Biology subjects

Goetz, J. G.

Publications and source records attributed to Goetz, J. G..

12 recordsLinked to original sources

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↗

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

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

Deciphering circulating tumor cells binding in a microfluidic system thanks to a parameterized mathematical model

The spread of metastases is a crucial process in which some questions remain unanswered. In this work, we focus on tumor cells circulating in the bloodstream, the so-called Circulating Tumor Cells (CTCs). Our aim is to characterize their trajectories under the influence of hemodynamic and adhesion forces. We focus on already available in vitro measurements performed with a microfluidic device corresponding to the trajectories of CTCs - without or with different protein depletions - interacting with an endothelial layer. A key difficulty is the weak knowledge of the fluid velocity that has to be reconstructed. Our strategy combines a differential equation model - a Poiseuille model for the fluid velocity and an ODE system for the cell adhesion model - and a robust and well-designed calibration procedure. The parameterized model quantifies the strong influence of fluid velocity on adhesion and confirms the expected role of several proteins in the deceleration of CTCs. Finally, it enables the generation of synthetic cells, even for unobserved experimental conditions, opening the way to a digital twin for flowing cells with adhesion.

cancer biology↗

bioRxiv2023

Targeting monocytic Occludin impairs monocyte transmigration and HIV neuroinvasion

Transmigration of circulating monocytes from the bloodstream toward the central nervous system (CNS) represents a hallmark of neuroinflammation and plays an important role during viral encephalitis and HIV-associated neurocognitive disorders (HAND). The molecular mechanisms involved in monocyte transmigration through endothelia has been extensively studied, but how monocytes locally unzip tight junction-associated proteins (TJAPs) of the endothelium composing the neurovascular unit (NVU) to reach the CNS remains poorly understood. Here, we show that human circulating monocytes express the TJAP Occludin (OCLN) to promote transmigration through cerebral microvessel endothelial cells. Silencing monocytic OCLN (mOCLN) impairs monocyte transmigration, while mOCLN overexpression increases transmigration. Using high-resolution live cell imaging, we observed that mOCLN clusters at the monocyte-endothelium interface during the transmigration process, forming a transient ring of mOCLN at the site of diapedesis. Furthermore, we designed OCLN-derived peptides targeting its extracellular loop (EL) 1 or 2 to prevent potential trans-homotypic interactions of mOCLN with endothelial OCLN. We found that transmigration of human monocytes was significantly inhibited upon treatment with the EL2 peptide in vitro and in zebrafish embryos, while preserving vascular integrity. Monocyte transmigration toward the brain is an important process for HIV neuroinvasion and here, we showed that the treatment of transmigrating monocytes with the EL2 peptide prevents the dissemination of HIV to cerebral organoids. In conclusion, our study identifies an important role for monocytic OCLN during transmigration and provides a proof-of-concept for the development of mitigation strategies to prevent HIV neuroinvasion.

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↗

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↗

bioRxiv2017

An Arf6- And Caveolae-Dependent Pathway Links Hemidesmosome Remodeling And Mechanoresponse

Hemidesmosomes (HDs) are epithelial-specific cell-matrix adhesions, which stably anchor the intracellular keratin network to the extracellular matrix. Although their main role is to protect the epithelial sheet from external mechanical strain, how HDs respond to mechanical stress remains poorly understood. Here we identify a pathway essential for HD remodeling, and outline its role with respect to 6{beta}4 integrin recycling. We find that 6{beta}4 integrin chains localize to the plasma membrane/caveolae and Arf6+ endocytic compartments. Based on FRAP and endocytosis assays, integrin recycling between both sites requires the small GTPase Arf6 and its co-regulators GIT1/{beta}PIX, but neither Caveolin1 (Cav1) nor Cavin1. Strikingly, when keratinocytes are stretched or hypo-osmotically shocked, 6{beta}4 integrin accumulates at cell edges, whereas Cav1 disappears from it. This process, which is isotropic relative to the orientation of stretch, depends on Arf6, Cav1 and Cavin1. We propose that mechanically-induced HD growth involves the isotropic flattening of caveolae (known for their mechanical buffering role) associated with integrin diffusion and turnover.

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↗