Biology subjects

Lefebvre, O.

Publications and source records attributed to Lefebvre, O..

16 recordsLinked to original sources

bioRxiv2026

Intravenously Delivered Lipid Nanoparticles Access Acute Spinal Cord Injury via Disrupted Vasculature

Trauma to the spinal cord disrupts the blood-spinal cord barrier and triggers a secondary injury cascade characterized by inflammation and progressive neuronal and glial cell death. Therapeutic cytokines and growth factors have shown promise as a treatment in preclinical studies, though their clinical translation is limited by short protein half-lives and the need for invasive intraspinal administration. Lipid nanoparticle-mediated delivery of mRNA offers an alternative strategy that enables transient protein production. Here, we investigated whether intravenously administered mRNA-lipid nanoparticles could leverage the injury-induced disruption of the blood-spinal cord barrier to access the injured spinal cord for local transgene expression. After spinal cord injury in a rat, lipid nanoparticles loaded with reporter mRNA were administered intravenously, and transgene expression was quantified in the spinal cord and peripheral organs. Intravenous delivery within a 6-hours post-injury resulted in local transgene expression in the injured spinal cord, demonstrating that mRNA-lipid nanoparticles cross the disrupted blood-spinal cord barrier. Transgene expression was observed in astrocytes, oligodendrocytes, microglia, and neurons, detected within 3 hours and remained elevated for up to 5 days post-injury. These findings demonstrate that systemic mRNA-lipid nanoparticles delivery exploit transient blood-spinal cord barrier disruption to achieve local gene expression in the injured spinal cord.

neuroscience↗

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

Functionalization of lipid nanoemulsions with humanized antibodies using plug-and-play cholesterol anchor for targeting cancer cells

Lipid NEs are promising green nanocarriers for diagnostic and therapeutic applications, but their functionalization with biomolecules, such as antibodies, remains a challenge due to liquid nature of their core. Here, we developed an original plug-and-play strategy to graft an antibody (trastuzumab) at the surface of NEs, using components generally recognized as safe (GRAS). We synthesized a reactive derivative of cholesterol and a Biotin-PEG3000-Lysine linker, which can react within one-pot formulation to form an amphiphilic carbamate Biotin-PEG3000-Cholesterol. The cholesterol ensures anchorage of the linker, which effectively exposes biotin moiety at the surface of NEs for further antibody grafting using a biotin-neutravidin coupling. The reaction between the Biotin-PEG3000-Lysine linker and NPC-Chol was confirmed by 1H-NMR and absorption spectroscopy. The obtained biotinylated 50-nm NEs loaded with a near-infrared dye were successfully targeted to neutravidin-coated glass surfaces and imaged at the single-droplet level. The biotinylated NEs bearing the trastuzumab antibody targeted specifically HER2-amplified breast cancer models HCC-1954 and SKBR3, in contrast to control MDA-MB-231 (HER2-low) cells. Altogether, our study proposes an efficient methodology for grafting antibodies to the surface of NEs, which offers new opportunities of application of these green nanocarriers in biomedicine.

pharmacology and toxicology↗

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↗

bioRxiv2025

Tumoral CD24 tunes platelets binding and pro-metastatic functions

One of the earliest steps of breast cancer metastasis occurs when tumor cells (TCs) disseminate through the bloodstream. There, they interact with several blood components. Among them, platelet favor TC survival and metastatic spread. While the binding of platelet to TC is highly variable, its molecular controls and downstream consequences remain unidentified. Here, we first document that high CD24 expression correlates with increased platelet binding and poorer survival in breast cancer. We further demonstrate that CD24-mediated platelet binding regulates TC cluster formation and resistance to anoikis in vitro. Depleting CD24 expression significantly reduces TC metastatic potential by rewiring the metastatic tumor microenvironment (mTME), affecting immune compartments and secreted factors. Overall, our work identifies CD24 as a molecular cue controlling TC-platelet interaction, dictating their metastatic potential. As such, it represents a druggable target to counteract platelet-TC collaboration in metastasis.

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

Nanomaterials trigger functional responses in primary human immune cells

Targeting the immune system with nanoparticles (NPs) to deliver immunomodulatory molecules emerged as a solution to address intra-tumoral immunosuppression and enhance therapeutic response. While the potential of nanoimmunotherapies in reactivating immune cells has been evaluated in several preclinical studies, the impact of drug-free nanomaterials on the immune system remains unknown. Here, we characterize the molecular and functional response of human NK cells and pan T cells to a selection of five NPs that are commonly used in biomedical applications. After a pre-screen to evaluate the toxicity of these nanomaterials on immune cells, we selected ultrasmall silica-based gadolinium (Si-Gd) NPs and poly(lactic-co-glycolic acid) (PLGA) NPs for further investigation. Bulk RNA-sequencing and flow cytometry analysis showcase that PLGA NPs trigger a transcriptional priming towards activation in NK and pan T cells. While PLGA NPs improved NK cells anti-tumoral functions in cytokines-deprived environment, Si-Gd NPs significantly impaired T cells activation as well as functional responses to a polyclonal antigenic stimulation. Altogether, we identified PLGAs NPs as suitable and promising candidates for further targeting approaches aiming to reactivate the immune system of cancer patients.

immunology↗

bioRxiv2024

Reinforced polymer-nanoparticle hydrogels for subcutaneous and sustained delivery of trastuzumab

In oncology, the advent of monoclonal antibody (mAbs) therapeutics represents a major breakthrough in various cancer diseases. However, these biotherapies often necessitate iterative hospital visits for intravenous infusion that can alter patients quality of life and contribute to the chronic saturation of hospitals. Interestingly, subcutaneous formulations of various mAbs offer a promising alternative facilitating faster administration compared with traditional intravenous methods, while still maintaining the same dosing schedule and providing time-saving advantages. Here, we developed an injectable mAb delivery platform using -cyclodextrin (CD)-reinforced polymer-nanoparticle hydrogels to perform a subcutaneous injection but also to delay the release of mAbs. By leveraging the versatility of our platform, we formulated hyaluronic acid- and alginate-based injectable drug depots by simply mixing components that are generally regarded as safe (GRAS). We used trastuzumab for the polymer-antibody complexation. The hydrogel depots delayed mAb release up to at least 3 days in both in vitro and in vivo mice models, outperforming clinically approved Herceptin subcutaneous formulation composed of trastuzumab with recombinant human hyaluronidase (rHuPH20).

bioengineering↗

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↗

bioRxiv2024

Endothelial calcium firing mediates extravasation of metastatic tumor cells.

Metastatic dissemination is driven by genetical, biochemical and biophysical cues that favor the distant colonization of organs and the formation of life-threatening secondary tumors. We have previously demonstrated that endothelial cells (ECs) actively remodel during extravasation by enwrapping arrested tumor cells (TCs) and extrude them from the vascular lumen while maintaining perfusion. In this work, we dissect the cellular and molecular mechanisms driving endothelial remodeling. Using high-resolution intravital imaging in zebrafish embryos, we demonstrate that the actomyosin network of ECs controls tissue remodeling and subsequent TC extravasation. Furthermore, we uncovered that this cytoskeletal remodeling is driven by altered endothelial- calcium (Ca2+) signaling caused by arrested TCs. Accordingly, we demonstrated that inhibition of voltage-dependent calcium L-type channels impairs extravasation. Lastly, we identified P2X4, TRP, and Piezo1 mechano-gated Ca2+ channels as key mediators of the process. These results further highlight the central role of endothelial remodeling during extravasation of TCs and open avenues for successful therapeutic targeting. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/587188v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@16fdc30org.highwire.dtl.DTLVardef@e0ff22org.highwire.dtl.DTLVardef@18e2311org.highwire.dtl.DTLVardef@a06320_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell 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↗

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↗

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

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↗

bioRxiv2017

Laminin α1 orchestrates VEGFA functions in the ecosystem of colorectal carcinoma

Tumor stroma remodeling is a key feature of malignant tumors and can promote cancer progression. Laminins are major constituents of basement membranes that physically separate the epithelium from the underlying stroma. By employing mouse models expressing high and low levels of the laminin 1 chain (LM1), we highlighted its implication in a tumorstroma crosstalk, thus leading to increased colon tumor incidence, angiogenesis and tumor growth. The underlying mechanism involves attraction of carcinoma-associated fibroblasts by LM1, VEGFA expression triggered by the complex integrin 2{beta}1-CXCR4 and binding of VEGFA to LM-111, which in turn promotes angiogenesis, tumor cell survival and proliferation. A gene signature comprising LAMA1, ITGB1, ITGA2, CXCR4 and VEGFA has negative predictive value in colon cancer. Together, this information opens novel opportunities for diagnosis and anti-cancer targeting.

cancer biology↗