Biology subjects

Hyenne, V.

Publications and source records attributed to Hyenne, V..

10 recordsLinked to original sources

bioRxiv2026

Quantitative Mapping of Organelle Positioning in Cultured Cells Using Semi-Automated Image Analysis Pipeline

Subcellular architecture is tightly controlled and contributes to the maintenance of cells homeostasis. Organelles are regulated in size, shape, number and position which respond to changes in extracellular environment. Lysosomes are of particular interest as they integrate various functions in the cells (nutrient sensing, metabolism, cell migration and adhesion), serving as signaling hubs. Their function is tightly linked to their subcellular position and deregulation of lysosome homeostasis leads to several diseases including cancer. Therefore, methods allowing precise analysis of organelle subcellular distribution can aid in fundamental, diagnostic and therapeutic approaches. Here, we provide a versatile image analysis pipeline using ImageJ and CellProfiler. This workflow allows to quantify subcellular lysosome distribution in living and fixed melanoma cells, and is applicable to other subcellular compartments and to various cell types.

cell biology↗

bioRxiv2026

Selective targeting of RalA with intrabodies impairs Triple Negative Breast Cancer metastasis

This study successfully developed and validated isoform-specific intrabodies targeting the highly homologous Ral oncoproteins, key effectors in cancer progression. Phage display was used to isolate single-chain variable fragment (scFv) clones that recognize specifically RalA (C1-A, G5-A), RalB (F6-B), or both paralogs (A12-AB). Using lentiviral transduction, these intrabodies were stably expressed as GFP fusions in murine breast cancer 4T1 cells. The anti-RalA clones C1-A and A12-AB demonstrated clear colocalization with RalA, confirming their binding activity inside the cell. We further confirmed their activity in cells by analyzing Ral-dependent pathways. All intrabodies targeting RalA (C1-A, G5-A, A12-AB) but not the one specific to RalB inhibited mitochondrial fission, a known RalA function. All clones, except the pan-Ral one, altered the endo-lysosome pathway by decreasing lysosome number. Furthermore, the RalA-specific C1-A clone reduced lysosomes size, and uniquely and strongly reduced extracellular vesicle secretion, highlighting its distinct inhibitory potential. In an orthotopic Triple-Negative Breast Cancer (TNBC) mouse model, The C1-A RalA specific clone significantly but weakly reduced primary tumor growth, but exerted a powerful anti-metastatic effect, dramatically reducing lung metastases, with a complete abolition of metastases observed in 2/5 mice. In summary, these potent, isoform-specific Ral intrabodies act as effective intracellular inhibitors, successfully modulating RalA-specific functions in cells and offering a promising therapeutic strategy for significantly suppressing tumor growth and metastasis in vivo.

cancer biology↗

bioRxiv2025

Therapeutic poxviruses induce the secretion of immunostimulating and anti-tumoral extracellular vesicles

Poxvirus-based vectors provide a versatile cancer immunotherapy platform, enabling the expression of immunostimulatory molecules and cancer-specific antigens. While infections with pathogenic viruses are well known to modulate extracellular vesicle (EV) biogenesis and function, the extent to which therapeutic poxviral vectors influence EV secretion by immune cells and thereby affect therapeutic efficacy remains underexplored. In this study, we showed that poxviruses, including the clinically relevant Modified Vaccinia Ankara (MVA), stimulate the secretion of small EVs (sEVs) containing viral proteins and immune-related signatures from peripheral blood mononuclear cells (PBMCs). Using an engineered MVA vector, we demonstrated the transfer of virus-encoded therapeutic payloads to sEVs, including the model ovalbumin (OVA)-derived peptide SIINFEKL presented by the class I major histocompatibility complex (MHC I) and the immune activators interleukin-12 (IL-12) and CD40 ligand (CD40L). Depending on the isolation method, these sEVs stimulated SIINFEKL-specific CD8 T cells with varying efficiencies in vitro. Remarkably, intravenous injection of these sEVs into E.G7-OVA lymphoma-bearing mice reduced tumor growth to an extent comparable to the virus itself. Taken together, our findings indicate that EVs released from immune cells infected with engineered therapeutic poxviruses exert potent antitumor activity. These vesicles represent actionable mediators whose secretion and functionalization can be harnessed to improve viral vector-based immunotherapies, as well as being considered as therapeutic vectors in their own. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/677320v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1eae18borg.highwire.dtl.DTLVardef@17d6fb5org.highwire.dtl.DTLVardef@311577org.highwire.dtl.DTLVardef@7839c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Serum albumin coated stellate mesoporous silica nanocomposites inhibit metastatic outgrowth in zebrafish embryos

Mesoporous silica-based nanoparticles (NPs) are promising tools for developing targeted therapeutic interventions in cancer. Endowed with a large pore silica shell suitable for drug encapsulation and with a responsive magnetic core, iron oxide stellate mesoporous silica (IO STMS) NPs stand out. Yet, their impact and potential toxicity on relevant in vivo models has not been carefully tested yet. Herein, we assessed the impact of these IO STMS nanocomposites in a syngeneic metastasis assay in zebrafish. NPs were surface-modified with human serum albumin (HSA) and loaded or not with the chemotherapeutic doxorubicin (DOX). In vitro, DOX-loaded NPs were expectedly more toxic to zebrafish melanoma (Zmel) cells than no-DOX NPs. In zebrafish embryos, the NPs were rapidly distributed through blood circulation and were found to colocalize over time with the vascular endothelium and local macrophages. Suprisingly, the NPs efficiently reduced the outgrowth of Zmel tumoral masses in an experimental metastasis assay in zebrafish embryos regardless of their loading with DOX. The anti-metastatic effect of these NPs was further improved by increasing the amount of HSA coating, also resulting in higher embryo survival. Altogether, IO STMS NPs showed promising cytotoxic effects on a relevant zebrafish metastasis model, inhibiting metastatic outgrowth in vivo independently of the drug loading. This opens the door to further testing for better exploiting their targeting and drug delivery potentialities.

cancer biology↗

bioRxiv2024

Depletion of all platelet integrins impacts hemostasis, thrombosis and tumor metastasis

Platelet integrins, in addition to other platelet receptors, are known to control hemostasis, thrombosis but also metastatic progression. Yet, their exclusive but combined deficiency has never been tested in these processes. Taking advantage of PF4Cre-{beta}1-/-/{beta}3-/- mouse strain, we show that platelets are exclusively depleted for all integrins. While they displayed impaired binding to fibrinogen and annexin-V, P-selectin exposure was normal. Platelet adhesion was abrogated on immobilized fibrinogen and fibrillar fibronectin under shear flow. PF4Cre-{beta}1-/- /{beta}3-/- mice presented an increased bleeding time and a profound defect in experimental models of arterial thrombosis. Platelet adhesion to tumor cells was also reduced, with a profound impact on tumor growth and metastatic burden in a model of triple negative breast cancer. Overall, these results confirm the central role of platelet integrins in hemostasis and thrombosis, and define their role in tumor growth and metastasis formation. 40-word summary: Depletion of all platelet integrins in PF4Cre-{beta}1-/-/{beta}3-/- mice leads to increased bleeding time and inhibits in vivo arterial thrombosis. Integrin-null platelets reduce tumor growth and metastatic burden in orthotopic and experimental metastasis models. Platelet integrins control hemostasis, thrombosis and metastasis.

cancer 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

Peripheral positioning of lysosomes supports melanoma aggressiveness

Emerging evidences suggest that function and position of organelles are pivotal for tumor cell dissemination. Among them, lysosomes stand out as they integrate metabolic sensing with gene regulation and secretion of proteases. Yet, how their function is linked to their position and how this controls metastasis remains elusive. Here, we analyzed lysosome subcellular distribution in patient-derived melanoma cells and patient biopsies and found that lysosome spreading scales with their aggressiveness. Peripheral lysosomes promote matrix degradation and invasion of melanoma cells which is directly linked to their lysosomal and cell transcriptional programs. When controlling lysosomal positioning using chemo-genetical heterodimerization in patient-derived melanoma cells, we demonstrated that perinuclear clustering impairs lysosomal secretion, matrix degradation and invasion. Impairing lysosomal spreading in two distinct in vivo models (mouse and zebrafish) significantly reduces invasive outgrowth. Our study provides a direct demonstration that lysosomal positioning controls cell invasion, illustrating the importance of organelle adaptation in carcinogenesis and suggesting that lysosome positioning could potentially be used for the diagnosis of metastatic melanoma.

cell biology↗

bioRxiv2022

Blood flow diverts extracellular vesicles from endothelial degradative compartments to promote angiogenesis

Extracellular vesicles released by tumors (tEVs) disseminate via circulatory networks and promote microenvironmental changes in distant organs favoring metastatic seeding. Despite their abundance in the bloodstream, how hemodynamics affect the function of circulating tEVs remains unsolved. We experimentally tuned flow profiles in vitro (microfluidics) and in vivo (zebrafish) and demonstrated that efficient uptake of tEVs occurs in endothelial cells subjected to capillary-like hemodynamics. Such flow profiles partially reroute internalized tEVs towards non-acidic and non-degradative Rab14-positive endosomes, at the expense of lysosomes, suggesting that endothelial mechanosensing diverts tEVs from degradation. Subsequently, tEVs promote the expression of pro-angiogenic transcription factors in flow-stimulated endothelial cells and favor vessel sprouting in zebrafish. Altogether, we demonstrate that capillary-like flow profiles potentiate the pro-tumoral function of circulating tEVs by promoting their uptake and rerouting their trafficking. We propose that tEVs contribute to pre-metastatic niche formation by exploiting endothelial mechanosensing in specific vascular regions with permissive hemodynamics.

cell biology↗

bioRxiv2021

The Necroptosis Effector MLKL drives Small Extracellular Vesicle Release and Tumour Growth in Glioblastoma

Extracellular vesicles (EVs) are lipid-based nano-sized particles that convey biological material from donor to recipient cells. They play key roles in tumour progression, notably in glioblastoma in which the subpopulation of Glioblastoma Stem-like Cells (GSCs) might represent a meaningful source of tumour-derived EVs. However, the mechanisms involved in the production and release of EVs by GSCs are still poorly understood. Here, we report the identification of MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of small EVs from GSCs. The targeting of MLKL by genetic, protein depletion or chemical approaches alters endosomal trafficking and EV release and reduces GSC expansion in vitro. This function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL triggers a reduction of both the tumour burden in xenografted mice and of the level of plasmatic EVs. This work reinforces the idea of a non-deadly role for MLKL in endosomal trafficking and suggests that interfering with EV biogenesis is a promising therapeutic option to sensitize glioblastoma cells to death.

cell biology↗

bioRxiv2018

Studying the fate of tumor extracellular vesicles at high spatio-temporal resolution using the zebrafish embryo

Tumor extracellular vesicles (tumor EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs have been reported to travel in the blood circulation, reach specific distant organs and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we show a new method for tracking individual circulating tumor EVs in a living organism: we combine novel, bright and specific fluorescent membrane probes, MemBright, with the transparent zebrafish embryo as an animal model. We provide the first description of tumor EVs hemodynamic behavior and document their arrest before internalization. Using transgenic lines, we show that circulating tumor EVs are uptaken by endothelial cells and blood patrolling macrophages, but not by leukocytes, and subsequently stored in acidic degradative compartments. Finally, we prove that the MemBright can be used to follow naturally released tumor EVs in vivo. Overall, our study demonstrates the usefulness and prospects of zebrafish embryo to track tumor EVs in vivo.\n\nHighlightsO_LIMemBright, a new family of membrane probes, allows for bright and specific staining of EVs\nC_LIO_LIZebrafish melanoma EVs are very similar to human and mouse melanoma EVs in morphology and protein content\nC_LIO_LIThe zebrafish embryo is an adapted model to precisely track tumor EVs dynamics and fate in a living organism from light to electron microscopy\nC_LIO_LICirculating tumor EVs are rapidly uptaken by endothelial cells and patrolling macrophages\nC_LIO_LICorrelated light and electron microscopy can be used in zebrafish to identify cells and compartments uptaking tumor EVs\nC_LI\n\nBlurbDispersion of tumor extracellular vesicles (EVs) throughout the body promotes tumor progression. However the behavior of tumor EVs in body fluids remains mysterious due to their small size and the absence of adapted animal model. Here we show that the zebrafish embryo can be used to track circulating tumor EVs in vivo and provide the first high-resolution description of their dissemination and uptake.

cell biology↗