Cancer Cell Biomechanical Properties Accompany Tspan8-Dependent Cutaneous Melanoma Invasion.

The intrinsic biomechanical properties of cancer cells remain poorly understood. To decipher whether cell stiffness modulation could increase melanoma cells' invasive capacity, we performed both in vitro and in vivo experiments exploring cell stiffness by atomic force microscopy (AFM). We correlated stiffness properties with cell morphology adaptation and the molecular mechanisms underlying epithelial-to-mesenchymal (EMT)-like phenotype switching. We found that melanoma cell stiffness reduction was systematically associated with the acquisition of invasive properties in cutaneous melanoma cell lines, human skin reconstructs, and Medaka fish developing spontaneous MAP-kinase-induced melanomas. We observed a systematic correlation of stiffness modulation with cell morphological changes towards mesenchymal characteristic gains. We accordingly found that inducing melanoma EMT switching by overexpressing the ZEB1 transcription factor, a major regulator of melanoma cell plasticity, was sufficient to decrease cell stiffness and transcriptionally induce tetraspanin-8-mediated dermal invasion. Moreover, ZEB1 expression correlated with Tspan8 expression in patient melanoma lesions. Our data suggest that intrinsic cell stiffness could be a highly relevant marker for human cutaneous melanoma development.

ßig-h3-structured collagen alters macrophage phenotype and function in pancreatic cancer.

Macrophages play an important role in immune and matrix regulation during pancreatic adenocarcinoma (PDAC). Collagen deposition massively contributes to the physical and functional changes of the tissue during pathogenesis. We investigated the impact of thick collagen fibers on the phenotype and function of macrophages. We recently demonstrated that the extracellular protein ßig-h3/TGFßi (Transforming growth factor-ß-induced protein) plays an important role in modulating the stiffness of the pancreatic stroma. By using atomic force microscopy, we show that ßig-h3 binds to type I collagen and establishes thicker fibers. Macrophages cultured on ßig-h3-structured collagen layers display a different morphology and a pro-tumoral M2 phenotype and function compared to those cultured on non-structured collagen layers. In vivo injection of those instructed CD206+CD163+ macrophages was able to suppress T cell responses. These results reveal for the first time that the collagen structure impacts the phenotype and function of macrophages by potentiating their immunosuppressive features.

Biomechanical Properties of Cancer Cells.

Since the crucial role of the microenvironment has been highlighted, many studies have been focused on the role of biomechanics in cancer cell growth and the invasion of the surrounding environment. Despite the search in recent years for molecular biomarkers to try to classify and stratify cancers, much effort needs to be made to take account of morphological and nanomechanical parameters that could provide supplementary information concerning tissue complexity adaptation during cancer development. The biomechanical properties of cancer cells and their surrounding extracellular matrix have actually been proposed as promising biomarkers for cancer diagnosis and prognosis. The present review first describes the main methods used to study the mechanical properties of cancer cells. Then, we address the nanomechanical description of cultured cancer cells and the crucial role of the cytoskeleton for biomechanics linked with cell morphology. Finally, we depict how studying interaction of tumor cells with their surrounding microenvironment is crucial to integrating biomechanical properties in our understanding of tumor growth and local invasion.

Stiffness measurement is a biomarker of skin ageing in vivo.

Human skin is particularly vulnerable to age-related deterioration and undergoes profound structural and functional changes, reflected in the external skin appearance. Skin ageing is characterized by features such as wrinkling or loss of elasticity. Even if research advances have been done concerning the molecular mechanisms that underlie these changes, very few studies have been conducted concerning the structure stiffness of the skin organ as a whole. In this study, we showed, thanks to human skin reconstructs and the Japanese Medaka fish model, that biomechanics is a new biomarker of skin ageing. We revealed that global stiffness measurement by Atomic Force Microscopy, since modulated through ageing in these models, can be a new biomarker of skin ageing, and reflects the profound reorganization of the dermis extracellular matrix, as shown by Transmission Electron Microscopy. Moreover, our data unveiled that the Japanese Medaka fish could represent a highly relevant integrated model to study skin ageing in vivo.

Gene profile of zebrafish fin regeneration offers clues to kinetics, organization and biomechanics of basement membrane.

How some animals regenerate missing body parts is not well understood. Taking advantage of the zebrafish caudal fin model, we performed a global unbiased time-course transcriptomic analysis of fin regeneration. Biostatistics analyses identified extracellular matrix (ECM) as the most enriched gene sets. Basement membranes (BMs) are specialized ECM structures that provide tissues with structural cohesion and serve as a major extracellular signaling platform. While the embryonic formation of BM has been extensively investigated, its regeneration in adults remains poorly studied. We therefore focused on BM gene expression kinetics and showed that it recapitulates many aspects of development. As such, the re-expression of the embryonic col14a1a gene indicated that col14a1a is part of the regeneration-specific program. We showed that laminins and col14a1a genes display similar kinetics and that the corresponding proteins are spatially and temporally controlled during regeneration. Analysis of our CRISPR/Cas9-mediated col14a1a knockout fish showed that collagen XIV-A contributes to timely deposition of laminins. As changes in ECM organization can affect tissue mechanical properties, we analyzed the biomechanics of col14a1a-/- regenerative BM using atomic force microscopy (AFM). Our data revealed a thinner BM accompanied by a substantial increase of the stiffness when compared to controls. Further AFM 3D-reconstructions showed that BM is organized as a checkerboard made of alternation of soft and rigid regions that is compromised in mutants leading to a more compact structure. We conclude that collagen XIV-A transiently acts as a molecular spacer responsible for BM structure and biomechanics possibly by helping laminins integration within regenerative BM.

Changes in nano-mechanical properties of human epidermal cornified cells depending on their proximity to the skin surface.

During formation of the stratum corneum (SC) barrier, terminally differentiated keratinocytes continue their maturation process within the dead superficial epidermal layer. Morphological studies of isolated human corneocytes have revealed differences between cornified envelopes purified from the deep and superficial SC. We used atomic force microscopy to measure the mechanical properties of native human corneocytes harvested by tape-stripping from different SC depths. Various conditions of data acquisition have been tested and optimized, in order to obtain exploitable and reproducible results. Probing at 200 nN allowed us to investigate the total stiffness of the cells (at 50 nm indentation) and that of the cornified envelopes (at 10 to15 nm), and lipid envelopes (at 5 to 10 nm). The obtained data indicated statistically significant differences between the superficial (more rigid) and deep (softer) corneocytes, thus confirming the existence of depth and maturation-related morphological changes within the SC. The proposed approach can be potentially used for minimally invasive evaluation of various skin conditions such as aging, skin hydration, and pathologies linked to SC.

Variations in basement membrane mechanics are linked to epithelial morphogenesis.

The regulation of morphogenesis by the basement membrane (BM) may rely on changes in its mechanical properties. To test this, we developed an atomic force microscopy-based method to measure BM mechanical stiffness during two key processes in Drosophila ovarian follicle development. First, follicle elongation depends on epithelial cells that collectively migrate, secreting BM fibrils perpendicularly to the anteroposterior axis. Our data show that BM stiffness increases during this migration and that fibril incorporation enhances BM stiffness. In addition, stiffness heterogeneity, due to oriented fibrils, is important for egg elongation. Second, epithelial cells change their shape from cuboidal to either squamous or columnar. We prove that BM softens around the squamous cells and that this softening depends on the TGFß pathway. We also demonstrate that interactions between BM constituents are necessary for cell flattening. Altogether, these results show that BM mechanical properties are modified during development and that, in turn, such mechanical modifications influence both cell and tissue shapes.

Transforming Growth Factor ß/activin signalling induces epithelial cell flattening during Drosophila oogenesis.

Although the regulation of epithelial morphogenesis is essential for the formation of tissues and organs in multicellular organisms, little is known about how signalling pathways control cell shape changes in space and time. In the Drosophila ovarian epithelium, the transition from a cuboidal to a squamous shape is accompanied by a wave of cell flattening and by the ordered remodelling of E-cadherin-based adherens junctions. We show that activation of the TGFß pathway is crucial to determine the timing, the degree and the dynamic of cell flattening. Within these cells, TGFß signalling controls cell-autonomously the formation of Actin filament and the localisation of activated Myosin II, indicating that internal forces are generated and used to remodel AJ and to promote cytoskeleton rearrangement. Our results also reveal that TGFß signalling controls Notch activity and that its functions are partly executed through Notch. Thus, we demonstrate that the cells that undergo the cuboidal-to-squamous transition produce active cell-shaping mechanisms, rather than passively flattening in response to a global force generated by the growth of the underlying cells. Thus, our work on TGFß signalling provides new insights into the mechanisms through which signal transduction cascades orchestrate cell shape changes to generate proper organ structure.