Evaluation of Vascularity, Blood Perfusion, and Oxygen Tension in Tumor Xenografts with Fluorescent Microscopy

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.