IDHwt gliomas. IDHmut 1p19q noncodeleted gliomas with higher CNV abundance (CNV-U) also show higher CBV when compared with those with lesser degree of CNVabundance (CNV-S)