Rapid vascular regrowth in tumors after reversal of VEGF inhibition
Michael R. Mancuso, Rachel Davis, Scott M. Norberg, Shaun O’Brien, Barbara Sennino, Tsutomu Nakahara, Virginia J. Yao, Tetsuichiro Inai, Peter Brooks, Bruce Freimark, David R. Shalinsky, Dana D. Hu-Lowe, Donald M. McDonald
Michael R. Mancuso, Rachel Davis, Scott M. Norberg, Shaun O’Brien, Barbara Sennino, Tsutomu Nakahara, Virginia J. Yao, Tetsuichiro Inai, Peter Brooks, Bruce Freimark, David R. Shalinsky, Dana D. Hu-Lowe, Donald M. McDonald
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Research Article Oncology

Rapid vascular regrowth in tumors after reversal of VEGF inhibition

Abstract

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 α–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.

Authors

Michael R. Mancuso, Rachel Davis, Scott M. Norberg, Shaun O’Brien, Barbara Sennino, Tsutomu Nakahara, Virginia J. Yao, Tetsuichiro Inai, Peter Brooks, Bruce Freimark, David R. Shalinsky, Dana D. Hu-Lowe, Donald M. McDonald

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Figure 6

Stability of vascular basement membrane during regression and regrowth of blood vessels in RIP-Tag2 tumors.

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Stability of vascular basement membrane during regression and regrowth o...
Fluorescence microscopic images of tumors showing CD31-positive endothelial cells (green), type IV collagen–positive basement membrane (red), and merged images at baseline (A), after AG-013736 treatment for 7 days (B) and after 7-day treatment and 7-day withdrawal (C). The 2 markers colocalized almost completely in untreated RIP-Tag2 tumors (A), but after AG-013736 treatment (B), CD31-positive vessels were sharply reduced but basement membrane was not, as reflected by abundant red strands (B, Merged). (C) By 7 days after treatment ended, tumor vascularity recovered, and most type IV collagen again colocalized with CD31-positive vessels (Merged). (D) Graph of area densities of CD31 and type IV collagen showing that basement membrane remained relatively constant during regression and regrowth of endothelial cells. (E) Graph showing similar rates of tumor vessel regrowth, reflected by CD31 area density, expressed as percent reduction compared with the value for untreated tumors, and disappearance of empty basement membrane sleeves, reflected by the fact that type IV collagen was unaccompanied by CD31, during 14 days after the end of AG-013736 treatment. *P < 0.05 compared with the untreated group. †P < 0.05 compared with the corresponding value for type IV collagen. Scale bar: 120 μm (A–C).