2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Abstract

SHORT ABSTRACT This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines. LONG ABSTRACT Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

Defective membrane repair machinery impairs survival of invasive cancer cells

“…All experiments were performed in 35-mm glass bottom dishes equipped with a square patterned coverslip (MatTek, Ashland, USA). Coating was performed as previously described 54 . Briefly, the glass coverslip was first coated for 20 min at RT with 1 mg/mL gelatin, which was fixed by 0.5% glutaraldehyde for 40 min at RT.…”

Dual role for CXCL12 signaling in semilunar valve development

“…Inhibition of PI3K affects orientation of CXCR4 + OFT mesenchymal cells in response to a CXCL12 gradient We further explored the role of CXCL12/CXCR4 in direction sensing in the OFT using a modified endoMT assay (Di Martino et al, 2017;Figures 4A-4C). Wild-type E10.5 OFTs were explanted onto collagen gels and cultured to allow ingrowth of explant cells (Figure 4A).…”

DDR2 induces linear invadosome to promote angiogenesis in a fibrillar type I collagen context

“…As VEGF plays a dual role in angiogenesis initiation, vessels branching and invadosome formation (Daubon et al, 2016;Guegan et al, 2008;Seano et al, 2014aSeano et al, , 2014b, we simultaneously tested the impact of VEGF treatment on DDR2 expression and on linear invadosome formation/activity. For this assay we established four cell conditions, in presence of gelatin (a denatured form of type I collagen, not recognized by DDR2) with or without VEGF or in the presence of type I collagen (on the top of a gelatin matrix) with or without VEGF (Di Martino et al, 2017). We first measured the impact of VEGF treatment on DDR2 expression ( Figure 2A).…”

“…This protocol was performed as described in Di Martino et al (Di Martino et al, 2017). The primary antibodies diluted 1/100 in PBS1X-4%BSA: DDR2 (Cell Signaling 12133S), Tks5…”