All the conclusions and observations are not surprising. The assay system they developed to analyze "second phase invadopodia" is very interesting (diagram shown above).
Chemoinvasion assay
Matrigel was purchased from BD. Laminin (Invitrogen) was labeled with Alexa Fluor 488 with a protein labeling kit (Invitrogen) according to the manufacturer’s instructions.
Transwell inserts with a 10.5-mm-diameter membrane covered with 1-μm diameter pores (BD) were coated with 80 μl with a 1:1 mix of 9–10 mg/ml Matrigel with 0.5 mg/ml of labeled or unlabeled laminin for 1 h at 4°C. 75 μl was then removed, and the inserts were put at 37°C for 30 min to allow polymerization of the Matrigel. Complete medium was added in both sides of the inserts to rehydrate the matrix before adding the cells. 4 × 10E4 MDA-MB-231 cells or 5 × 10E4 HCT116 cells were resuspended in 1 ml DME and added to the upper side of the filters. DME supplemented with 15% FCS and 40 ng/ml HGF was used as a chemoattractant. After 15-h incubation at 37°C, cells were washed with PBS and fixed with 4% paraformaldehyde. For immunostaining, the membrane was cut out of the inserts and stained from both sides according to the protocol described in Immunofluorescence. Cells were imaged with a 63× objective of an LSM 510 META laser-scanning confocal microscope. The images and z sections were processed with ImageJ and Photoshop CS6. Knockdown experiments were performed in duplicates at least three times. The length of each protrusion was calculated with Image Browser software based on phalloidin staining. Approximately 150 protrusions were analyzed per duplicate and per experiment. For each experiment, protrusions were classified into two groups according to their length: short (<5 μm) and long protrusions (> 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.
Here is the paper abstract
J Cell Biol. 2010 May 3;189(3):541-56. Epub 2010 Apr 26.
Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia.
Schoumacher M, Goldman RD, Louvard D, Vignjevic DM.Unité Mixte de Recherche 144, Centre National de la Recherche Scientifique, Paris, Cedex 05, France. marie.schoumacher@curie.fr
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.
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