Effect of ADAM28 depletion on angiogenesis and lymphangiogenesis processes

Introduction
ADAM28, a protease of the adamalysin family, is overexpressed by carcinoma cells in various cancers including non-small cell lung carcinoma (NSCLC) and breast cancer. In vivo studies reported that knockdown of ADAM28 reduced primary tumor growth and lung metastatic formation suggesting a potential pro-tumoral role of ADAM28. Although cellular mechanism remain poorly described, ADAM28 is thought to stimulate angiogenesis and thus, promoting tumor progression. Indeed, ADAM28 can cleave CTGF from the CTGF/VEGF165 complex leading to the release of VEGF165. Another known substrate of ADAM28 is von Willebrand factor (vWF) which deficiency is associated with an increased angiogenesis and vasculature density. Therefore, we could hypothesize that ADAM28 induces angiogenesis through the cleavage of vWF. All these findings suggest that ADAM28 contributes to cancer development and progression by stimulating angiogenesis.

Aim
This project aims to investigate ADAM28 functions in the lung tumor microenvironment especially in the angiogenesis using an ADAM28 conditional knockout mouse model.

Results
Angiogenesis and lymphangiogenesis were assessed in lung metastasis of both ADAM28 KO and WT mice after intravenous injection of LLC cells. Immunofluorescence staining indicated that blood vessel density was increased within lung metastasis of WT mice as compared to ADAM28 KO mice. In contrast, lymphatic vessel density was similar between both groups. In addition, endothelial cell migration was evaluated in tumor-free mice using the ex vivo aortic ring assay. Result indicated that endothelial cells from ADAM28 KO mice migrated more than endothelial cells from WT mice. We also showed that blood vessel permeability was similar in WT and ADAM28 KO mice suggesting that ADAM28 depletion don’t affect blood vessel structure. Moreover, we assessed the expression of adhesion markers (ICAM-1, VCAM-1) and found no difference between both groups.

Perspectives
All these data need to be confirmed before drawing further conclusions. Indeed, we will confirm the increased blood vessel density in lung metastasis of WT mice using other tumor mouse models. It could also be interesting to evaluate angiogenesis in primary tumor of WT and ADAM28 KO mice. We will repeat the aortic ring experiment and perform immunostaining of pericytes and endothelial cells to study in more details the structure of the newly formed blood vessels. Angiogenesis and lymphangiogenesis will be investigated using different in vivo models such as the ear sponge assay and the corneal neovascularization model which are well-established in the laboratory. Furthermore, we would like to isolate endothelial cells from the lung of WT and ADAM28 KO mice to determine ADAM28 expression level in these cells and then, perform different in vitro assays (proliferation, migration, activation).