Biology subjects

Follain, G.

Publications and source records attributed to Follain, G..

17 recordsLinked to original sources

bioRxiv2026

Pancreatic cancer cells breach endothelial barriers through protrusion-driven invasion or endothelial retraction

Extravasation, the exit of circulating cancer cells from blood vessels, is a critical yet poorly understood step in metastatic dissemination. Here we show that pancreatic ductal adenocarcinoma (PDAC) cells can breach endothelial barriers through two mechanistically distinct modes of extravasation. MIA PaCa-2 cells breach endothelial junctions via filopodia-like protrusions, enabling access to and spread across the basal extracellular matrix (ECM). By contrast, AsPC-1 cells remain rounded atop the endothelium and cross the barrier by triggering rapid retraction of neighbouring endothelial cells. These distinct extravasation modes were also observed in zebrafish larvae. In the mouse lung, AsPC-1 cells arrest, survive, induce endothelial detachment from the basal lamina, and extravasate through this retraction mechanism before metastatic outgrowth. Mechanistically, AsPC-1-secreted factors are sufficient to destabilise endothelial monolayers, and AsPC-1 cells also induce endothelial apoptosis; however, blocking apoptosis does not prevent barrier disruption. By contrast, treatment with saracatinib, a Src-family kinase inhibitor, protects endothelial barriers, limits early vascular disruption in the lung, and delays metastatic outgrowth. Together, these findings reveal that PDAC cells can extravasate via mechanistically distinct routes, suggesting that effective anti-metastatic strategies may need to target multiple modes of endothelial barrier breach rather than a single pathway.

cell biology↗

bioRxiv2026

Scaffold protein SHANK3 regulates endothelial cell motility and tissue mechanics

SHANK3 is a multidomain scaffolding protein critical for neuronal function, which has been linked to neurodevelopmental disorders such as autism spectrum disorder. More recently, SHANK3 has been shown to play a role in cell survival and actin dynamics outside the nervous system. Here, we show that SHANK3 is widely expressed in endothelial cells across different tissues, where its role is not well understood. SHANK3 localised to endothelial cell-cell junctions in cultured endothelial cells, and its depletion compromised endothelial barrier function. SHANK silencing altered cell mechanics including elongated cell morphology, reduced cell-matrix traction forces and alteration of cell migration rate. It further triggered dynamic heterogeneity in endothelial monolayers, with regions of coordinated long-range migration interspersed with areas exhibiting only local velocity fluctuations, consistent with a transition toward more fluid-like tissue behaviour. This change in collective dynamics was accompanied by increased spheroid spreading and fusion, suggestive of altered tissue viscosity, and coincided with disrupted cell-cell junction morphology and mechanical forces in SHANK3-depleted cells. In vivo, SHANK3 depletion impaired endothelial cell migration, resulting in delayed sprouting of intersegmental vessels and disruption of the vascular network in zebrafish embryos. Furthermore, inducible endothelial-specific deletion of SHANK3 in postnatal mice impaired angiogenic sprouting and reduced vascular complexity in the developing retina. Overall, we demonstrate that SHANK3 plays a role in endothelial cell motility and tissue mechanics, with implications for vascular processes during development.

cell biology↗

bioRxiv2026

Tissue-scale mechanics controls differentiation strategy and dynamics of epithelial multilayering

Generating and maintaining multilayered epithelia requires coordinated cell division, differentiation, and tissue architecture, yet the precise mechanisms of multilayering remain unclear. Using the developing mouse epidermis, we show that basal stem cells adopt distinct multilayering strategies depending on tissue mechanics. Combining quantitative morphometry, embryo live imaging and physical modeling, we observe that early in development, the epidermis is fluid-like, allowing undifferentiated cells to move suprabasally through perpendicular divisions or basal detachment before differentiating. As the tissue matures and rigidifies, a mechanical barrier is established that only allows upward movement of basal cells that have committed to differentiation. The final step of this commitment is delamination that requires Notch signaling, triggered by increased tissue stiffness and jamming. This mechanical regulation orchestrates a feedback loop that induces cell upward motion precisely when the basal layer becomes crowded. Together, our findings identify tissue mechanics as the key determinant of how tissues drive multilayering and reveal mechanically regulated Notch signaling as a driver of epidermal delamination.

cell biology↗

bioRxiv2024

Signaling downstream of tumor-stroma interaction regulates mucinous colorectal adenocarcinoma apicobasal polarity

Mucinous colorectal carcinoma (MUC CRC) dissemination into the tumor stroma and metastasis to multiple organs, including the peritoneum, is associated with poor prognosis. Disseminating MUC CRCs exhibit either a conventional apical-in or an inverted apical-out polarity phenotype that influence patient outcome. Identifying the mechanisms controlling MUC CRC polarity is critical to understand disease progression. Here, we analyze patient-derived MUC CRC xenografts, with apical-in or apical-out polarity, ex vivo or within collagen gels to mimic the peritumoral stroma. Single-cell analyses reveal 2{beta}1-integrin as a key collagen-binding receptor in these models. Collagen-2{beta}1-integrin interaction activates Src and ERK/MAPK signaling and upregulates the expression of SorLA, an endosomal sorting receptor. SorLA supports apical-in polarity and carcinoma-stroma interactions by promoting integrin recycling to the plasma membrane and HER2/HER3 expression through a positive feedback mechanism. Accordingly, we observe positive correlation between HER2, HER3 and SorLA in patient samples with the highest HER2 expression in apical-in-presenting tissues. Treatment of tumor spheres with clinically relevant HER2/HER3-targeting antibodies reverts sphere polarity and impedes collagen remodeling and adhesion to mouse peritoneum. This SorLA--integrin--HER2/HER3 signaling axis may represent a basis for MUC CRC-patient stratification and shed light on other carcinomas with similar apical-out phenotypes.

cancer biology↗

bioRxiv2024

Fast label-free live imaging reveals key roles of flow dynamics and CD44-HA interaction in cancer cell arrest on endothelial monolayers

Leukocyte extravasation is a key component of the innate immune response, while circulating tumor cell extravasation is a critical step in metastasis formation. Despite their importance, the mechanisms underlying leukocyte and tumor cell extravasation remain incompletely understood. Here, we developed an imaging pipeline that integrates fast, label-free live-cell imaging with deep learning-based image analysis to quantitatively track and compare the initial steps of extravasation--cell landing and arrest on an endothelial monolayer--under physiological flow conditions. We find that pancreatic ductal adenocarcinoma (PDAC) cells exhibit variable adhesion strength and flow sensitivity. Remarkably, some PDAC cells demonstrate comparable endothelial engagement as leukocytes, preferentially arresting at endothelial junctions, potentially due to increased stiffness at these sites, which leads to exposure to the underlying basal extracellular matrix. PDAC cells also tend to cluster in regions with high, heterogeneous expression of the endothelial CD44 receptor. Simulations suggest that clustering results from the combination of CD44-mediated attachment and localized flow disturbances that facilitate subsequent cell attachment. Targeting CD44 using siRNA or function-blocking antibodies, or degrading its ligand hyaluronic acid (HA), almost completely abolishes PDAC cell attachment. Overall, we demonstrate that cancer and immune cells share both common and unique features in endothelial adhesion under flow, and identify CD44 and HA as key mediators of PDAC cell arrest.

cell biology↗

bioRxiv2024

Restoring mechanophenotype reverts malignant properties of ECM-enriched vocal fold cancer

Increased extracellular matrix (ECM) and matrix stiffness promote solid tumor progression. However, mechanotransduction in cancers arising in mechanically active tissues remains underexplored. Here, we report upregulation of multiple ECM components accompanied by tissue stiffening in vocal fold cancer (VFC). We compare non-cancerous (NC) and patient- derived VFC cells - from early (mobile, T1) to advanced-stage (immobile, T3) cancers - revealing an association between VFC progression and cell-surface receptor heterogeneity, reduced laminin-binding integrin cell-cell junction localization and a flocking mode of collective cell motility. Mimicking physiological movement of healthy vocal fold tissue (stretching/vibration), decreases oncogenic nuclear {beta}-catenin and YAP levels in VFC. Multiplex immunohistochemistry of VFC tumors uncovered a correlation between ECM content, nuclear YAP and patient survival, concordant with VFC sensitivity to YAP-TEAD inhibitors in vitro. Our findings present evidence that VFC is a mechanically sensitive malignancy and restoration of tumor mechanophenotype or YAP/TAZ targeting, represents a tractable anti-oncogenic therapeutic avenue for VFC.

cell biology↗

bioRxiv2024

Cell viscosity influences hematogenous dissemination and metastatic extravasation of tumor cells

Metastases arise from a multi-step process during which tumor cells change their mechanics in response to microenvironmental cues. While such mechanical adaptability could influence metastatic success, how tumor cell mechanics directly impacts intravascular behavior of circulating tumor cells (CTCs) remains poorly understood. In the present study, we demonstrate how the deformability of CTCs affects hematogenous dissemination and identify the mechanical profiles that favor metastatic extravasation. Combining intravital microscopy with CTC-mimicking elastic beads and mechanically-tuned tumor cells, we demonstrate that the inherent properties of circulating objects dictate their ability to enter constraining vessels. We identify cellular viscosity as the key property that governs CTC circulation and arrest patterns. We further demonstrate that cellular viscosity is required for efficient extravasation and find that properties that favor extravasation and subsequent metastatic outgrowth can be opposite. Altogether, we identify CTC viscosity as a key biomechanical parameter that shapes several steps of metastasis.

cancer biology↗

bioRxiv2023

TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is primarily expressed in the vasculature in vivo and that its depletion leads to vascular abnormalities in vivo and loss of barrier integrity in cultured endothelial cells. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct, high-affinity interaction with CCM2. Modeling and functional testing of TLNRD1 and CCM2 mutants reveal that their interaction is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the 4-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Notably, a CCM2 pathogenic mutation linked to vascular dementia in patients maps to the interface and disrupts the interaction. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity by controlling TLNRD1 bundling activity.

cell biology↗

bioRxiv2023

NanoPyx: super-fast bioimage analysis powered by adaptive machine learning

To overcome the challenges posed by large and complex microscopy datasets, we have developed NanoPyx, an adaptive bioimage analysis framework designed for high-speed processing. At the core of NanoPyx is the Liquid Engine, an agent-based machine-learning system that predicts acceleration strategies for image analysis tasks. Unlike traditional single-algorithm methods, the Liquid Engine generates multiple CPU and GPU code variations using a meta-programming system, creating a competitive environment where different algorithms are benchmarked against each other to achieve optimal performance under the user"s computational environment. In initial experiments focusing on super-resolution analysis methods, the Liquid Engine demonstrated an over 10-fold computational speed improvement by accurately predicting the ideal scenarios to switch between algorithmic implementations. NanoPyx is accessible to users through a Python library, code-free Jupyter notebooks, and a napari plugin, making it suitable for individuals regardless of their coding proficiency. Furthermore, the optimisation principles embodied by the Liquid Engine have broader implications, extending their applicability to various high-performance computing fields.

bioinformatics↗

bioRxiv2022

Platelets favor the outgrowth of established metastases

Despite abundant evidence demonstrating that platelets foster metastasis, a therapeutic approach based on anti-platelet agents is not an option due to the risk of hemorrhages. In addition, whether platelets can regulate metastasis at the late stages of the disease remains unknown. In this study, we subjected syngeneic models of metastasis to various thrombocytopenic regimes to show that platelets provide a biphasic contribution to metastasis. While potent intravascular binding of platelets to tumor cells efficiently promotes metastasis, platelets further support the outgrowth of established metastases. Genetic depletion and pharmacological targeting of the platelet-specific receptor GPVI in humanized mouse models efficiently reduced the growth of established metastases, independently of active platelet binding to tumor cells in the bloodstream. Our study is the first to demonstrate therapeutic efficacy when targeting animals carrying growing metastases. It further identifies GPVI as the first molecular target whose inhibition can impair metastasis without inducing collateral hemostatic perturbations.

cancer biology↗

bioRxiv2022

Fast4DReg: Fast registration of 4D microscopy datasets

Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal quantification of biological processes. While multiple methods and tools exist to correct images post-acquisition, performing drift correction of large 3D videos using open-source solutions remains challenging and time-consuming. Here we present a new tool developed for ImageJ/Fiji called Fast4DReg that can quickly correct axial and lateral drift in 3D video microscopy datasets. Fast4DReg works by creating intensity projections along multiple axes and estimating the drift between frames using 2D cross-correlations. Using synthetic and acquired datasets, we demonstrate that Fast4DReg performs better than other state-of-the-art open-source drift correction tools and significantly outperforms them in speed (5x to 60x). We also demonstrate that Fast4DReg can be used to register misaligned channels in 3D using either calibration slides or misaligned images directly. Altogether Fast4DReg provides a quick and easy-to-use method to correct 3D imaging data before further visualization and analysis. Fast4DReg is available on GitHub.

bioinformatics↗

bioRxiv2022

High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation

In recent years, the development of new image analysis approaches has highlighted the possibility of recovering superresolution information from short sequences of wide-field images. Our recently developed method, SRRF (Super-Resolution Radial Fluctuations), enables long-term live-cell imaging beyond the resolution limit without specialized hardware. Here, we present eSRRF (enhanced-SRRF), a significant improvement over our initial method, enhancing image fidelity to the underlying structure and resolution. Especially, eSRRF uses automated data-driven parameter optimization, including an estimation of the number of frames necessary for optimal reconstruction. We demonstrate the improved fidelity of the images reconstructed with eSRRF and highlight its versatility and ease of use over a wide range of microscopy techniques and biological systems. We also extend eSRRF to 3D super-resolution microscopy by combining it with multi-focus microscopy (MFM), obtaining volumetric super-resolution imaging of live cells with acquisition speed of ~1 volume/second.

biophysics↗

bioRxiv2020

Automated cell tracking using StarDist and TrackMate

The ability of cells to migrate is a fundamental physiological process involved in embryonic development, tissue homeostasis, immune surveillance, and wound healing. Therefore, the mechanisms governing cellular locomotion have been under intense scrutiny over the last 50 years. One of the main tools of this scrutiny is live-cell quantitative imaging, where researchers image cells over time to study their migration and quantitatively analyze their dynamics by tracking them using the recorded images. Despite the availability of computational tools, manual tracking remains widely used among researchers due to the difficulty setting up robust automated cell tracking and large-scale analysis. Here we provide a detailed analysis pipeline illustrating how the deep learning network StarDist can be combined with the popular tracking software TrackMate to perform 2D automated cell tracking and provide fully quantitative readouts. Our proposed protocol is compatible with both fluorescent and widefield images. It only requires freely available and open-source software (ZeroCostDL4Mic and Fiji), and does not require any coding knowledge from the users, making it a versatile and powerful tool for the field. We demonstrate this pipelines usability by automatically tracking cancer cells and T cells using fluorescent and brightfield images. Importantly, we provide, as supplementary information, a detailed step-by-step protocol to allow researchers to implement it with their images.

cell biology↗

bioRxiv2018

Intravascular arrest of circulating tumor cells is a two-step process exploiting their adhesion repertoire.

Cancer metastasis is a process whereby a primary tumor spreads to distant organs. We have previously demonstrated that blood flow controls the intravascular arrest of circulating tumor cells (CTCs), through stable adhesion to endothelial cells. We now aim at defining the contribution of cell adhesive potential and at identifying adhesion receptors at play. Early arrest is mediated by the formation of weak adhesion depending on CD44 and integrin v{beta}3. Stabilization of this arrest uses integrin 5{beta}1-dependent adhesions with higher adhesion strength, which allows CTCs to stop in vascular regions with lower shear forces. Moreover, blood flow favors luminal deposition of fibronectin on endothelial cells, an integrin 5{beta}1 ligand. Finally, we show that only receptors involved in stable adhesion are required for subsequent extravasation and metastasis. In conclusion, we identified the molecular partners that are sequentially exploited by CTCs to arrest and extravasate in vascular regions with permissive flow regimes.

cancer biology↗

bioRxiv2018

Live tracking of inter-organ communication by endogenous exosomes in vivo

Extracellular vesicles (EVs) are released by most cell types but the definitive demonstration of their functional relevance remains challenging due to the lack of appropriate model organisms. Here we developed an in vivo model to study EV physiology by expressing CD63-pHluorin in zebrafish embryos. A combination of microscopy techniques and proteomic analysis allowed us to study the biogenesis, composition, transfer, uptake and fate of individual endogenous EVs in vivo. We identified an exosome population released in a syntenin-dependent manner from the Yolk Syncytial Layer into the blood circulation. These exosomes were specifically captured, endocytosed and degraded by patrolling macrophages and endothelial cells in the Caudal Vein Plexus (CVP) in a scavenger receptor and dynamin-dependent manner. Interference with exosome secretion affected CVP growth, supporting their trophic role. Altogether, our work provides a unique model to track in vivo inter-organ communication by endogenous exosomes at individual vesicle level and high spatio-temporal accuracy.\n\nHighlights- Single endogenous EVs can be live-visualized in the whole embryo with CD63-pHluorin\n- In the YSL, syntenin regulates exosome release into the blood for their propagation\n- YSL exosomes reach the tail to be taken up by macrophages and endothelial cells\n- Uptake is scavenger receptor and dynamin-dependent and provides trophic support\n\n\nBlurbWe propose zebrafish embryos expressing a fluorescent reporter for exosomes as a relevant model organism to live-track production, journey and fate of individual extracellular vesicles in vivo. Our model allows investigation of the composition of EVs and the molecular mechanisms controlling their biogenesis and fate and functions in receiving cells.

cell biology↗

bioRxiv2017

Hemodynamic forces tune the arrest, adhesion and extravasation of circulating tumor cells

Metastatic seeding is driven by cell-intrinsic and environmental cues, yet the contribution of biomechanics is poorly known. We aim to elucidate the impact of blood flow on the arrest and the extravasation of circulating tumor cells (CTCs) in vivo. Using the zebrafish embryo, we show that arrest of CTCs occurs in vessels with favorable flow profiles where flow forces control the adhesion efficacy of CTCs to the endothelium. We biophysically identified the threshold values of flow and adhesion forces allowing successful arrest of CTCs. In addition, flow forces fine-tune tumor cell extravasation by impairing the remodeling properties of the endothelium. Importantly, we also observe endothelial remodeling at arrest sites of CTCs in mouse brain capillaries. Finally, we observed that human supratentorial brain metastases preferably develop in areas with low perfusion. Altogether, these results demonstrate that hemodynamic profiles at metastatic sites regulate key steps of extravasation preceding metastatic outgrowth.

cancer biology↗

bioRxiv2017

Hemodynamic forces can be accurately measured in vivo with optical tweezers

Force sensing and generation at the tissular and cellular scale is central to many biological events. There is a growing interest in modern cell biology for methods enabling force measurements in vivo. Optical trapping allows non-invasive probing of pico-Newton forces and thus emerged as a promising mean for assessing biomechanics in vivo. Nevertheless, the main obstacles rely in the accurate determination of the trap stiffness in heterogeneous living organisms, at any position where the trap is used. A proper calibration of the trap stiffness is thus required for performing accurate and reliable force measurements in vivo. Here, we introduce a method that overcomes these difficulties by accurately measuring hemodynamic profiles in order to calibrate the trap stiffness. Doing so, and using numerical methods to assess the accuracy of the experimental data, we measured flow profiles and drag forces imposed to trapped red blood cells of living zebrafish embryos. Using treatments enabling blood flow tuning, we demonstrated that such method is powerful in measuring hemodynamic forces in vivo with high accuracy and confidence. Altogether, this study demonstrates the power of optical tweezing in measuring low range hemodynamic forces in vivo and offers an unprecedented tool in both cell and developmental biology.

biophysics↗