IH tumor sphere specifically secrete VEGF and Rapamycin, an mTOR/VEGF inhibitor inhibits proliferation of IH tumor stem cells. A, Concentration of VEGF secreted from tumor spheres at different days of cell culture. Every 5 days media was collected and the concentration of VEGF was evaluated by multiplex Luminex assay. Three independent measurements were performed. The result indicates that IH tumor spheres continuously secreted VEGF and that the secreted growth factor may be involved in the proliferation of the tumor spheres. B, Inhibition of growth of tumor spheres by Rapamycin. Three different concentration of Rapamycin, 1 nM, 2 nM and 4 nM was used. At days 0, 3, 6 and 10, a sample of tumor spheres was collected, dissociated with collagenase IV and number of cells were counted.