HypoxamiRs Profiling Identify miR-765 as a Regulator of the Early Stages of Vasculogenic Mimicry in SKOV3 Ovarian Cancer Cells

Vasculogenic mimicry (VM) is a novel cancer hallmark in which malignant cells develop matrix-associated 3D tubular networks with a lumen under hypoxia to supply nutrients needed for tumor growth. Recent studies showed that microRNAs (miRNAs) may have a role in VM regulation. In this study, we examined the relevance of hypoxia-regulated miRNAs (hypoxamiRs) in the early stages of VM formation. Data showed that after 48 h hypoxia and 12 h incubation on matrigel SKOV3 ovarian cancer cells undergo the formation of matrix-associated intercellular connections referred hereafter as 3D channels-like structures, which arose previous to the apparition of canonical tubular structures representative of VM. Comprehensive profiling of 754 mature miRNAs at the onset of hypoxia-induced 3D channels-like structures showed that 11 hypoxamiRs were modulated (FC>1.5; p < 0.05) in SKOV3 cells (9 downregulated and 2 upregulated). Bioinformatic analysis of the set of regulated miRNAs showed that they might impact cellular pathways related with tumorigenesis. Moreover, overall survival analysis in a cohort of ovarian cancer patients (n = 485) indicated that low miR-765, miR-193b, miR-148a and high miR-138 levels were associated with worst patients outcome. In particular, miR-765 was severely downregulated after hypoxia (FC < 32.02; p < 0.05), and predicted to target a number of protein-encoding genes involved in angiogenesis and VM. Functional assays showed that ectopic restoration of miR-765 in SKOV3 cells resulted in a significant inhibition of hypoxia-induced 3D channels-like formation that was associated with a reduced number of branch points and patterned tubular-like structures. Mechanistic studies confirmed that miR-765 decreased the levels of VEGFA, AKT1 and SRC-α transducers and exerted a negative regulation of VEGFA by specific binding to its 3‘UTR. Finally, overall survival analysis of a cohort of ovarian cancer patients (n = 1435) indicates that high levels of VEGFA, AKT1 and SRC-α and low miR-765 expression were associated with worst patients outcome. In conclusion, here we reported a novel hypoxamiRs signature which constitutes a molecular guide for further clinical and functional studies on the early stages of VM. Our data also suggested that miR-765 coordinates the formation of 3D channels-like structures through modulation of VEGFA/AKT1/SRC-α axis in SKOV3 ovarian cancer cells

Diffraction techniques and vibrational spectroscopy opportunities to characterise bones

From a histological point of view, bones that allow body mobility and protection of internal organs consist not only of different organic and inorganic tissues but include vascular and nervous elements as well. Moreover, due to its ability to host different ions and cations, its mineral part represents an important reservoir, playing a key role in the metabolic activity of the organism. From a structural point of view, bones can be considered as a composite material displaying a hierarchical structure at different scales. At the nanometre scale, an organic part, i.e. collagen fibrils and an inorganic part, i.e. calcium phosphate nanocrystals are intimately mixed to assure particular mechanical properties

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold

An engineered three dimensional (3D) in vitro cell culture system was designed with the goal of inducing and controlling in vitro osteogenesis in a reproducible manner under conditions more similar to the in vivo bone microenvironment than traditional two-dimensional (2D) models. This bioreactor allows efficient mechanical loading and perfusion of an original cubic calcium phosphate bioceramic of highly controlled composition and structure. This bioceramic comprises an internal portion containing homogeneously interconnected macropores surrounded by a dense layer, which minimises fluid flow bypass around the scaffold. This dense and flat layer permits the application of a homogeneous loading on the bioceramic while also enhancing its mechanical strength. Numerical modelling of constraints shows that the system provides direct mechanical stimulation of cells within the scaffold. Experimental results establish that under perfusion at a steady flow of 2 µL/min, corresponding to 3 ≤ Medium velocity ≤ 23 µm/s, mouse calvarial cells grow and differentiate as osteoblasts in a reproducible manner, and lay down a mineralised matrix. Moreover, cells respond to mechanical loading by increasing C-fos expression, which demonstrates the effective mechanical stimulation of the culture within the scaffold. In summary, we provide a “proof-of-concept” for osteoblastic cell culture in a controlled 3D culture system under perfusion and mechanical loading. This model will be a tool to analyse bone cell functions in vivo, and will provide a bench testing system for the clinical assessment of bioactive bone-targeting molecules under load

Physico-Chemical Characterization and In Vitro Biological Evaluation of Pure SiHA for Bone Tissue Engineering Application

International audienc

Physico-Chemical Characterization and In Vitro Biological Evaluation of Pure SiHA for Bone Tissue Engineering Application

International audienc

Evaluation of Cu2+ doping potential to control the calcium-phosphate particles-induced acute inflammatory response in bone site.

International audienc

The acute inflammatory response to copper(II)-doped biphasic calcium phosphates

International audienceInfection and inflammation are two key features to consider to avoid septic or aseptic loosening of bone-implanted biomaterials. In this context, various approaches to fine-tune the biomaterial's properties have been studied in order to modulate the crosstalk between immune and skeletal cells. Cation-doping strategies for tuning of calcium phosphates properties has been evidenced as a promising way to control the biomaterial-induced inflammatory process, and thus improving their osteoimmunomodulatory properties. Copper(II) ions are recognized for their antibacterial potential, but the literature on their impact on particulate material-induced acute inflammation is scarce. We synthesized copper(II) ions-doped biphasic calcium phosphate (BCP), intended to exhibit osteoimmunomodulatory properties. We addressed in vitro, for the first time, the inflammatory response of human primary polymorphonuclear neutrophils (PMNs) to copper(II) ions-doped or undoped (BCP) powders, synthesized by an original and robust wet method, in the presence or absence of LPS as a costimulant to mimic an infectious environment. ELISA and zymography allowed us to evidence, in vitro, a specific increase in IL-8 and GRO-α secretion but not MIP-1β, TNF-α, or MMP-9, by PMNs. To assess in vivo relevance of these findings, we used a mouse air pouch model. Thanks to flow cytometry analysis, we highlighted an increased PMN recruitment with the copper(II) ions-doped samples compared to undoped samples. The immunomodulatory effect of copper(II) ions-doped BCP powders and the consequent induced moderate level of inflammation may promote bacterial clearance by PMNs in addition to the antimicrobial potential of the material. Copper(II) doping provides new insights into calcium phosphate (CaP)-based biomaterials for prosthesis coating or bone reconstruction by effectively modulating the inflammatory environment

Three-Dimensional Organotypic Cultures Reshape the microRNAs Transcriptional Program in Breast Cancer Cells

The 3D organotypic cultures, which depend on the growth of cells over the extracellular matrix (ECM) used as a scaffold, can better mimic several characteristics of solid cancers that influence tumor biology and the response to drug therapies. Most of our current knowledge on cancer is derived from studies in 2D cultures, which lack the ECM-mediated microenvironment. Moreover, the role of miRNAs that is critical for fine-tuning of gene expression is poorly understood in 3D cultures. The aim of this study was to compare the miRNA expression profiles of breast cancer cells grown in 2D and 3D conditions. On an on-top 3D cell culture model using a basement membrane matrix enriched with laminin, collagen IV, entactin, and heparin-sulfate proteoglycans, the basal B (Hs578T) and luminal (T47D) breast cancer cells formed 3D spheroid-like stellate and rounded mass structures, respectively. Morphological changes in 3D cultures were observed as cell stretching, cell–cell, and cell–ECM interactions associated with a loss of polarity and reorganization on bulk structures. Interestingly, we found prolongations of the cytoplasmic membrane of Hs578T cells similar to tunneled nanotubes contacting between neighboring cells, suggesting the existence of cellular intercommunication processes and the possibility of fusion between spheroids. Expression profiling data revealed that 354 miRNAs were differentially expressed in 3D relative to 2D cultures in Hs578T cells. Downregulated miRNAs may contribute to a positive regulation of genes involved in hypoxia, catabolic processes, and focal adhesion, whereas overexpressed miRNAs modulate genes involved in negative regulation of the cell cycle. Target genes of the top ten modulated miRNAs were selected to construct miRNA/mRNA coregulation networks. Around 502 interactions were identified for downregulated miRNAs, including miR-935/HIF1A and miR-5189-3p/AKT that could contribute to cell migration and the response to hypoxia. Furthermore, the expression levels of miR-935 and its target HIF1A correlated with the expression found in clinical tumors and predicted poor outcomes. On the other hand, 416 interactions were identified for overexpressed miRNAs, including miR-6780b-5p/ANKRD45 and miR-7641/CDK4 that may result in cell proliferation inhibition and cell cycle arrest in quiescent layers of 3D cultures. In conclusion, 3D cultures could represent a suitable model that better resembles the miRNA transcriptional programs operating in tumors, with implications not only in the understanding of basic cancer biology in 3D microenvironments, but also in the identification of novel biomarkers of disease and potential targets for personalized therapies in cancer