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

Summary

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.

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.

Introduction

Extracellular matrix (ECM) remodeling occurs during physiological and pathological processes, such as angiogenesis and tumor cell invasion. In physiological conditions, many cell types, mostly from the hematopoietic lineage, are able to degrade ECM elements. For example, macrophages are able to cross anatomical barriers to reach tissues, and osteoclasts degrade bone matrix to ensure calcium homeostasis. More globally, all matrices in the body renew to maintain their physical and chemical properties. In cancer tissues, the tumor microenvironment composition is altered. For example, in breast and lung cancer, type I collagen is overexpressed and accumulates around the t....

Protocol

NOTE: Prior to fixation, all steps are completed in a sterile laminar flow hood.

1. 2D matrix

NOTE: 2D matrices are used to determine the percentage of cells forming invadosomes and the matrix degradation area per cell.

Figure 1: Protocol. Summary of the protocol used to prepare gelatin and collagen I matrices. It is possible to....

Results

Using a combination of two types of matrices, gelatin and type I collagen (Figures 1 and 4), we have highlighted a new type of invadosome, known as linear invadosomes. The labelling of these matrices allows for the observation of linear invadosome formation along collagen I fibers and of their degradation capabilities (Figure 5). The number of invasive structures can then be quantified by the macro described previously (step 4.2), and the.......

Discussion

Classically, invadosomes are studied in vitro without regard to the microenvironment and the matrix on which the cells are plated. Several types of matrices are currently used, including gelatin, fibronectin, vitronectin, or high-density fibrillar collagen (HDFC)^7,11; however, these are often not representative of the microenvironment in which cells reside and are not physiologically relevant. Here, a novel type of matrix, which consists of an associatio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gelatin solution	Sigma	G1393	Stock 20 mg/ml used at 1 mg/ml
Gelatin from pig skin Oregon green 488 conjugate	Molecular probe life technologies	G13186	5mg dilute at 1 mg/ml in sterile water
Microscope cover glasses	Marlenfeld GmbH & Co	111520	Ø12mm No.1
2.5% Glutaraldehyd in 0.1M sodium cacodylate buffer pH 7.4	Electron microscopy science	15960	Dilute at 0.5 % in sterile water
1x PBS pH 7.4	Gibco by life technologies	10010-015	Use this PBS in all steps before fixation
Collagen I Rat tail	Corning	354236
5 carboxy X rhodamin siccinimidyl ester	Life technologies	C-6125
DPBS 1x + calcium + magnesium	Gibco by life technologies	14040-091
Paraformaldehyde 16% solution	Electron microscopy science	15710	Dilute at 4% in 1X PBS
Triton X 100	Sigma	T9284
10x PBS buffer pH 7.4	Ambion	AM9625	Dilute at 1X in water Use in steps after fixation
Tks5 antibody	Santa Cruz	sc-30122	Invadosome markers Dilution : 1/100
Cortactin 4F11 antibody	Millipore	5180	Invadosome markers Dilution :1/100
DDR1 antibody	Cell signaling	5583	Linear invadosome receptor
Dilution :1/100
Phalloidin FluoProbes	Interchim	FT-AZ0330	Fibrillary actin marker Dilution :1/200
Hoechst	Sigma	33258	Nucleus marker Dilution :1/200
Secondary antibodies FluoProbes	Interchim	FP-488 FP-547H or FP-647H
Albumin from bovine serum	Sigma	A2153	Dilute at 4% in 1X PBS
Fluoromount G mounting medium	Interchim	FP 483331
ImageJ software	Public domain		http://www.macbiophotonics.ca/imagej/
Cell culture insert	Corning	353097	8.0µm pore size / 24 wells
Sodium hydroxide (NaOH)	Sigma	221465