Danijela Matic Vignjevic

Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia

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

Fascin, a Novel Target of β-Catenin-TCF Signaling, Is Expressed at the Invasive Front of Human Colon Cancer

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 beta-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 beta-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 beta-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 beta-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.

Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro

Significance Tumor growth intrinsically generates pressure onto the surrounding tissues, which conversely compress the tumor. These mechanical forces have been suggested to contribute to tumor growth regulation. We developed a microfluidic technique to produce 3D cell-based assays and to interrogate the interplay between tumor growth and mechanics in vitro. Multicellular spheroids are grown in permeable elastic capsules. Capsule deformation provides a direct measure of the exerted pressure. By simultaneously imaging the spheroid by confocal microscopy, we show that confinement induces a drastic cellular reorganization, including increased motility of peripheral cells. We propose that compressive stress has a beneficial impact on slowing down tumor evolution but may have a detrimental effect by triggering cell invasion and metastasis. 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.

Compressive Stress Inhibits Proliferation in Tumor Spheroids through a Volume Limitation

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.

In vivo Tumor Targeting Using a Novel Intestinal Pathogen-Based Delivery Approach

Efficient methods for tumor targeting are eagerly awaited and must satisfy several challenges: molecular specificity, transport through physiologic barriers, and capacity to withstand extracellular or intracellular degradation and inactivation by the immune system. Through interaction with its hosts, the intestinal pathogen-produced Shiga toxin has evolved molecular properties that are of interest in this context. Its nontoxic B-subunit binds to the cellular toxin receptor, glycosphingolipid Gb3, which is highly expressed on human cancers and has recently been reported to be involved in the formation of metastasis in colorectal cancers. Its function as a target for cancer therapy has already been addressed in xenograft experiments. We here show that after oral or i.v. injections in mice, the B-subunit targets spontaneous digestive Gb3-expressing adenocarcinomas. The nontumoral mucosa is devoid of labeling, with the exception of rare enteroendocrine and CD11b-positive cells. As opposed to other delivery tools that are often degraded or recycled on cancer cells, the B-subunit stably associates with these cells due to its trafficking via the retrograde transport route. This can be exploited for the in vivo delivery of contrast agents to tumors, as exemplified using fibered confocal fluorescence endoscopy and positron emission tomography (PET) imaging. In conclusion, the data presented in this manuscript lay the groundwork for a novel delivery technology that, in addition to its use for molecular imaging applications such as noninvasive PET, could also be exploited for targeted tumor therapies.

Assembly, heterogeneity, and breaching of the basement membranes

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.

Enterocyte loss of polarity and gut wound healing rely upon the F-actin–severing function of villin

Significance Intestinal epithelium damage is common but becomes recurrent in chronic intestinal disorders. Healing implies cell migration, which necessitates extensive cellular reorganization. We demonstrate that intestinal epithelial cells completely disassemble their apical actin-based microvilli upon migration, and we identify the protein villin and its actin-severing function as responsible for this physiological process. We show that this apical pole effacement is required for the acquisition of a motile phenotype and efficient wound healing. These findings demonstrate how intestinal epithelial cells acquired a mechanism at the level of the actin cytoskeleton to convert efficiently from a highly differentiated to a motile polarity. Efficient wound healing is required to maintain the integrity of the intestinal epithelial barrier because of its constant exposure to a large variety of environmental stresses. This process implies a partial cell depolarization and the acquisition of a motile phenotype that involves rearrangements of the actin cytoskeleton. Here we address how polarized enterocytes harboring actin-rich apical microvilli undergo extensive cell remodeling to drive injury repair. Using live imaging technologies, we demonstrate that enterocytes in vitro and in vivo rapidly depolarize their microvilli at the wound edge. Through its F-actin–severing activity, the microvillar actin-binding protein villin drives both apical microvilli disassembly in vitro and in vivo and promotes lamellipodial extension. Photoactivation experiments indicate that microvillar actin is mobilized at the lamellipodium, allowing optimal migration. Finally, efficient repair of colonic mechanical injuries requires villin severing of F-actin, emphasizing the importance of villin function in intestinal homeostasis. Thus, villin severs F-actin to ensure microvillus depolarization and enterocyte remodeling upon injury. This work highlights the importance of specialized apical pole disassembly for the repolarization of epithelial cells initiating migration.

Cancer-associated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane

At the stage of carcinoma in situ, the basement membrane (BM) segregates tumor cells from the stroma. This barrier must be breached to allow dissemination of the tumor cells to adjacent tissues. Cancer cells can perforate the BM using proteolysis; however, whether stromal cells play a role in this process remains unknown. Here we show that an abundant stromal cell population, cancer-associated fibroblasts (CAFs), promote cancer cell invasion through the BM. CAFs facilitate the breaching of the BM in a matrix metalloproteinase-independent manner. Instead, CAFs pull, stretch, and soften the BM leading to the formation of gaps through which cancer cells can migrate. By exerting contractile forces, CAFs alter the organization and the physical properties of the BM, making it permissive for cancer cell invasion. Blocking the ability of stromal cells to exert mechanical forces on the BM could therefore represent a new therapeutic strategy against aggressive tumors.Stromal cells play various roles in tumor establishment and metastasis. Here the authors, using an ex-vivo model, show that cancer-associated fibroblasts facilitate colon cancer cells invasion in a matrix metalloproteinase-independent manner, likely by pulling and stretching the basement membrane to form gaps.

The hallmarks of CAFs in cancer invasion.

The ability of cancer cells to move out of the primary tumor and disseminate through the circulation to form metastases is one of the main contributors to poor patient outcome. The tumor microenvironment provides a niche that supports cancer cell invasion and proliferation. Carcinoma-associated fibroblasts (CAFs) are a highly enriched cell population in the tumor microenvironment that plays an important role in cancer invasion. However, it remains unclear whether CAFs directly stimulate cancer cell invasion or they remodel the extracellular matrix to make it more permissive for invasion. Here we discuss paracrine communication between cancer cells and CAFs that promotes tumor invasion but also stimulates CAFs to remodel the matrix increasing cancer dissemination.

Cancer-associated fibroblasts lead tumor invasion through integrin-β3–dependent fibronectin assembly

Cancer-associated fibroblasts (CAFs) are the most abundant cells of the tumor stroma. Their capacity to contract the matrix and induce invasion of cancer cells has been well documented. However, it is not clear whether CAFs remodel the matrix by other means, such as degradation, matrix deposition, or stiffening. We now show that CAFs assemble fibronectin (FN) and trigger invasion mainly via integrin-&agr;v&bgr;3. In the absence of FN, contractility of the matrix by CAFs is preserved, but their ability to induce invasion is abrogated. When degradation is impaired, CAFs retain the capacity to induce invasion in an FN-dependent manner. The level of expression of integrins &agr;v and &bgr;3 and the amount of assembled FN are directly proportional to the invasion induced by fibroblast populations. Our results highlight FN assembly and integrin-&agr;v&bgr;3 expression as new hallmarks of CAFs that promote tumor invasion.