Do cancer cells have distinct adhesions in 3D collagen matrices and in vivo

European Journal of Cell Biology 91 (2012) 930–937

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European Journal of Cell

Biology

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /e j c

b

Do cancer cells have distinct adhesions in 3D collagen matrices and in vivo?

Sara Geraldo a ,b ,Anthony Simon a ,b ,Nadia Elkhatib a ,b ,Daniel Louvard a ,b ,Luc Fetler a ,c ,Danijela M.Vignjevic a ,b ,∗

a

Institut Curie,Paris 75005,France b

UMR144/CNRS,France c

UMR168/CNRS,France

a r t i c l e

i n f o

Article history:

Received 9March 2012

Received in revised form 24July 2012Accepted 25July 2012

Keywords:

Focal adhesions 3D matrix Vinculin Collagen

Intravital imaging

a b s t r a c t

During metastasis,cancer cells breach the basement membrane and migrate through the stroma mostly composed of a network of collagen I fibers.Cell migration on 2D is initiated by protrusion of the cell membrane followed by formation of adhesions that link the actin cytoskeleton to the extracellular matrix (ECM).Cells then move forwards by exerting traction forces on the adhesions at its front and by disassem-bling adhesions at the rear.In 2D,only the ventral surface of a migrating cell is in contact with the ECM,where cell–matrix adhesions are assembled.In 3D matrices,even though the whole surface of a migrating cell is available for interacting with the ECM,it is unclear whether discrete adhesion structures actually ing high-resolution confocal microscopy we imaged the endogenous adhesome proteins in three different cancer cell types embedded in non-pepsinized collagen type I,polymerized at a slow rate,to allow the formation of a network that resembles the organization of EMC observed in vivo.Vinculin aggregates were detected in the cellular protrusions,frequently colocalizing with collagen fibers,imply-ing they correspond to adhesion structures in 3D.As the distance from the substrate bottom increases,adhesion aggregates become smaller and almost undetectable in some cell ing intravital imaging we show here,for the first time,the existence of adhesome proteins aggregates in vivo.These aggregates share similarities with the ones found in 3D collagen matrices.It still remains to be determined if adhe-sions assembled in 3D and in vivo share functional similarities to the well-described adhesions in 2D.This will provide a major step forward in understanding cell migration in more physiological environments.

© 2012 Elsevier GmbH. All rights reserved.

Introduction

In order to escape the primary tumor and reach the blood ves-sels,cancer cells encounter different ECMs during their course of invasive migration.First they breach the basement membrane,a thin and dense sheet-like structure composed of a network of col-lagen IV and laminin (Yurchenco,2011).Then they migrate through the stroma composed of fibrillar collagens,proteoglycans and var-ious glycoproteins (Boot-Handford and Tuckwell,2003;Egeblad et al.,2010;Naba et al.,2012).

Cell migration is a continuous process,but for simplicity,it is often described in four individual steps.First,actin polymerization drives protrusions at the cell leading edge;second,those newly extended protrusions become stabilized by adhering to the extra-cellular matrix (ECM);third,the cell moves forward by exerting

∗Corresponding author at:Equipe de Morphogenese et Signalisation cellulaires,UMR 144CNRS/Institut Curie,Institut Curie,25rue d’Ulm,75248Paris cedex 05,France.Tel.:+330142346366;fax:+330142346377.

E-mail address:danijela.vignjevic@curie.fr (D.M.Vignjevic).

traction forces at the adhesion points;and fourth,adhesions at the cell rear are released enabling translocation of the cell body.

However,most normal or cancer cells migrating in two-dimensional (2D)and three-dimensional (3D)substrates differ in their morphology and modes of migration (Petrie et al.,2009;Meyer et al.,2012).Cells migrating on planar 2D substrates,either plain or coated with ECM proteins such as laminin,collagen I or fibronectin,develop broad and flat protrusions called lamellipo-dia and finger-like protrusions called filopodia (Pollard and Borisy,2003;Vignjevic and Montagnac,2008).The same cells growing in 3D matrices are more elongated and while cellular protrusions that resemble lamellipodia and filopodia are still observed,they are smaller in size and in fewer numbers (Wirtz et al.,2011).This change in morphology is partly due to differences in dimensional-ity of 2D and 3D substrates.While in 2D only the ventral surface of a cell migrating is in contact with the ECM,in 3D the whole surface of a migrating cell is contacting ECM proteins.Those differences in cell morphology and cell–matrix interfaces have probably a major role in determining the mechanism and rates of cells migrating in different environments.

Adhesion to the ECM is mostly achieved by cell surface recep-tors called integrins.Intracellularly,integrins are connected to the

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