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Cell migration and invasion

Keywords : cell migration, actin cytoskeleton, mouse models, cancer cell invasion, tumor microenvironment, gut, wound healing, intravital imaging, cancer-associated fibroblasts, fascin. 3D in vitro assays

Group leader: Danijela Matic Vignjevic

Cell migration  and invasion

The broad objective of our research is to understand how epithelial cells interact with their microenvironment during migration, focusing on the mechanism of cell migration and the role of actin cytoskeleton in this process. We use a gut as model system to understand cell migration in homeostasis, wound healing and cancer invasion. Our research strategy combines molecular and cell biology techniques with live-cell imaging. In particular, we use 2D and 3D in vitro cell cultures; tissue slices cultured ex vivo; and different transgenic mouse models to study cell migration in the living animal.

Cell migration in gut homeostasis and during wound healing.
The entire intestinal epithelium is renewed every week due to cell division in the crypts coupled with cell migration towards the villi and loss of cells by apoptosis at the tip of villi. However, the mechanism responsible for the migration of intestinal cells remains largely unknown. Our goal is to determine if epithelial cells migrate passively as a consequence of the pushing force generated by cell division in the crypts, actively using cellular protrusions or if they are transported by underlying fibroblasts.

Role of actin cytoskeleton in cell invasion.
Uncoupling cell proliferation from apoptosis and possibly from cell migration can lead to pathologies such as cancer. In carcinoma in situ, the basement membrane (BM) represents a physical barrier that prevents spreading of the primary tumor to adjacent tissues. We have shown that breaching of the BM is a three-stage process: specialized, finger-like protrusions, called invadopodia first perforate BM, then elongate into mature invadopodia that finally guide the cell towards the stromal compartment (Figure 1A). Formation of invadopodia requires only the actin cytoskeleton, while invadopodia elongation also depends on intact microtubules and vimentin intermediate filament networks (Figure 1B). We are investigating the role of different actin binding proteins in invadopodia formation. For example, by stabilizing actin filaments into bundles, we found that fascin is required for formation and maintenance of invadopodia (Schoumacher et al. 2010). In agreement with its role in invasion, we found that fascin is highly expressed at the invasive front of human colon adenocarcinoma while completely absent from normal epithelial cells (Vignjevic et al, 2007) (Figure 1C). To test if fascin expression can induce metastasis, we have generated a conditional fascin transgenic mouse and crossed it with mice that develop spontaneous benign tumors in the gut (Schoumacher et al. in preparation).


Figure 1. Actin cytoskeleton in cell invasion.: A) Colon cancer cells (green) invading native BM (red): invadopodia form (top), elongate (middle) and cell protrude (bottom). Image: M. Schoumacher. B) Electron micrograph of invadopodia: actin (yellow), microtubules (red) and intermediate filaments (blue). Image: D. Vignjevic. C) Fascin expression (brown) in normal human colon tissue and adenocarcinoma. Image: D. Vignjevic.Figure 1. Actin cytoskeleton in cell invasion.: A) Colon cancer cells (green) invading native BM (red): invadopodia form (top), elongate (middle) and cell protrude (bottom). Image: M. Schoumacher. B) Electron micrograph of invadopodia: actin (yellow), microtubules (red) and intermediate filaments (blue). Image: D. Vignjevic. C) Fascin expression (brown) in normal human colon tissue and adenocarcinoma. Image: D. Vignjevic.


Role of actin cytoskeleton in cell migration.

Cells initiate migration by extending membrane protrusions, lamellipodia and filopodia, that are driven by actin polymerization. We are investigating if filopodia are sensory organelles responsible for directed cell migration using 2D and 3D chemotactic chambers. Newly extended cellular protrusions are then stabilized by adhesions that link the actin cytoskeleton to the underlying extracellular matrix. Cells move forward by exerting traction forces on these adhesions at the cell front while adhesions at the cell rear must be released to allow cell translocation. We are interested in how tensile forces generated by stress fiber contraction, strengthen adhesions at the cell front but disassemble adhesions at the cell rear. Finally, using two-photon microscopy we are studying how cells migrate in and interact with complex environments in the living mice (Figure 2A). For example, recently we have explored the appearance of focal adhesions assembled in vitro and in vivo (Geraldo et al, 2012) (Figure 2B).


Figure 2. Actin cytoskeleton in cell migration.: A) Cancer cells (green) invade extracellular matrix (pink) in the living mouse observed by two-photon microscopy. Image: S. Geraldo. B) Focal adhesions (green) in cancer cells migrating in vitro in 2D and 3D collagen I matrices (pink) or in vivo. Image: S. Geraldo and A. Simon.Figure 2. Actin cytoskeleton in cell migration.: A) Cancer cells (green) invade extracellular matrix (pink) in the living mouse observed by two-photon microscopy. Image: S. Geraldo. B) Focal adhesions (green) in cancer cells migrating in vitro in 2D and 3D collagen I matrices (pink) or in vivo. Image: S. Geraldo and A. Simon.


Cooperation of cancer cells and fibroblasts during invasion.

It is believed that cancer cells perforate BM, but stromal cells such as carcinoma-associated fibroblasts (CAFs) also secrete matrix proteinases (Figure 3A). Our objective is to understand who is invading whom – do cancer cells invade the stroma or is the stroma invading tumor cells? We are studying if cancer cells and fibroblasts have overlapping or distinct functions that need to be combined to perforate the BM (Figure 3B). Once the BM becomes compromised, cancer cells migrate through the stroma towards the blood vessels, allowing dissemination of the tumor and formation of metastasis. Using multicellular spheroids of cancer cells embedded in 3D collagen matrices, we are investigating how CAFs stimulate invasion of cancer cells (Figure 3C). Finally, using orthotopic implantation of cancer cells into mouse colon wall we are investigating if CAFs can promote formation of metastasis.
Figure 3. Cooperation of CAFs and cancer cells in invasion.: A) Cancer cells and CAFs (red and blue) separated by native BM (green). Image: A. Glentis and V. Gurchenkov. B) Multicellular cancer spheroids (actin in blue). Image: V. Gurchenkov and K. Alessandri. C) Multicellular cancer spheroids (red) embeaded in collagen I containing CAFs (yellow). Image: A. Glentis.Figure 3. Cooperation of CAFs and cancer cells in invasion.: A) Cancer cells and CAFs (red and blue) separated by native BM (green). Image: A. Glentis and V. Gurchenkov. B) Multicellular cancer spheroids (actin in blue). Image: V. Gurchenkov and K. Alessandri. C) Multicellular cancer spheroids (red) embeaded in collagen I containing CAFs (yellow). Image: A. Glentis.


Invasion of cancer cells induced by mechanical pressure.

In addition to cellular and biochemical tumor microenvironment, mechanical stress also plays a crucial role in tumor growth. In collaboration with physicists P. Nassoy (Institut d’Optique Graduate School, Talence) and G. Cappello (UMR168, Institute Curie) we are investigating if pressure imposed by stroma can have an additional role in stimulating cancer invasion.

Current fundingCurrent funding

Key publications

  • Year of publication : 2014

  • While absent from normal epithelia, an actin bundling protein, fascin, becomes expressed in invasive carcinoma of different origins. It is highly enriched at the tumors' invasive front suggesting that it could play a role in cancer invasion. Multiple studies have shown that fascin, through its role in formation of cellular protrusions such as filopodia and invadopodia, enhances cancer cell migration and invasion in vitro. However, the role of fascin in vivo remains unknown. We have generated a compound transgenic mouse model that allows expression of fascin in the intestinal epithelium in the Apc-mutated background. Conditional expression of fascin led to decrease in mice survival and increase in tumor burden compared to control animals. Induction of fascin expression in adult tumor-bearing animals accelerated tumor progression and led to formation of invasive adenocarcinoma. Altogether, our study shows that fascin can promote tumor progression in vivo, but also unravels an unexpected role of fascin in tumor initiation.

  • Migrating cells nucleate focal adhesions (FAs) at the cell front and disassemble them at the rear to allow cell translocation. FAs are made of a multiprotein complex, the adhesome, which connects integrins to stress fibers made of mixed-polarity actin filaments [1-5]. Myosin II-driven contraction of stress fibers generates tensile forces that promote adhesion growth [6-9]. However, tension must be tightly controlled, because if released, FAs disassemble [3, 10-12]. Conversely, excess tension can cause abrupt cell detachment resulting in the loss of a major part of the adhesion [9, 12]. Thus, both adhesion growth and disassembly depend on tensile forces generated by stress fiber contraction, but how this contractility is regulated remains unclear. Here, we show that the actin-bundling protein fascin crosslinks the actin filaments into parallel bundles at the stress fibers' termini. Fascin prevents myosin II entry at this region and inhibits its activity in vitro. In fascin-depleted cells, polymerization of actin filaments at the stress fiber termini is slower, the actin cytoskeleton is reorganized into thicker stress fibers with a higher number of myosin II molecules, FAs are larger and less dynamic, and consequently, traction forces that cells exert on their substrate are larger. We also show that fascin dissociation from stress fibers is required to allow their severing by cofilin, leading to efficient disassembly of FAs.

  • Basement membranes are thin sheets of self-assembled extracellular matrices that are essential for embryonic development and for the homeostasis of adult tissues. They play a role in structuring, protecting, polarizing, and compartmentalizing cells, as well as in supplying them with growth factors. All basement membranes are built from laminin and collagen IV networks stabilized by nidogen/perlecan bridges. The precise composition of basement membranes, however, varies between different tissues. Even though basement membranes represent physical barriers that delimit different tissues, they are breached in many physiological or pathological processes, including development, the immune response, and tumor invasion. Here, we provide a brief overview of the molecular composition of basement membranes and the process of their assembly. We will then illustrate the heterogeneity of basement membranes using two examples, the epithelial basement membrane in the gut and the vascular basement membrane. Finally, we examine the different strategies cells use to breach the basement membrane.

  • Year of publication : 2013

  • Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell-cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.

  • To escape the primary tumor and infiltrate stromal compartments, invasive cancer cells must traverse the basement membrane (BM). To break this dense matrix, cells develop finger-like protrusions, called invadopodia, at their ventral surface. Invadopodia secrete proteases to degrade the BM, and then elongate which allows the cell to invade the subjacent tissue. Here, we describe two complementary invasion assays. The native BM invasion assay, based on BM isolated from rat or mouse mesentery, is a physiologically significant approach for studying the stages of BM crossing at the cellular level. The Matrigel-based chemoinvasion assay is a powerful technique for studying invadopodia's molecular composition and organization at the subcellular level.

  • Year of publication : 2012

  • 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 disassembling 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 exist. Using 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, implying 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 lines. Using 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 adhesions 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.

  • Year of publication : 2010

  • Invasive cancer cells are believed to breach the basement membrane (BM) using specialized protrusions called invadopodia. We found that the crossing of a native BM is a three-stage process: invadopodia indeed form and perforate the BM, elongate into mature invadopodia, and then guide the cell toward the stromal compartment. We studied the remodeling of cytoskeleton networks during invadopodia formation and elongation using ultrastructural analysis, spatial distribution of molecular markers, and RNA interference silencing of protein expression. We show that formation of invadopodia requires only the actin cytoskeleton and filopodia- and lamellipodia-associated proteins. In contrast, elongation of invadopodia is mostly dependent on filopodial actin machinery. Moreover, intact microtubules and vimentin intermediate filament networks are required for further growth. We propose that invadopodia form by assembly of dendritic/diagonal and bundled actin networks and then mature by elongation of actin bundles, followed by the entry of microtubules and vimentin filaments. These findings provide a link between the epithelial to mesenchymal transition and BM transmigration.