Liver Metastasis Is Facilitated by the Adherence of Circulating Tumor Cells to Vascular Fibronectin Deposits.

The interaction between circulating tumor cells (CTC) and endothelial cells during extravasation is a critical process during metastatic colonization, but its mechanisms remain poorly characterized. Here we report that the luminal side of liver blood vessels contains fibronectin deposits that are enriched in mice bearing primary tumors and are also present in vessels from human livers affected with metastases. Cancer cells attached to endothelial fibronectin deposits via talin1, a major component of focal adhesions. Talin1 depletion impaired cancer cell adhesion to the endothelium and transendothelial migration, resulting in reduced liver metastasis formation in vivo Talin1 expression levels in patient CTC's correlated with prognosis and therapy response. Together, our findings uncover a new mechanism for liver metastasis formation involving an active contribution of hepatic vascular fibronectin and talin1 in cancer cells. Cancer Res; 77(13); 3431-41. ©2017 AACR.

How cells flow in the spreading of cellular aggregates.

Like liquid droplets, cellular aggregates, also called "living droplets," spread onto adhesive surfaces. When deposited onto fibronectin-coated glass or polyacrylamide gels, they adhere and spread by protruding a cellular monolayer (precursor film) that expands around the droplet. The dynamics of spreading results from a balance between the pulling forces exerted by the highly motile cells at the periphery of the film, and friction forces associated with two types of cellular flows: (i) permeation, corresponding to the entry of the cells from the aggregates into the film; and (ii) slippage as the film expands. We characterize these flow fields within a spreading aggregate by using fluorescent tracking of individual cells and particle imaging velocimetry of cell populations. We find that permeation is limited to a narrow ring of width ξ (approximately a few cells) at the edge of the aggregate and regulates the dynamics of spreading. Furthermore, we find that the subsequent spreading of the monolayer depends heavily on the substrate rigidity. On rigid substrates, the migration of the cells in the monolayer is similar to the flow of a viscous liquid. By contrast, as the substrate gets softer, the film under tension becomes unstable with nucleation and growth of holes, flows are irregular, and cohesion decreases. Our results demonstrate that the mechanical properties of the environment influence the balance of forces that modulate collective cell migration, and therefore have important implications for the spreading behavior of tissues in both early development and cancer.

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.

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

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.

Basement membrane invasion assays: native basement membrane and chemoinvasion assay.

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.

Light-sheet microscopy in thick media using scanned Bessel beams and two-photon fluorescence excitation.

In this study we show that it is possible to successfully combine the benefits of light-sheet microscopy, self-reconstructing Bessel beams and two-photon fluorescence excitation to improve imaging in large, scattering media such as cancer cell clusters. We achieved a nearly two-fold increase in axial image resolution and 5-10 fold increase in contrast relative to linear excitation with Bessel beams. The light-sheet penetration depth could be increased by a factor of 3-5 relative to linear excitation with Gaussian beams. These finding arise from both experiments and computer simulations. In addition, we provide a theoretical description of how these results are composed. We investigated the change of image quality along the propagation direction of the illumination beams both for clusters of spheres and tumor multicellular spheroids. The results reveal that light-sheets generated by pulsed near-infrared Bessel beams and two photon excitation provide the best image resolution, contrast at both a minimum amount of artifacts and signal degradation along the propagation of the beam into the sample.

Self-reconstructing sectioned Bessel beams offer submicron optical sectioning for large fields of view in light-sheet microscopy.

One of main challenges in light-sheet microscopy is to design the light-sheet as extended and thin as possible--extended to cover a large field of view, thin to optimize resolution and contrast. However, a decrease of the beam's waist also decreases the illumination beam's depth of field. Here, we introduce a new kind of beam that we call sectioned Bessel beam. These beams can be generated by blocking opposite sections of the beam's angular spectrum. In combination with confocal-line detection the optical sectioning performance of the light-sheet can be decoupled from the depth of field of the illumination beam. By simulations and experiments we demonstrate that these beams exhibit self-reconstruction capabilities and penetration depths into thick scattering media equal to those of conventional Bessel beams. We applied sectioned Bessel beams to illuminate tumor multicellular spheroids and prove the increase in contrast. Sectioned Bessel beams turn out to be highly advantageous for the investigation of large strongly scattering samples in a light-sheet microscope.

Wavelet-based image fusion in multi-view three-dimensional microscopy.

MOTIVATION: Multi-view microscopy techniques such as Light-Sheet Fluorescence Microscopy (LSFM) are powerful tools for 3D + time studies of live embryos in developmental biology. The sample is imaged from several points of view, acquiring a set of 3D views that are then combined or fused in order to overcome their individual limitations. Views fusion is still an open problem despite recent contributions in the field. RESULTS: We developed a wavelet-based multi-view fusion method that, due to wavelet decomposition properties, is able to combine the complementary directional information from all available views into a single volume. Our method is demonstrated on LSFM acquisitions from live sea urchin and zebrafish embryos. The fusion results show improved overall contrast and details when compared with any of the acquired volumes. The proposed method does not need knowledge of the system's point spread function (PSF) and performs better than other existing PSF independent fusion methods. AVAILABILITY AND IMPLEMENTATION: The described method was implemented in Matlab (The Mathworks, Inc., USA) and a graphic user interface was developed in Java. The software, together with two sample datasets, is available at http://www.die.upm.es/im/software/SPIMFusionGUI.zip A public release, free of charge for non-commercial use, is planned after the publication of this article.

Nodal cis-regulatory elements reveal epiblast and primitive endoderm heterogeneity in the peri-implantation mouse embryo.

Nodal, a secreted factor known for its conserved functions in cell-fate specification and the establishment of embryonic axes, is also required in mammals to maintain the pluripotency of the epiblast, the tissue that gives rise to all fetal lineages. Although Nodal is expressed as early as E3.5 in the mouse embryo, its regulation and functions at pre- and peri-implantation stages are currently unknown. Sensitive reporter transgenes for two Nodal cis-regulatory regions, the PEE and the ASE, exhibit specific expression profiles before implantation. Mutant and inhibitor studies find them respectively regulated by Wnt/ß-catenin signaling and Activin/Nodal signaling, and provide evidence for localized and heterogeneous activities of these pathways in the inner cell mass, the epiblast and the primitive endoderm. These studies also show that Nodal and its prime effector, FoxH1, are not essential to preimplantation Activin/Nodal signaling. Finally, a strong upregulation of the ASE reporter in implanting blastocysts correlates with a downregulation of the pluripotency factor Nanog in the maturing epiblast. This study uncovers conservation in the mouse blastocyst of Wnt/ß-catenin and Activin/Nodal-dependent activities known to govern Nodal expression and the establishment of polarity in the blastula of other deuterostomes. Our results indicate that these pathways act early on to initiate distinct cell-specification processes in the ICM derivatives. Our data also suggest that the activity of the Activin/Nodal pathway is dampened by interactions with the molecular machinery of pluripotency until just before implantation, possibly delaying cell-fate decisions in the mouse embryo.