Ascites microenvironment conditions the peritoneal pre-metastatic niche to promote the implantation of ovarian tumor spheroids: Involvement of fibrinogen/fibrin and αV and α5ß1 integrins.

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood. Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5ß1 integrins are involved. In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium.

The Balance Between Shear Flow and Extracellular Matrix in Ovarian Cancer-on-Chip.

Ovarian cancer is the most lethal gynecologic cancer in developed countries. In the tumor microenvironment, the extracellular matrix (ECM) and flow shear stress are key players in directing ovarian cancer cells invasion. Artificial ECM models based only on ECM proteins are used to build an ovarian tumor-on-chip to decipher the crosstalk between ECM and shear stress on the migratory behavior and cellular heterogeneity of ovarian tumor cells. This work shows that in the shear stress regime of the peritoneal cavity, the ECM plays a major role in driving individual or collective ovarian tumor cells migration. In the presence of basement membrane proteins, migration is more collective than on type I collagen regardless of shear stress. With increasing shear stress, individual cell migration is enhanced; while, no significant impact on collective migration is measured. This highlights the central position that ECM and flow shear stress should hold in in vitro ovarian cancer models to deepen understanding of cellular responses and improve development of ovarian cancer therapeutic platforms. In this frame, adding flow provides significant improvement in biological relevance over the authors' previous work. Further steps for enhanced clinical relevance require not only multiple cell lines but also patient-derived cells and sera.

Detection of invisible biological traces in relation to the physicochemical properties of substrates surfaces in forensic casework.

Touch DNA, which can be found at crime scenes, consists of invisible biological traces deposited through a person's skin's contact with an object or another person. Many factors influence touch DNA transfer, including the "destination" substrate's surface. The latter's physicochemical characteristics (wettability, roughness, surface energy, etc.) will impact touch DNA deposition and persistence on a substrate. We selected a representative panel of substrates from objects found at crime scenes (glass, polystyrene, tiles, raw wood, etc.) to investigate the impact of these characteristics on touch DNA deposition and detection. These were shown to impact cell deposition, morphology, retention, and subsequent touch DNA genetic analysis. Interestingly, cell-derived fragments found within keratinocyte cells and fingermarks using in vitro touch DNA models could be successfully detected whichever the substrates' physicochemistry by targeting cellular proteins and carbohydrates for two months, indoors and outdoors. However, swabbing and genetic analyses of such mock traces from different substrates produced informative profiles mainly for substrates with the highest surface free energy and therefore the most hydrophilic. The substrates' intrinsic characteristics need to be considered to better understand both the transfer and persistence of biological traces, as well as their detection and collection, which require an appropriate methodology and sampling device to get informative genetic profiles.

Targeting cell-derived markers to improve the detection of invisible biological traces for the purpose of genetic-based criminal identification.

At a crime scene, investigators are faced with a multitude of traces. Among them, biological traces are of primary interest for the rapid genetic-based identification of individuals. "Touch DNA" consists of invisible biological traces left by the simple contact of a person's skin with objects. To date, these traces remain undetectable with the current methods available in the field. This study proposes a proof-of-concept for the original detection of touch DNA by targeting cell-derived fragments in addition to DNA. More specifically, adhesive-structure proteins (laminin, keratin) as well as carbohydrate patterns (mannose, galactose) have been detected with keratinocyte cells derived from a skin and fingermark touch-DNA model over two months in outdoor conditions. Better still, this combinatory detection strategy is compatible with DNA profiling. This proof-of-concept work paves the way for the optimization of tools that can detect touch DNA, which remains a real challenge in helping investigators and the delivery of justice.

In Vitro Models of Ovarian Cancer: Bridging the Gap between Pathophysiology and Mechanistic Models.

Ovarian cancer (OC) is a disease of major concern with a survival rate of about 40% at five years. This is attributed to the lack of visible and reliable symptoms during the onset of the disease, which leads over 80% of patients to be diagnosed at advanced stages. This implies that metastatic activity has advanced to the peritoneal cavity. It is associated with both genetic and phenotypic heterogeneity, which considerably increase the risks of relapse and reduce the survival rate. To understand ovarian cancer pathophysiology and strengthen the ability for drug screening, further development of relevant in vitro models that recapitulate the complexity of OC microenvironment and dynamics of OC cell population is required. In this line, the recent advances of tridimensional (3D) cell culture and microfluidics have allowed the development of highly innovative models that could bridge the gap between pathophysiology and mechanistic models for clinical research. This review first describes the pathophysiology of OC before detailing the engineering strategies developed to recapitulate those main biological features.

Three-Dimensional Collagen Topology Shapes Cell Morphology, beyond Stiffness.

Cellular heterogeneity is associated with many physiological processes, including pathological ones, such as morphogenesis and tumorigenesis. The extracellular matrix (ECM) is a key player in the generation of cellular heterogeneity. Advances in our understanding rely on our ability to provide relevant in vitro models. This requires obtainment of the characteristics of the tissues that are essential for controlling cell fate. To do this, we must consider the diversity of tissues, the diversity of physiological contexts, and the constant remodeling of the ECM along these processes. To this aim, we have fabricated a library of ECM models for reproducing the scaffold of connective tissues and the basement membrane by using different biofabrication routes based on the electrospinning and drop casting of biopolymers from the ECM. Using a combination of electron microscopy, multiphoton imaging, and AFM nanoindentation, we show that we can vary independently protein composition, topology, and stiffness of ECM models. This in turns allows one to generate the in vivo complexity of the phenotypic landscape of ovarian cancer cells. We show that, while this phenotypic shift cannot be directly correlated with a unique ECM feature, the three-dimensional collagen fibril topology patterns cell shape, beyond protein composition and stiffness of the ECM. On this line, this work is a further step toward the development of ECM models recapitulating the constantly remodeled environment that cells face and thus provides new insights for cancer model engineering and drug testing.

A biomimetic model of 3D fluid extracellular macromolecular crowding microenvironment fine-tunes ovarian cancer cells dissemination phenotype.

An early fundamental step in ovarian cancer progression is the dissemination of cancer cells through liquid environments, one of them being cancer ascites accumulated in the peritoneal cavity. These biological fluids are highly crowded with a high total macromolecule concentration. This biophysical property of fluids is widely used in tissue engineering for a few decades now, yet is largely underrated in cancer biomimetic models. To unravel the role of fluids extracellular macromolecular crowding (MMC), we exposed ovarian cancer cells (OCC) to high molecular weight inert polymer solutions. High macromolecular composition of extracellular liquid presented a differential effect: i) it impeded non-adherent OCC aggregation in suspension and, decreased their adhesion; ii) it promoted adherent OCC migration by decreasing extracellular matrix deposition. Besides, there seemed to be a direct link between the extracellular MMC and intracellular processes, especially the actin cytoskeleton organization and the nucleus morphology. In conclusion, extracellular fluid MMC orients OCC dissemination phenotype. Integrating MMC seems crucial to produce more relevant mimetic 3D in vitro fluid models to study ovarian dissemination but also to screen drugs.

Synthesis and antitumor activities investigation of a C-nucleoside analogue of ribavirin.

SRO-91 is a non-natural ribofuranosyl-1,2,3-triazole C-nucleoside obtained by a synthetic sequence involving a C-alkynyl glycosylation mediated by metallic indium and a Huisgen cycloaddition for the construction of the triazole. Its structure is close to the one of ribavirin, a drug presenting a broad-spectrum against viral infections. SRO-91 antitumor activities were investigated on 9 strains of tumor cells and IC50 of the order of 1 µM were obtained on A431 epidermoid carcinoma cells and B16F10 skin melanoma cells. In addition, studies of ovarian tumor cell inhibitions show an interesting activity in regard to the need for new drugs for this pathology. Finally, cytotoxicity and mouse toxicity studies reveal a favorable therapeutic index for SRO-91.

Evaluation of the potential of a new ribavirin analog impairing the dissemination of ovarian cancer cells.

Epithelial ovarian cancers are insidious pathologies that give a poor prognosis due to their late discovery and the increasing emergence of chemoresistance. Development of small pharmacological anticancer molecules remains a major challenge. Ribavirin, usually used in the treatment of hepatitis C virus infections and more recently few cancers, has been a suggestion. However, Ribavirin has many side-effects, suggesting that the synthesis of analogs might be more appropriate. We have investigated the effect of a Ribavirin analog, SRO-91, on cancer cell behavioral characteristics considered as some of the hallmarks of cancer. Two human ovarian adenocarcinoma cell lines (SKOV3 and IGROV1) and normal cells (mesothelial and fibroblasts) have been used to compare the effects of SRO-91 with those of Ribavirin on cell behavior underlying tumor cell dissemination. SRO-91, like Ribavirin, inhibits proliferation, migration, clonogenicity and spheroids formation of cancer cells. Unlike Ribavirin, SRO-91 is preferentially toxic to cancer compared with normal cells. An in vitro physiologically relevant model showed that SRO-91, like Ribavirin or cisplatin, inhibits cancer cell implantation onto peritoneal mesothelium. In conclusion, SRO-91 analog effects on tumor dissemination and its safety regarding non-cancerous (normal) cells are encouraging findings a promising drug for the treatment of ovarian cancer.

Studies of membranotropic and fusogenic activity of two putative HCV fusion peptides.

Over the past decades, membranotropic peptides such as positively charged cell-penetrating peptides (CPPs) or amphipathic antimicrobial peptides (AMPs) have received increasing interest in order to improve therapeutic agent cellular uptake. As far as we are concerned, we were interested in studying HCV fusion peptides as putative anchors. Two peptides, HCV6 and HCV7, were identified and conjugated to a fluorescent tag NBD and tested for their interaction with liposomes as model membranes. DSC and spectrofluorescence analyses demonstrate HCV7 propensity to insert or internalize in vesicles containing anionic lipids DMPG whereas no activity was observed with zwitterionic DMPC. This behavior could be explained by the peptide sequence containing a cationic arginine residue. On the contrary, HCV6 did not exhibit any membranotropic activity but was the only sequence able to induce liposomes' fusion or aggregation monitored by spectrofluorescence and DLS. This two peptides mild activity was related to their inefficient structuration in contact with membrane mimetics, which was demonstrated by CD and NMR experiments. Altogether, our data allowed us to identify two promising membrane-active peptides from E1 and E2 HCV viral proteins, one fusogenic (HCV6) and the other membranotropic (HCV7). The latter was also confirmed by fluorescence microscopy with CHO cells, indicating that HCV7 could cross the plasma membrane via an endocytosis process. Therefore, this study provides new evidences supporting the identification of HCV6 as the HCV fusion peptide as well as insights on a novel membranotropic peptide from the HCV-E2 viral protein.

Fibronectin amyloid-like aggregation alters its extracellular matrix incorporation and promotes a single and sparsed cell migration.

Fibronectin (Fn) is an extracellular matrix (ECM) multifunctional glycoprotein essential for regulating cells behaviors. Within ECM, Fn is found as polymerized fibrils. Apart from fibrils, Fn could also form other kind of supramolecular assemblies such as aggregates. To gain insight into the impact of Fn aggregates on cell behavior, we generated several Fn oligomeric assemblies. These assemblies displayed various amyloid-like properties but were not cytotoxic. In presence of the more amyloid-like structured assemblies of Fn, the cell-ECM networks were altered and the cell shapes shifted toward extended mesenchymal morphologies. Additionnaly, the Fn amyloid-like aggregates promoted a single-cell and sparsed migration of SKOV3 cancer cells, which was associated with a relocalization of αv integrins from plasma membrane to perinuclear vesicles. These data pointed out that the features of supramolecular Fn assemblies could represent a higher level of fine-tuning cell phenotype, and especially migration of cancer cells.

Regulation of Extracellular Matrix Synthesis by Shell Extracts from the Marine Bivalve Pecten maximus in Human Articular Chondrocytes- Application for Cartilage Engineering.

The shells of the bivalve mollusks are organo-mineral structures predominantly composed of calcium carbonate, but also of a minor organic matrix, a mixture of proteins, glycoproteins, and polysaccharides. These proteins are involved in mineral deposition and, more generally, in the spatial organization of the shell crystallites in well-defined microstructures. In this work, we extracted different organic shell extracts (acid-soluble matrix, acid-insoluble matrix, water-soluble matrix, guanidine HCl/EDTA-extracted matrix, referred as ASM, AIM, WSM, and EDTAM, respectively) from the shell of the scallop Pecten maximus and studied their biological activities on human articular chondrocytes (HACs). We found that these extracts differentially modulate the biological activities of HACs, depending on the type of extraction and the concentration used. Furthermore, we showed that, unlike ASM and AIM, WSM promotes maintenance of the chondrocyte phenotype in monolayer culture. WSM increased the expression of chondrocyte-specific markers (aggrecan and type II collagen), without enhancing that of the main chondrocyte dedifferentiation marker (type I collagen). We also demonstrated that WSM could favor redifferentiation of chondrocyte in collagen sponge scaffold in hypoxia. Thus, this study suggests that the organic matrix of Pecten maximus, particularly WSM, may contain interesting molecules with chondrogenic effects. Our research emphasizes the potential use of WSM of Pecten maximus for cell therapy of cartilage.

Shell extracts of the edible mussel and oyster induce an enhancement of the catabolic pathway of human skin fibroblasts, in vitro.

Mollusc shells are composed of more than 95% calcium carbonate and less than 5% organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. In this study, we investigated the effects of matrix macromolecular components extracted from the shells of two edible molluscs of economic interest, i.e., the blue mussel Mytilus edulis and the Pacific oyster Crassostrea gigas. The potential biological activities of these organic molecules were analysed on human dermal fibroblasts in primary culture. Our results demonstrate that shell extracts of the two studied molluscs modulate the metabolic activities of the cells. In addition, the extracts caused a decrease of type I collagen and a concomitant increase of active MMP-1, both at the mRNA and the protein levels. Therefore, our results suggest that shell extracts from M. edulis and C. gigas contain molecules that promote the catabolic pathway of human dermal fibroblasts. This work emphasises the potential use of these shell matrices in the context of anti-fibrotic strategies, particularly against scleroderma. More generally, it stresses the usefulness to valorise bivalve shells that are coproducts of shellfish farming activity.

New synthesis method of HA/P(D,L)LA composites: study of fibronectin adsorption and their effects in osteoblastic behavior for bone tissue engineering.

A novel synthetic method to synthesize hydroxyapatite/poly (D,L) lactic acid biocomposite is presented in this study by mixing only the precursors hydroxyapatite and (D,L) LA monomer without adding neither solvent nor catalyst. Three compositions were successfully synthesized with the weight ratios of 1/1, 1/3, and 3/5 (hydroxyapatite/(D,L) lactic acid), and the grafting efficiency of poly (D,L) lactic acid on hydroxyapatite surface reaches up to 84 %. Scanning electron microscopy and Fourier transform infrared spectroscopy showed that the hydroxyapatite particles were successfully incorporated into the poly (D,L) lactic acid polymer and X ray diffraction analysis showed that hydroxyapatite preserved its crystallinity after poly (D,L) lactic acid grafting. Differential scanning calorimetry shows that Tg of hydroxyapatite/poly (D,L) lactic acid composite is less than Tg of pure poly (D,L) lactic acid, which facilitates the shaping of the composite obtained. The addition of poly (D,L) lactic acid improves the adsorption properties of hydroxyapatite for fibronectin extracellular matrix protein. Furthermore, the presence of poly (D,L) lactic acid on hydroxyapatite surface coated with fibronectin enhanced pre-osteoblast STRO-1 adhesion and cell spreading. These results show the promising potential of hydroxyapatite/poly (D,L) lactic acid composite as a bone substitute material for orthopedic applications and bone tissue engineering.

Application of APTES-Anti-E-cadherin film for early cancer monitoring.

Cancer staging is a way to classify cancer according to the extent of the disease in the body. The stage is usually determined by several factors such as the location of the primary tumor, the tumor size, the degree of spread in the surrounding tissues, etc. The study of E-cadherin (EC) expression on cancerous cells of patients has revealed variations in the molecular expression patterns of primary tumors and metastatic tumors. The detection of these cells requires a long procedure involving conventional techniques, thus, the requirement for development of new rapid devices that permit direct and highly sensitive detection stimulates the sensing field progress. Here, we explore if E-cadherin could be used as a biomarker to bind and detect epithelial cancer cells. Hence, the sensitive and specific detection of E-cadherin expressed on epithelial cells is approached by immobilizing anti-E-cadherin antibody (AEC) onto aminosilanized indium-tin oxide (ITO) surface. The immunosensing surfaces have been characterized by electrochemical measurements, wettability and confocal microscopy and their performance has been assessed in the presence of cancer cell lines. Under optimal conditions, the resulting immunosensor displayed a selective detection of E-cadherin expressing cells, which could be detected either by fluorescence or electrochemical techniques. The developed immunosensing surface could provide a simple tool that can be applied to cancer staging.

Glioma cell dispersion is driven by α5 integrin-mediated cell-matrix and cell-cell interactions.

Glioblastoma multiform (GBM) is the most common and most aggressive primary brain tumor. The fibronectin receptor, α5 integrin is a pertinent novel therapeutic target. Despite numerous data showing that α5 integrin support tumor cell migration and invasion, it has been reported that α5 integrin can also limit cell dispersion by increasing cell-cell interaction. In this study, we showed that α5 integrin was involved in cell-cell interaction and gliomasphere formation. α5-mediated cell-cell cohesion limited cell dispersion from spheroids in fibronectin-poor microenvironment. However, in fibronectin-rich microenvironment, α5 integrin promoted cell dispersion. Ligand-occupied α5 integrin and fibronectin were distributed in fibril-like pattern at cell-cell junction of evading cells, forming cell-cell fibrillar adhesions. Activated focal adhesion kinase was not present in these adhesions but was progressively relocalized with α5 integrin as cell migrates away from the spheroids. α5 integrin function in GBM appears to be more complex than previously suspected. As GBM overexpressed fibronectin, it is most likely that in vivo, α5-mediated dissemination from the tumor mass overrides α5-mediated tumor cell cohesion. In this respect, α5-integrin antagonists may be useful to limit GBM invasion in brain parenchyma.

Effects of human fibronectin and human serum albumin sequential adsorption on preosteoblastic cell adhesion.

Fibronectin (Fn) is widely reported to promote cell adhesion and spreading, and recent reports attest to the synergistic effect of coadsorbed albumin (unexpected due to the passivating character of the latter protein). In this study, the sequential adsorption of fibronectin and albumin, and the morphology of cultured MC3T3-E1 preosteoblastic cells are investigated on three important biomaterial surfaces: silicon oxide, poly(styrene) (PS), and hydroxyapatite (HA). Using quartz crystal microgravimetry with dissipation analysis, the adsorbed protein composition and mechanics are determined. Interestingly, cell morphological changes correlate neither with the amount of Fn nor the rigidity of the protein layer. On the PS surface, Alb is seen to significantly diminish cell spreading, possibly due to Alb aggregation with a partially denatured initially placed Fn layer. HA appears to be a particularly favorable substrate for osteoblast adhesion, despite having low Fn adsorption and protein layer rigidity.

Shell extracts from the marine bivalve Pecten maximus regulate the synthesis of extracellular matrix in primary cultured human skin fibroblasts.

Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the -112/-61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications.

Cell cycle arrest or survival signaling through αv integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids.

Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation. The above data indicate that ovarian cancer cells can resist anoikis in the spheroid state by arrest in the cell cycle or through activation of αv-integrin-ERK-mediated survival signals. Such signaling might result in the selection of resistant cells within disseminating spheroids, favoring further relapse in ovarian cancers.

Ovarian cancer ascites-derived vitronectin and fibronectin: combined purification, molecular features and effects on cell response.

BACKGROUND: Intra-abdominal ascites is a complication of ovarian cancers and constitutes a permissive microenvironment for metastasis. Since fibronectin and vitronectin are key actors in ovarian cancer progression, we investigated their occurrence and molecular characteristics in various ascites fluids and the influence of these ascites-derived proteins on cell behavior. METHODS: Fibronectin and vitronectin were investigated by immunoblotting within various ascites fluids. A combined affinity-based protocol was developed to purify both proteins from the same sample. Each purified protein was characterized with regard to its molecular features (molecular mass of isoforms, tryptophan intramolecular environment, hydrodynamic radii), and its influence on cell adhesion. RESULTS: Fibronectin and vitronectin were found in all tested ascites. Several milligrams of purified proteins were obtained from ascites of varying initial volumes. Molecular mass isoforms and conformational lability of proteins differed according to the ascites of origin. When incorporated into the cancer cell environment, ascites-derived fibronectin and vitronectin supported cell adhesion and migration with various degrees of efficiency, and induced the recruitment of integrins into focal contacts. CONCLUSIONS: To our knowledge, this is the first combined purification of two extracellular matrix proteins from a single pathological sample containing a great variety of bioactive molecules. This study highlights that ascites-derived fibronectin and vitronectin exhibit different properties depending on the ascites. GENERAL SIGNIFICANCE: Investigating the relationships between the molecular properties of ascites components and ovarian cancer cell phenotype according to the ascites may be critical for a better understanding of the recurrence of this lethal disease and for further biomarker identification.