Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells. Filopodial bundles arose not by a specific nucleation event, but by reorganization of the lamellipodial dendritic network analogous to fusion of established filopodia but occurring at the level of individual filaments. Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Λ-precursors. An early marker of initiation was the gradual coalescence of GFP-vasodilator-stimulated phosphoprotein (GFP-VASP) fluorescence at the leading edge into discrete foci. The GFP-VASP foci were associated with Λ-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of Λ-precursors initiated filament bundling and completed formation of the nascent filopodium. We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t 1/2 < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.

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.tissue mechanics | microfluidics | tumor growth | mechanotransduction

Metastasis begins with the invasion of tumor cells into the stroma and migration toward the blood stream. Human pathology studies suggest that tumor cells invade collectively as strands, cords and clusters of cells into the stroma, which is dramatically reorganized during cancer progression. Cancer cells in intravital mouse models and in vitro display many 'modes' of migration, from single isolated cells with round or elongated phenotypes to loosely-/non-adherent 'streams' of cells or collective migration of cell strands and sheets. The tumor microenvironment, and in particular stroma organization, influences the mode and dynamics of invasion. Future studies will clarify how the combination of stromal network structure, tumor cell signaling and extracellular signaling cues influence cancer cell migration and metastasis.

Microtubules and intermediate filaments cooperate with actin and other components of filopodia during invadopodia maturation.

In most instances, the growth of solid tumors occurs in constrained environments and requires a competition for space. A mechanical crosstalk can arise from this competition. In this article, we dissect the biomechanical sequence caused by a controlled compressive stress on multicellular spheroids (MCSs) used as a tumor model system. On timescales of minutes, we show that a compressive stress causes a reduction of the MCS volume, linked to a reduction of the cell volume in the core of the MCS. On timescales of hours, we observe a reversible induction of the proliferation inhibitor, p27Kip1, from the center to the periphery of the spheroid. On timescales of days, we observe that cells are blocked in the cell cycle at the late G1 checkpoint, the restriction point. We show that the effect of pressure on the proliferation can be antagonized by silencing p27Kip1. Finally, we quantify a clear correlation between the pressure-induced volume change and the growth rate of the spheroid. The compression-induced proliferation arrest that we studied is conserved for five cell lines, and is completely reversible. It demonstrates a generic crosstalk between mechanical stresses and the key players of cell cycle regulation. Our results suggest a role of volume change in the sensitivity to pressure, and that p27Kip1 is strongly influenced by this change.

Cancer cells become metastatic by acquiring a motile and invasive phenotype. This step requires remodeling of the actin cytoskeleton and the expression of exploratory, sensory organelles known as filopodia. Aberrant B-catenin-TCF target gene activation plays a major role in colorectal cancer development. We identified fascin1, a key component of filopodia, as a target of B-catenin-TCF signaling in colorectal cancer cells. Fascin1 mRNA and protein expression were increased in primary cancers in a stage-dependent manner. Fascin1 was exclusively localized at the invasive front of tumors also displaying nuclear B-catenin. Forced expression of fascin1 in colorectal cancer cells increased their migration and invasion in cell cultures and caused cell dissemination and metastasis in vivo, whereas suppression of fascin1 expression by small interfering RNA reduces cell invasion. Although expression of fascin1 in primary tumors correlated with the presence of metastases, fascin1 was not expressed in metastases. Our studies show that fascin1 expression is tightly regulated during development of colon cancer metastases and is a novel target of B-catenin-TCF signaling. We propose that transient up-regulation of fascin1 in colorectal cancer promotes the acquisition of migratory and invasive phenotypes that lead to metastasis. Moreover, the expression of fascin1 is down-regulated when tumor cells reach their metastatic destination where migration ceases and proliferation is enhanced. Although metastasis to vital organs is often the cause of mortality, only limited success has been attained in developing effective therapeutics against metastatic disease. We propose that genes involved in cell migration and invasion, such as fascin1, could serve as novel targets for metastasis prevention. [Cancer Res 2007;67(14):6844-53]

The precise role of the microenvironment on tumor growth is poorly understood. Whereas the tumor is in constant competition with the surrounding tissue, little is known about the mechanics of this interaction. Using a novel experimental procedure, we study quantitatively the effect of an applied mechanical stress on the long-term growth of a spheroid cell aggregate. We observe that a stress of 10kPa is sufficient to drastically reduce growth by inhibition of cell proliferation mainly in the core of the spheroid. We compare the results to a simple numerical model developed to describe the role of mechanics in cancer progression.