Faculty Bibliography

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.

The resection of deep-seated brain tumors has been associated with morbidity due to injury to critical neural structures during the approach. Recent technological advancements in navigation and stereotaxy, surgical planning, brain tractography and minimal-access brain ports present the opportunity to overcome such limitations. Here, we present the case of a pediatric patient with a left thalamic/midbrain juvenile pilocytic astrocytoma (JPA). The tumor displaced the corticospinal fibers posteriorly and resulted in hemiparesis. Using whole brain tractography to plan a corridor for the approach, neuronavigation, a tubular retractor and an exoscope for visualization, we obtained gross total resection of the tumor, while minimizing injury to white matter bundles, including the corticospinal fibers. We propose that surgical planning with whole brain tractography is essential for reducing morbidity while accessing deep-lying brain lesions via retractor tubes, by means of sparing critical fiber tracts.