Characterization of IH tumor spheres. (A) Determination of the self renewal and differentiation capabilities for IH tumor spheres. 1) Dissociation of secondary tumor spheres to single cell colonies. 2) Tertiary tumor sphere formed from a single cell of a dissociated secondary tumor sphere. 3) Phase contrast image of differentiating tumor sphere in the presence of VEGF and its spider like morphology exhibited after 24 hours. 4) Tumor spheres differentiating into endothelial like structures after 5 to 6 days of culture in the presence of VEGF. Magnification: 100x. (B) Immunostaining of differentiating tumor spheres over time with anti-GLUT1 and anti-CD133 antibodies (top panel). The bottom panel represents fully differentiated tumor spheres. Magnification: 100 ×.