Introduction The formation of metastases in distant organs is a critical step in cancer progression and is the major cause of mortality. To escape from the primary tumor and invade adjacent tissues, cancer cells must degrade the basement membrane (BM) that separates the epithelial and stromal compartments (Thiery, 2002). The degradation of the BM is performed by matrix metalloproteinases (MMPs). In cell culture assays, MMPs accumulate in fingerlike membrane protrusions, termed invadopodia, that form on the ventral surface of cancer cells (Chen, 1989; Linder, 2007; Poincloux et al., 2009). Invadopodia are actin-rich structures, and the actin polymerization machinery is critical for both their formation and function (Buccione et al., 2004; Lorenz et al., 2004; Yamaguchi et al., 2005; Artym et al., 2006; Baldassarre et al., 2006; Bowden et al., 2006; Weaver, 2006; Clark et al., 2007; Philippar et al., 2008; Sakurai-Yageta et al., 2008; Lizárraga et al., 2009). On a two-dimensional substratum, protrusion of the cell leading edge is driven by polymerization of actin within two structures, filopodia and lamellipodia. In lamellipodia, actin organizes into a mesh of unbundled filaments, often described as dendritic or diagonal networks (Svitkina and Borisy, 1999; Koestler et al., 2008), whereas in filopodia, actin filaments organize into parallel bundles (Gupton and Gertler, 2007; Mattila and Lappalainen, 2008). These two different types of organization rely on the action of specific actin-organizing proteins. In lamellipodia, the formin mDia2 is targeted to the plasma membrane where it may nucleate mother filaments, which then serve as a base for Arp2/3-dependent nucleation of actin branches (Yang et al., 2007), which are further stabilized by cortactin (Higgs and Pollard, 2001; Weaver et al., 2001). At the same time, any unnecessary growing barbed ends are capped by capping protein (Wear and Cooper, 2004). If barbed ends are protected from capping by mDia2 itself (Yang et al., 2007) or VASP (vasodilator-stimulated phosphoprotein; Bear et al., 2002; Trichet et al., 2008), actin filaments continue to elongate persistently and gradually converge to form filopodia. Behind the leading edge of the lamellipodium, longer unbranched filaments are cross-linked by the actin-binding protein (ABP) α-actinin, whereas in filopodia, long parallel actin filaments are tightly bundled by fascin and to some degree T-fimbrin (Svitkina et al., 2003; Vignjevic et al., 2006). Molecular traffic and signaling along filopodial shafts are mediated by the molecular motor myosinX (Sousa and Cheney, 2005; Pi et al., 2007; Mattila and Lappalainen, 2008). These two types of actin organization have distinct roles in the cell: the dendritic network in a lamellipodium produces a force that is sufficient to drive membrane protrusion and cell crawling on a planar substrate, whereas the tight actin bundle in a filopodium produces the required stiffness to form a rodlike projection that is believed to be used by the cell to explore the environment and infiltrate between small gaps. Therefore, actin bundling and the subsequent formation of fingerlike protrusions could be a general mechanism for penetration of the substratum. For example, invasive cancer cells could exploit actin dynamics by using actin bundles to form invadopodia that penetrate the BM and invade the stroma (Vignjevic and Montagnac, 2008). In support of this hypothesis, fascin, an actin-bundling protein, is widely overexpressed in invasive cancers of different cellular origins (Hashimoto et al., 2005), demonstrating specific up-regulation at the invasive tumor front (Vignjevic et al., 2007). Invadopodia formation appears to depend both on proteins involved in the generation of a dendritic actin network such as Arp2/3 (Yamaguchi et al., 2005; Baldassarre et al., 2006) and cortactin (Artym et al., 2006; Bowden et al., 2006; Clark et al., 2007) and also on proteins involved in the generation of an unbranched actin network such as VASP (Philippar et al., 2008) and mDia2 (Lizárraga et al., 2009). Consequently, the regulation of actin organization in invadopodia remains a contentious issue. The majority of studies on invadopodia have focused on the actin cytoskeleton, with comparatively little research into the other two major cytoskeletal networks consisting of microtubules and intermediate filaments. Intact microtubules are known to be required for the function of invadopodia-related structures, podosomes, formed in monocytes and osteoclasts (Kopp et al., 2006; Linder, 2007), but they do not play a role in invadopodia formation (Kikuchi and Takahashi, 2008). A connection between microtubules and MMP trafficking in invadopodia may exist because the exocytosis of MMP-2 and -9 in melanoma cells is microtubule dependent (Schnaeker et al., 2004). There is also evidence supporting fimbrin-mediated interactions between actin and vimentin filaments in the formation of podosomes (Correia et al., 1999). Studies into the formation and dynamics of invadopodia have been performed mostly on thin layers of ECM that have been coated directly onto glass (Weaver, 2006, 2008; Linder, 2007). These conditions do not accurately reflect the physiological environment because the extension of the invadopodia beyond the thin matrix layer is blocked by glass. Determining the organization and the role of the cytoskeleton during invasion is not feasible in these topologically restricted experimental conditions. Therefore, we have developed a new assay that more closely mimics the process of cancer cell invasion, permitting us to study the remodeling of the actin cytoskeleton during the formation and maturation of invadopodia. This assay has also enabled us to assess the role of other two cytoskeleton networks, microtubules and intermediate filaments, during the early stages of cancer cell invasion. Results The dissolution of the BM is a three-stage process To visualize how invasive cancer cells pass through the BM to infiltrate the subjacent stroma, we isolated a native BM from the rat peritoneum as previously described (Hotary et al., 2006). In the absence of cancer cells, no holes were present in the BM, confirming that the membranes obtained during the isolation procedure were intact (Fig. 1 A). Next, we tested the ability of different types of cancer cells to penetrate the BM. The colon cancer cell line HT29 with poor metastatic potential was unable to degrade the BM even after 4 d of culture, confirming their incapacity to invade (Fig. 1 B). On the contrary, the metastatic breast cancer cells MDA-MB-231 and colon cancer cells HCT116 succeeded in breaking the BM. The invading cells developed actin-rich protrusions on their ventral side, which corresponded to the holes in the BM (Fig. 1 C and not depicted). These protrusions were from 1 to 7 µm long and ranged from 0.5 to 2 µm in diameter (on average 1.15 ± 0.88 µm; n = 117; stages 1 and 2). In the later stages of invasion, a large area of the cell protruded through the hole (stage 3). Although most cells were in stage 1 within 1 d after the incubation, they reached stage 2 between 3 and 5 d and stage 3 after 5 d (Fig. 1 D). After 7 d, the majority of cells had moved across the BM, as previously reported (Hotary et al., 2006). Because the protrusions developed in stages 1 and 2 appeared similar to invadopodia, we determined whether they contained the same molecular components (Fig. 2). Cortactin and MT1-MMP, the two best-described invadopodial markers (Nakahara et al., 1997; Bowden et al., 1999, 2006), were also localized in the cellular protrusions developed on the native BM; although cortactin (Fig. 2 A) was present all along, MT1-MMP was located primarily at the base of these protrusions (Fig. 2 B). An active form of c-src was also detected in these protrusions, as it has been reported for invadopodia (Fig. 2 C; Chen, 1989; Buccione et al., 2004; Weaver, 2006; Linder, 2007). These observations suggest the following sequence of events as invasive cancer cells penetrate into the underlying stroma: short protrusions (henceforth referred to as invadopodia) degrade the BM (stage 1) and then elongate into longer protrusions (mature invadopodia; stage 2) that then guide the cell body to deform through the hole and enter the stromal compartment (stage 3). Figure 1. Stages of BM breakage by invasive cancer cells. (A) Mesothelial BM. (left panels) x–y images showing the top of the BM immunostained for laminin (left) or collagen IV (right). (right panels) x–z sections of the corresponding x–y projection revealing the two BM layers present in the rat peritoneum. (B and C) Cells cultured atop of peritoneal BM stained with phalloidin–Alexa Fluor 488 (green) and DAPI (blue). BM was detected by laminin staining (red). Merged images are shown in the right column. HT29 (B) and HCT116 (C) cells are shown. (top) Stage 1, early stage of invasion characterized by degradation of the BM and formation of short invasive protrusions (invadopodia). (middle) Stage 2, intermediate stage of invasion and formation of long invasive protrusions (mature invadopodia). (bottom) Stage 3, late stage of invasion and infiltration of the cell on the other side of the membrane. Asterisks indicate sites of degradation and localization of invasive protrusions. (D) Time requirement for BM penetration by HCT116 cells. Black bars show the percentage of cells in stage 1. Light gray bars show the percentage of cells in stage 2. Dark gray bars show the percentage of cells in stage 3. Bars: (A and C) 5 µm; (B) 10 µm. Figure 2. Invasive cancer cells form invadopodia on the native BM. (A–C) Localization of invadopodia markers in invasive protrusions: cortactin (A), MT1-MMP (B), and c-src (C). From left to right, columns show actin revealed by phalloidin-Cy3 (red), invadopodia markers as indicated (green), BM detected by immunostaining for laminin and collagen IV (gray), and merged images. Asterisks indicate invasive protrusions. Bars, 5 µm. In vitro assays used to mimic the formation and elongation of invadopodia To mimic stage 1 of invasion, we used a typical invadopodia assay, whereby cancer cells are cultured on a thin layer of fluorescently labeled gelatin (Artym et al., 2006, 2009). Within 5–6 h, MDA-MB-231 cells degraded the fluorescent ECM, producing dark spots in the fluorescent background. Invadopodia were defined as an accumulation of F-actin at the ventral surface of the cells that localized to the sites of matrix degradation (Fig. S1). The thickness of the gelatin layer covering the glass coverslip limited the length of the invadopodia. Thus, to mimic the second stage of invasion, we developed a new experimental technique: the chemoinvasion assay. All experiments were performed using two cell lines, MDA-MB-231 and HCT116. Cells were plated on filters containing 1-µm-diameter pores, covered by a thin layer of fluorescently labeled Matrigel to mimic the BM, and chemoattracted using serum and hepatocyte growth factor (HGF)–enriched medium. Only the first 2 µm of the pores were reliably covered and filled with Matrigel, but deeper parts were mostly open-spaced, as in all commercially coated filters (unpublished data). The pore size chosen corresponded to the mean diameter of invadopodia but was smaller than the cell nucleus. As a consequence, cells formed invadopodia without passing through the filter. In addition, the thickness of the filter (∼11 µm) permitted the elongation of invadopodia inside the pores (Fig. 3 A). In this assay, within 15 h, the cells remodeled the matrix and formed actin-rich protrusions that were an average of 6.9 ± 0.5 µm long, reaching a maximum length of ∼12 µm (Fig. 3 B). To verify that the formation of these invasive protrusions was dependent on matrix degradation, MDA-MB-231 cells were treated with a general MMP inhibitor, GM6001. After GM6001 treatment, the number of cells able to degrade the gelatin matrix was reduced by 67 ± 1%. Similarly, the number of cells forming invasive protrusions in the chemoinvasion assay was decreased by 59 ± 2% compared with control cells (Fig. 3 C), as previously shown (Ayala et al., 2008). In addition, the noninvasive HT29 cancer cells were neither able to degrade the gelatin matrix nor able to form mature invadopodia in the chemoinvasion assay (unpublished data). Therefore, the activity of MMPs was required in both assays for the formation of invadopodia. Figure 3. Invadopodia elongation and chemoinvasion assay. (A) Schematic diagram of the chemoinvasion assay. Blue lines correspond to the x–y planes shown in B. (B) HCT116 cells in the chemoinvasion assay. Fluorescent Matrigel, actin stained with phalloidin-Cy3, cortactin, and a merged image of actin and Matrigel are shown. (top) x–y projections presenting the cell on the top of the filter. (middle) x–y projections presenting the cell below the focal plane of the filter. Arrows and arrowheads indicate a short and long protrusion, respectively. On the merged picture, the white rectangular corresponds to the longitudinal cut shown in the bottom panels. (bottom) x–z projections showing longitudinal cut through the cell at the level of the protrusions. (C) Effect of the metalloprotease inhibitor GM6001 on the formation of invadopodia. (left) Gelatin assay shows the percentage of cells that degraded the gelatin. (right) Chemoinvasion assay shows the percentage of cells that formed mature invadopodia. The results were normalized to the cells treated with DMSO. Light gray bars correspond to the cells treated with 10 µM GM6001. Dark gray bars correspond to control DMSO-treated cells. Error bars indicate SEM. *, P 2 h (i.e., long-lived invadopodia; Video 1). Depletion of fascin and, even more strikingly, depletion of the Arp2/3 complex inhibited the formation of long-lived invadopodia. Most of the invadopodia from Arp2/3-depleted cells had a lifetime of 20–40 min (Video 2), whereas invadopodia from fascin-depleted cells persisted for 40–60 min. In contrast, depletion of myosinX did not significantly affect the lifetime of invadopodia. Together, these results indicate that both branched and bundled actin structures have a role in the stabilization of invadopodia. Protein distribution in mature invadopodia We hypothesize that lamellipodia- and filopodia-associated molecules cooperate in the formation of invadopodia: the Arp2/3 complex and mDia2 could be required for the generation of actin filaments, which are further elongated by mDia2, VASP, and myosinX and ultimately bundled by fascin and T-fimbrin. This would permit invadopodia to protrude through the degraded BM to reach the stroma. The increase in the spatial resolution gained using the chemoinvasion assay made it possible to analyze the distribution of actin-associated molecules along mature invadopodia (stage 2) by immunostaining or expression of GFP-tagged proteins (Fig. 6 and Fig. S2). We verified that soluble GFP was not enriched in mature invadopodia, indicating that enrichment of the GFP-tagged proteins in such protrusions was not simply a result of diffusion (unpublished data). The length of mature invadopodia was determined by staining for actin. The protrusions were classified into two groups according to their length: short ( 5 µm). We first examined the distribution of lamellipodia markers. Cortactin was localized throughout the protrusions independently of their length. In HCT116 cells, Arp2/3 colocalized with actin all along the length of short protrusions (n = 25), whereas in the majority of long protrusions (75%; n = 26), it was limited to their base (Fig. 6 A and Fig. S3 B). In MDA-MB-231 cells, the Arp2/3 complex was localized along the shafts of the majority of mature invadopodia (90%; n = 34; Figs. S2 and S3 A). Although the extension of the actin branched network generated by the Arp2/3 complex remained variable and cell type dependent, the Arp2/3 complex was never enriched at the tip of the protrusions. We next analyzed the localization of α-actinin, VASP, and mDia2 as common markers of lamellipodia and filopodia (Fig. 6, A and B). α-Actinin and VASP were present along the shaft of mature invadopodia independently of their length. Because VASP can bind to actin barbed ends, its distribution could reveal fluctuations in the length of actin filaments within the protrusion. Although full-length mDia2 was mostly cytoplasmic, as reported previously (Alberts, 2001; Yang et al., 2007), careful analysis showed that it was localized all along the shafts of invadopodia (Fig. 6 B) and sometimes enriched at their tips. However, an active form of mDia2 (ΔGBD-mDia2) that exhibited relatively low cytoplasmic fluorescence localized at the tips of the majority of long mature invadopodia in MDA-MB-231 cells (Figs. S2 C and S3 A). In HCT116 cells, the distribution of ΔGBD-mDia2 was heterogeneous (Figs. S2 D and S3 B). Figure 6. The elongation of invadopodia relies on lamellipodial and filopodial machinery. (A and B) Immunofluorescence analysis of HCT116 cells in the chemoinvasion assay: spatial distribution of lamellipodial and filopodial markers in mature invadopodia. (left) x–y projections of the cell at the focal plane of the filter. The image is merged, showing actin revealed by phalloidin-Cy3 and the specified ABPs (green). Insets show the x–y projections of the ventral surface of the cell at the focal planes below the filter. Arrows indicate invasive protrusions. (right) z projections of the indicated protrusion. A, actin (red); ABP, ABP as specified in the left panels (green); M, merge. (A) ABPs associated with lamellipodia revealed by immunostaining (cortactin and p34 subunit of Arp2/3 complex) or by expression of GFP fusion proteins (VASP and α-actinin). (B) ABPs associated with filopodia visualized by immunostaining (myosinX) or by expression of GFP fusion proteins (fascin, T-fimbrin, and mDia2). For the visualization of fascin, the nonphosphorylatable mutant (S39A) was used. (C and D) Effect of the depletion of lamellipodial and filopodial machinery on the elongation of invadopodia in MDA-MB-231 cells in the chemoinvasion assay. (C, left) Immunoblot analysis after siRNA treatment. Scrambled siRNA served as a control for the nonspecific cell response (si-control). Tubulin served as a loading control. (right) mRNA expression levels of mDia2 in the cells transfected with two independent siRNA against mDia2 and control siRNA. β-Actin mRNA levels were used as a loading control. (D) Repartition of the protrusions according to their length in cells transfected with siRNA as indicated. Light gray bars show short protrusions ( 5 µm). The dashed line (50%) is shown to compare the distribution of protrusion lengths between control and siRNA-treated cells. *, P 2.5 µm). *, P 5 µm). *, P 80% depleted of vimentin. Both approaches demonstrated that vimentin filaments are required for the elongation of invadopodia. Finally, we asked whether keratin filaments have an overlapping function with vimentin in invadopodia elongation. We found that vimentin depletion from a keratin-free cell line, MDA-MB-435, did not have an additional effect on invadopodia length (Fig. S5, D–F). In conclusion, elongation of invadopodia is dependent on an intact vimentin network. Figure 9. Localization and role of intermediate filaments during invadopodia formation and elongation. (A) Gelatin degradation assay using MDA-MB-231 cells. (top) Localization of cytokeratins. From left to right, fluorescently labeled gelatin, actin (phalloidin-Cy3), cortactin, and cytokeratin filaments are shown. (bottom) Localization of vimentin. From left to right, fluorescently labeled gelatin, actin (phalloidin-Cy3), the Arp2/3 complex, and vimentin filaments are shown. Insets show higher magnification images of the boxed regions. (B) Chemoinvasion assay and localization of intermediate filaments in mature invadopodia in HCT116 cells. (top) Cytokeratin filaments. (bottom) Vimentin filaments. (left) x–y projections of the cell above the focal plane of the filter. Merged images of actin and intermediate filaments (green) are shown. Insets show x–y projections of the cell below the focal plane of the filter. Arrows and asterisks indicate protrusions shorter and longer than 5 µm, respectively. (middle) z projections of the indicated protrusions. A, actin (red); B, intermediate filaments (green); M, merged image. (right) Presence or absence of intermediate filaments in invasive protrusions according to their length. (C, top) Immunofluorescence analysis after depletion of vimentin filaments by siRNA in MDA-MB-231 cells. DAPI (left), vimentin (middle), and a merged image (right) are shown. si-control indicates cells treated with a scrambled siRNA; si-vim1 and si-vim2 indicate cells treated with siRNAs against vimentin. (bottom) Immunoblot analysis after siRNA treatment. Scrambled siRNA served as a control for the nonspecific cell response (si-control). Tubulin served as a loading control. (D) Repartition of the protrusions according to their length in MDA-MB-231 cells treated with the indicated siRNA. Light gray bars show short protrusions ( 5 µm). *, P 5 µm). *, P 5 µm). The mean repartition between all experiments was then calculated. Statistical analyses were performed with SigmaStat 3.0 software. Kruskal-Wallis one-way analysis of variance on ranks was used, and differences between the groups were considered significant if the p-value was <0.001. Multiple comparisons versus si-control cells were checked by the Holm-Sidak method with an overall significance level of 0.05. EM For EM experiments, cell density was increased to 2 × 105 cells per insert (1-µm pore filters). All chemical components were purchased from Electron Microscopy Sciences. Cells were fixed for at least 2 h in 2% glutaraldehyde in cacodylate acid, pre- or postextracted for 30 s in the extraction buffer with 1% Triton X-100, and postfixed for 20 min with 1 mg/ml tannic acid in H2O. Cells were stained with 0.2% osmium in H2O for 20 min on ice and 20 min with 2 mg/ml uranyl acid in H2O. To detect cells in semithin sections, cells were colored with 0.6% toluidine blue. Membranes were then progressively dehydrated in ethanol, poststained for 20 min with 2 mg/ml uranyl acid in 100% ethanol, and then washed in 100% ethanol and embedded in epon (Araldite502/Embed-812 embedding media; Electron Microscopy Sciences) according to the manufacturer’s recommendations. Blocks were cut as ultrathin longitudinal sections and put on 100-mesh grids coated with 0.8% Formvar. Samples were contrasted with a solution of lead-citrate, rinsed with H2O, and observed with a transmission electron microscope (Philips CM120; FEI Company). Online supplemental material Fig. S1 shows that lamellipodial and filopodial markers are present in invadopodia. Fig. S2 shows the spatial distribution of lamellipodial and filopodial markers in mature invadopodia. Fig. S3 shows the frequency of localization of lamellipodial and filopodial markers and their role in mature invadopodia. Fig. S4 shows additional electron micrographs of mature invadopodia. Fig. S5 shows localization and a role of microtubules and vimentin in mature invadopodia. Videos 1 and 2 show F-actin dynamics in MDA-MB-231 cells plated on fluorescent gelatin and treated with control or p34 siRNA, respectively. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200909113/DC1.
