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A4200 - Endothelial Autophagy Is Necessary for Tumor Angiogenesis and Tumor Growth
Author Block: C. D. Ochoa, G. Fu, R. Wu, C. Liao, L. S. Terada; Division of Pulmonary and Critical Care, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States.
Background: Lung cancer remains the most common cause of cancer-related deaths worldwide. Lung cancer growth, invasion, and metastasis are contingent on angiogenesis; however, current anti-angiogenesis therapies offer dramatic but short-lived effects on tumor growth and are limited by systemic side effects. Thus, the discovery of novel therapeutic targets that are effective and specific against tumor angiogenesis is of the utmost necessity. Autophagy is a conserved evolutionary process by which the cell sequesters damaged macromolecules and organelles into autophagosomes and delivers them to lysosomes for degradation and recycling. Autophagy is linked to tumor development, but its role is complex and incompletely understood. More specifically, endothelial autophagy’s role in lung tumor angiogenesis is largely unknown. Methods: To address this gap in knowledge, we engineered a mouse model of defective endothelial autophagy by knocking out the autophagy-essential gene Atg5 specifically in endothelium. We crossed Atg5fl/fl mice, harboring a floxed Atg5 exon 3, with VE-cadherin-Cre mice, both on C57BL/6 backgrounds. This generated Atg5fl/fl;VE-Cre (Atg5fl/fl) and Atg5+/fl;VE-Cre (Atg5+/fl) animals. We examined tumor angiogenesis by heterotopic implantation of Lewis Lung Carcinoma 1 (LLC1) cells in the right flank of both Atg5fl/fl and Atg5+/fl mice. After 21 days, tumors were resected, and tumor weight and tumor growth rates were determined. Malignancies were subsequently evaluated for vessel density and vessel maturity following the established immunohistochemistry protocols. As Collagen I is not present in normal basement membranes but is expressed by lung cancers among other tumors, we next investigated lung endothelial Collagen I matrix invasion. Finally, we tested the lung endothelial secretion of angiogenic factors including IL-6, a known modulator of pathological angiogenesis. Results: First, adult Atg5fl/fl were phenotypically normal. Second - interestingly - at 21 days post-implantation, LLC1 tumor weight was decreased, and tumor growth rate was blunted in Atg5fl/fl mice when compared to Atg5+/fl controls. Third, tumor microvascular density was reduced in Atg5fl/fl mice when compared to Atg5+/fl littermates. Fourth, albeit the reduced angiogenesis, Atg5fl/fl tumor vessels displayed higher pericyte association indicating greater maturity. Fifth, isolated primary Atg5fl/fl lung endothelial cells demonstrated loss of invasion into Collagen I matrices when compared to Atg5+/fl mice. And sixth, Atg5fl/fl lung endothelial cells secreted less IL-6 when grown in Collagen I coated plates. Conclusion: Endothelial autophagy is not required for normal vascular homeostasis. However, tumors need endothelial autophagy for angiogenesis and growth. Additionally, autophagy is necessary for lung endothelial cell secretion of IL-6 and invasion of Collagen I matrices.