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Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less alpha-SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.

We have previously shown that forskolin and 3-isobutyl-1-methylxanthine (IBMX) increased accumulation of cisplatin (DDP) in DDP-sensitive 2008 human ovarian carcinoma cells in proportion to their ability to increase cAMP. Since the major function of cAMP is to activate protein kinase A, it was conjectured that the stimulation of DDP accumulation was mediated by a protein kinase A substrate. We now show that exposure of 2008 cells to forskolin resulted in phosphorylation of a prominent 52-kD membrane protein. Microsequencing of the band demonstrated it to be human beta-tubulin. Similarly, pretreatment of 2008 cells with the microtubule stabilizing drug taxol increased platinum accumulation in a dose-dependent manner. In 11-fold DDP-resistant 2008/C13*5.25 cells, decreased DDP accumulation was associated with enhanced spontaneous formation of microtubule bundles and decreased expression of beta-tubulin and the tubulin-associated p53 antioncogene relative to 2008 cells. 2008/C13*5.25 cells had altered sensitivity to tubulin-binding drugs, being hypersensitive to taxol and cross-resistant to colchicine. We conclude that pharmacologic alterations of tubulin enhance accumulation of DDP, and that the DDP-resistant phenotype in 2008/C13*5.25 cells is associated with tubulin abnormalities.