Enrichment, Characterization, and Proteomic Profiling of Small Extracellular Vesicles Derived from Human Limbal Mesenchymal Stromal Cells and Melanocytes.

Limbal epithelial progenitor cells (LEPC) rely on their niche environment for proper functionality and self-renewal. While extracellular vesicles (EV), specifically small EVs (sEV), have been proposed to support LEPC homeostasis, data on sEV derived from limbal niche cells like limbal mesenchymal stromal cells (LMSC) remain limited, and there are no studies on sEVs from limbal melanocytes (LM). In this study, we isolated sEV from conditioned media of LMSC and LM using a combination of tangential flow filtration and size exclusion chromatography and characterized them by nanoparticle tracking analysis, transmission electron microscopy, Western blot, multiplex bead arrays, and quantitative mass spectrometry. The internalization of sEV by LEPC was studied using flow cytometry and confocal microscopy. The isolated sEVs exhibited typical EV characteristics, including cell-specific markers such as CD90 for LMSC-sEV and Melan-A for LM-sEV. Bioinformatics analysis of the proteomic data suggested a significant role of sEVs in extracellular matrix deposition, with LMSC-derived sEV containing proteins involved in collagen remodeling and cell matrix adhesion, whereas LM-sEV proteins were implicated in other cellular bioprocesses such as cellular pigmentation and development. Moreover, fluorescently labeled LMSC-sEV and LM-sEV were taken up by LEPC and localized to their perinuclear compartment. These findings provide valuable insights into the complex role of sEV from niche cells in regulating the human limbal stem cell niche.

MIBlood-EV: Minimal information to enhance the quality and reproducibility of blood extracellular vesicle research.

Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor-dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre-analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood-EV), a tool to record and report information about pre-analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre-analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence-based optimization of pre-analytical protocols through meta-analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter-laboratory comparative EV studies, and (iv) improve the peer review process.

Tspan6 stimulates the chemoattractive potential of breast cancer cells for B cells in an EV- and LXR-dependent manner.

The immune microenvironment in breast cancer (BCa) is controlled by a complex network of communication between various cell types. Here, we find that recruitment of B lymphocytes to BCa tissues is controlled via mechanisms associated with cancer cell-derived extracellular vesicles (CCD-EVs). Gene expression profiling identifies the Liver X receptor (LXR)-dependent transcriptional network as a key pathway that controls both CCD-EVs-induced migration of B cells and accumulation of B cells in BCa tissues. The increased accumulation oxysterol ligands for LXR (i.e., 25-hydroxycholesterol and 27-hydroxycholesterol) in CCD-EVs is regulated by the tetraspanin 6 (Tspan6). Tspan6 stimulates the chemoattractive potential of BCa cells for B cells in an EV- and LXR-dependent manner. These results demonstrate that tetraspanins control intercellular trafficking of oxysterols via CCD-EVs. Furthermore, tetraspanin-dependent changes in the oxysterol composition of CCD-EVs and the LXR signaling axis play a key role in specific changes in the tumor immune microenvironment.

Airway basal cells show a dedifferentiated KRT17highPhenotype and promote fibrosis in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. In this study, we focus on the properties of airway basal cells (ABC) obtained from patients with IPF (IPF-ABC). Single cell RNA sequencing (scRNAseq) of bronchial brushes revealed extensive reprogramming of IPF-ABC towards a KRT17high PTENlow dedifferentiated cell type. In the 3D organoid model, compared to ABC obtained from healthy volunteers, IPF-ABC give rise to more bronchospheres, de novo bronchial structures resembling lung developmental processes, induce fibroblast proliferation and extracellular matrix deposition in co-culture. Intratracheal application of IPF-ABC into minimally injured lungs of Rag2-/- or NRG mice causes severe fibrosis, remodeling of the alveolar compartment, and formation of honeycomb cyst-like structures. Connectivity MAP analysis of scRNAseq of bronchial brushings suggested that gene expression changes in IPF-ABC can be reversed by SRC inhibition. After demonstrating enhanced SRC expression and activity in these cells, and in IPF lungs, we tested the effects of saracatinib, a potent SRC inhibitor previously studied in humans. We demonstrate that saracatinib modified in-vitro and in-vivo the profibrotic changes observed in our 3D culture system and novel mouse xenograft model.

Highly Sensitive Nanomagnetic Quantification of Extracellular Vesicles by Immunochromatographic Strips: A Tool for Liquid Biopsy.

Extracellular vesicles (EVs) are promising agents for liquid biopsy-a non-invasive approach for the diagnosis of cancer and evaluation of therapy response. However, EV potential is limited by the lack of sufficiently sensitive, time-, and cost-efficient methods for their registration. This research aimed at developing a highly sensitive and easy-to-use immunochromatographic tool based on magnetic nanoparticles for EV quantification. The tool is demonstrated by detection of EVs isolated from cell culture supernatants and various body fluids using characteristic biomarkers, CD9 and CD81, and a tumor-associated marker-epithelial cell adhesion molecules. The detection limit of 3.7 × 105 EV/µL is one to two orders better than the most sensitive traditional lateral flow system and commercial ELISA kits. The detection specificity is ensured by an isotype control line on the test strip. The tool's advantages are due to the spatial quantification of EV-bound magnetic nanolabels within the strip volume by an original electronic technique. The inexpensive tool, promising for liquid biopsy in daily clinical routines, can be extended to other relevant biomarkers.

Efficient Small Extracellular Vesicles (EV) Isolation Method and Evaluation of EV-Associated DNA Role in Cell-Cell Communication in Cancer.

Small extracellular vesicles (sEVs) play essential roles in intercellular signaling both in normal and pathophysiological conditions. Comprehensive studies of dsDNA associated with sEVs are hampered by a lack of methods, allowing efficient separation of sEVs from free-circulating DNA and apoptotic bodies. In this work, using controlled culture conditions, we enriched the reproducible separation of sEVs from free-circulated components by combining tangential flow filtration, size-exclusion chromatography, and ultrafiltration (TSU). EV-enriched fractions (F2 and F3) obtained using TSU also contained more dsDNA derived from the host genome and mitochondria, predominantly localized inside the vesicles. Three-dimensional reconstruction of high-resolution imaging showed that the recipient cell membrane barrier restricts a portion of EV-DNA. Simultaneously, the remaining EV-DNA overcomes it and enters the cytoplasm and nucleus. In the cytoplasm, EV-DNA associates with dsDNA-inflammatory sensors (cGAS/STING) and endosomal proteins (Rab5/Rab7). Relevant to cancer, we found that EV-DNA isolated from leukemia cell lines communicates with mesenchymal stromal cells (MSCs), a critical component in the BM microenvironment. Furthermore, we illustrated the arrangement of sEVs and EV-DNA at a single vesicle level using super-resolution microscopy. Altogether, employing TSU isolation, we demonstrated EV-DNA distribution and a tool to evaluate the exact EV-DNA role of cell-cell communication in cancer.

Rapid Capture of Cancer Extracellular Vesicles by Lipid Patch Microarrays.

Extracellular vesicles (EVs) contain various bioactive molecules such as DNA, RNA, and proteins, and play a key role in the regulation of cancer progression. Furthermore, cancer-associated EVs carry specific biomarkers and can be used in liquid biopsy for cancer detection. However, it is still technically challenging and time consuming to detect or isolate cancer-associated EVs from complex biofluids (e.g., blood). Here, a novel EV-capture strategy based on dip-pen nanolithography generated microarrays of supported lipid membranes is presented. These arrays carry specific antibodies recognizing EV- and cancer-specific surface biomarkers, enabling highly selective and efficient capture. Importantly, it is shown that the nucleic acid cargo of captured EVs is retained on the lipid array, providing the potential for downstream analysis. Finally, the feasibility of EV capture from patient sera is demonstrated. The demonstrated platform offers rapid capture, high specificity, and sensitivity, with only a small need in analyte volume and without additional purification steps. The platform is applied in context of cancer-associated EVs, but it can easily be adapted to other diagnostic EV targets by use of corresponding antibodies.

Surface-Enhanced Raman Spectroscopy to Characterize Different Fractions of Extracellular Vesicles from Control and Prostate Cancer Patients.

Extracellular vesicles (EVs) are membrane-enclosed structures ranging in size from about 60 to 800 nm that are released by the cells into the extracellular space; they have attracted interest as easily available biomarkers for cancer diagnostics. In this study, EVs from plasma of control and prostate cancer patients were fractionated by differential centrifugation at 5000× g, 12,000× g and 120,000× g. The remaining supernatants were purified by ultrafiltration to produce EV-depleted free-circulating (fc) fractions. Spontaneous Raman and surface-enhanced Raman spectroscopy (SERS) at 785 nm excitation using silver nanoparticles (AgNPs) were employed as label-free techniques to collect fingerprint spectra and identify the fractions that best discriminate between control and cancer patients. SERS spectra from 10 µL droplets showed an enhanced Raman signature of EV-enriched fractions that were much more intense for cancer patients than controls. The Raman spectra of dehydrated pellets of EV-enriched fractions without AgNPs were dominated by spectral contributions of proteins and showed variations in S-S stretch, tryptophan and protein secondary structure bands between control and cancer fractions. We conclude that the AgNPs-mediated SERS effect strongly enhances Raman bands in EV-enriched fractions, and the fractions, EV12 and EV120 provide the best separation of cancer and control patients by Raman and SERS spectra.

Wnt5A modulates integrin expression in a receptor-dependent manner in ovarian cancer cells.

Wnt5A signals through various receptors that confer versatile biological functions. Here, we used Wnt5A overexpressing human ovarian SKOV-3 and OVCAR-3 stable clones for assessing integrin expression, cell proliferation, migration, invasion, and the ability of multicellular aggregates (MCAs) formation. We found here, that Wnt5A regulates differently the expression of its receptors in the stable Wnt5A overexpressing clones. The expression levels of Frizzled (FZD)-2 and -5, were increased in different clones. However ROR-1, -2 expression levels were differently regulated in clones. Wnt5A overexpressing clones showed increased cell proliferation, migration, and clonogenicity. Moreover, Wnt5A overexpressing SKOV-3 clone showed increased MCAs formation ability. Cell invasion had been increased in OVCAR-3-derived clones, while this was decreased in SKOV-3-derived clone. Importantly, αv integrin expression levels were increased in all assessed clones, accompanied by increased cell attachment to fibronectin and focal adhesion kinase activity. Moreover, the treatment of clones with Box5 as a Wnt5A/FZD5 antagonist abrogates ITGAV increase, cell proliferation, migration, and their attachment to fibronectin. Accordingly, we observed significantly higher expression levels of ITGAV and ITGB3 in human high-grade serous ovarian cancer specimens and ITGAV correlated positively with Wnt5A in metastatic serous type ovarian cancer. In summary, we hypothesize here, that Wnt5A/FZD-5 signaling modulate αv integrin expression levels that could be associated with ovarian cancer cell proliferation, migration, and fibronectin attachment.

The CD151-midkine pathway regulates the immune microenvironment in inflammatory breast cancer.

The immune microenvironment in inflammatory breast cancer (IBC) is poorly characterised, and molecular and cellular pathways that control accumulation of various immune cells in IBC tissues remain largely unknown. Here, we discovered a novel pathway linking the expression of the tetraspanin protein CD151 in tumour cells with increased accumulation of macrophages in cancerous tissues. It is notable that elevated expression of CD151 and a higher number of tumour-infiltrating macrophages correlated with better patient responses to chemotherapy. Accordingly, CD151-expressing IBC xenografts were characterised by the increased infiltration of macrophages. In vitro migration experiments demonstrated that CD151 stimulates the chemoattractive potential of IBC cells for monocytes via mechanisms involving midkine (a heparin-binding growth factor), integrin α6ß1, and production of extracellular vesicles (EVs). Profiling of chemokines secreted by IBC cells demonstrated that CD151 increases production of midkine. Purified midkine specifically stimulated migration of monocytes, but not other immune cells. Further experiments demonstrated that the chemoattractive potential of IBC-derived EVs is blocked by anti-midkine antibodies. These results demonstrate for the first time that changes in the expression of a tetraspanin protein by tumour cells can affect the formation of the immune microenvironment by modulating recruitment of effector cells to cancerous tissues. Therefore, a CD151-midkine pathway can be considered as a novel target for controlled changes of the immune landscape in IBC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Three-dimensional cell models for extracellular vesicles production, isolation, and characterization.

Extracellular vesicles (EVs) play a pivotal role in cancer progression. However, the majority of functional studies performed so far relies on data acquired in traditional 2D cultures. Because the spatial architecture of tissue is decisive for the cell fate, new cell models to study EV functions in the 3D environment must approximate in vitro models to the physiological conditions. Several models were developed during the last years, which may be suitable to serve as 3D models to study EVs; among them are hydrogels, solid scaffolds, bioreactors, and 3D CoSeedis™ inserts. We present in this chapter a protocol for a 3D cell model based on the 3D CoSeedis™ agarose inserts, allowing for a long-term culture of cells of different origins under serum-free conditions and easy EV recovery. Additionally, information on individual culture conditions in 3D CoSeedis™ for different cell lines, protocols for model evaluation, and quality controls are included. We hope that our suggestions and experience will be useful to carry out EV study under more physiological conditions and contribute to the EV research field's progress.

Biodegradable Nanocarriers Resembling Extracellular Vesicles Deliver Genetic Material with the Highest Efficiency to Various Cell Types.

Efficient delivery of genetic material to primary cells remains challenging. Here, efficient transfer of genetic material is presented using synthetic biodegradable nanocarriers, resembling extracellular vesicles in their biomechanical properties. This is based on two main technological achievements: generation of soft biodegradable polyelectrolyte capsules in nanosize and efficient application of the nanocapsules for co-transfer of different RNAs to tumor cell lines and primary cells, including hematopoietic progenitor cells and primary T cells. Near to 100% efficiency is reached using only 2.5 × 10-4 pmol of siRNA, and 1 × 10-3 nmol of mRNA per cell, which is several magnitude orders below the amounts reported for any of methods published so far. The data show that biodegradable nanocapsules represent a universal and highly efficient biomimetic platform for the transfer of genetic material with the utmost potential to revolutionize gene transfer technology in vitro and in vivo.

Extracellular Vesicles: Recent Developments in Technology and Perspectives for Cancer Liquid Biopsy.

Extracellular micro- and nanoscale membrane vesicles produced by different cells progressively attract the attention of the scientific community. They function as mediators of intercellular communication and transport genetic material and signaling molecules between the cells. In the context of keeping homeostasis, the extracellular vesicles contribute to the regulation of various systemic and local processes. Vesicles released by the tumor and activated stromal cells exhibit multiple functions including support of tumor growth, preparation of the pre-metastatic niches, and immune suppression. Considerable progress has been made regarding the criteria of classification of the vesicles according to their origin, content, and function: Exosomes, microvesicles, also referred to as microparticles or ectosomes, and large oncosomes were defined as actively released vesicles. Additionally, apoptotic bodies represented by a highly heterogeneous population of particles produced during apoptosis, the programmed cell death, should be considered. Because the majority of isolation techniques do not allow the separation of different types of vesicles, a joined term "extracellular vesicles" (EVs) was recommended by the ISEV community for the definition of vesicles isolated from either the cell culture supernatants or the body fluids. Because EV content reflects the content of the cell of origin, multiple studies on EVs from body fluids in the context of cancer diagnosis, prediction, and prognosis were performed, actively supporting their high potential as a biomarker source. Here, we review the leading achievements in EV analysis from body fluids, defined as EV-based liquid biopsy, and provide an overview of the main EV constituents: EV surface proteins, intravesicular soluble proteins, EV RNA including mRNA and miRNA, and EV DNA as potential biomarkers. Furthermore, we discuss recent developments in technology for quantitative EV analysis in the clinical setting and future perspectives toward miniaturized high-precision liquid biopsy approaches.

Tspan8 is expressed in breast cancer and regulates E-cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles.

Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8- tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up-regulation of E-cadherin and down-regulation of Twist, p120-catenin, and ß-catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal-epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell-cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several-fold increase in EV number in cell culture and the circulation of tumour-bearing animals. We observed increased protein levels of E-cadherin and p120-catenin in these EVs; furthermore, Tspan8 and p120-catenin were co-immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

3D Cellular Architecture Affects MicroRNA and Protein Cargo of Extracellular Vesicles.

The success of malignant tumors is conditioned by the intercellular communication between tumor cells and their microenvironment, with extracellular vesicles (EVs) acting as main mediators. While the value of 3D conditions to study tumor cells is well established, the impact of cellular architecture on EV content and function is not investigated yet. Here, a recently developed 3D cell culture microwell array is adapted for EV production and a comprehensive comparative analysis of biochemical features, RNA and proteomic profiles of EVs secreted by 2D vs 3D cultures of gastric cancer cells, is performed. 3D cultures are significantly more efficient in producing EVs than 2D cultures. Global upregulation of microRNAs and downregulation of proteins in 3D are observed, indicating their dynamic coregulation in response to cellular architecture, with the ADP-ribosylation factor 6 signaling pathway significantly downregulated in 3D EVs. The data strengthen the biological relevance of cellular architecture for production and cargo of EVs.

Porphyria cutanea tarda and hepatoerythropoietic porphyria: Identification of 19 novel uroporphyrinogen III decarboxylase mutations.

Porphyria Cutanea Tarda (PCT) is a cutaneous porphyria that results from the hepatic inhibition of the heme biosynthetic enzyme uroporphyrinogen decarboxylase (UROD), and can occur either in the absence or presence of an inherited heterozygous UROD mutation (PCT subtypes 1 and 2, respectively). A heterozygous UROD mutation causes half-normal levels of UROD activity systemically, which is a susceptibility factor but is not sufficient alone to cause type 2 PCT. In both Types 1 and 2 PCT, the cutaneous manifestations are precipitated by additional factors that lead to generation of an inhibitor that more profoundly reduces hepatic UROD activity. PCT is an iron-related disorder, and many of its known susceptibility factors, which include infections (e.g. hepatitis C virus, HIV), high alcohol consumption, smoking, estrogens, and genetic traits (e.g. hemochromatosis mutations) can increase hepatic iron accumulation. Hepatoerythropoietic Porphyria (HEP) is a rare autosomal recessive disease that results from homozygosity or compound heterozygosity for UROD mutations and often causes infantile or childhood onset of both erythropoietic and cutaneous manifestations. During the 11-year period from 01/01/2007 through 12/31/2017, the Mount Sinai Porphyrias Diagnostic Laboratory provided molecular diagnostic testing for 387 unrelated patients with PCT and four unrelated patients with HEP. Of the 387 unrelated individuals tested for Type 2 PCT, 79 (20%) were heterozygous for UROD mutations. Among 26 family members of mutation-positive PCT patients, eight (31%) had the respective family mutation. Additionally, of the four unrelated HEP patients referred for UROD mutation analyses, all had homozygosity or compound heterozygosity for UROD mutations, and all eight asymptomatic family members were heterozygotes for UROD mutations. Of the UROD mutations identified, 19 were novel, including nine missense, two nonsense, one consensus splice-site, and seven insertions and deletions. These results expand the molecular heterogeneity of PCT and HEP by adding a total of 19 novel UROD mutations. Moreover, the results document the usefulness of molecular testing to confirm a genetic susceptibility trait in Type 2 PCT, confirm a diagnosis in HEP, and identify heterozygous family members.

Extracellular vesicles or free circulating DNA: where to search for BRAF and cKIT mutations?

Clinical evidence in oncology argues for the advantages of performing molecular analysis of blood biomarkers to provide information about systemic changes and tumor heterogeneity. Whereas the diagnostic value of cell-free circulating DNA (fcDNA) has successfully been demonstrated in several studies, DNA enclosed in extracellular vesicles (EV) has only recently been described, and its potential diagnostic value is unclear. We established a protocol for separation of EV and fc fractions and tested for presence of mutant BRAFV600E mediating resistance to Vemurafenib and cKITD816V mediating resistance to Imatinib in blood of patients with melanoma and mastocytosis. Our results show that EV contain significantly higher amounts of total DNA as compared to the fc fraction. However, about ten-fold higher copy numbers of the wild type and mutant BRAF and cKIT were detected in the fcDNA fraction supporting its diagnostic value and pointing to differences in fc and EV DNA content.

A specific spectral signature of serum and plasma-derived extracellular vesicles for cancer screening.

In cancer, extracellular vesicles (EV) contribute to tumor progression by regulating local and systemic effects. Being released into body fluids, EV may be used in nanomedicine as a valuable source for diagnostic biomarkers. In this work, infrared and Raman spectroscopy were used for comprehensive comparative analysis of cancer versus non-cancer EV and patient screening. Two different EV fractions enriched in exosomes and microvesicles were isolated by differential centrifugation from serum and plasma of cancer and non-cancer patients and from serum and plasma of a healthy donor. The EV fractions were then subjected to drop-coating deposition and drying on calcium fluoride substrates. Reduction of alpha-helix-rich proteins and enhancement of beta-sheet-rich proteins as a cancer-specific blood EV signature was determined, and subsequently this feature was applied for a pilot study aiming to detect prostate cancer in a test cohort of patients with high-grade prostate carcinoma and benign hypoplasia.

Collaborative Action of Surface Chemistry and Topography in the Regulation of Mesenchymal and Epithelial Markers and the Shape of Cancer Cells.

Malignant transformation is associated with enhancement of cell plasticity, which allows cancer cells to survive under different conditions by adapting to their microenvironment during growth and metastatic spread. Much effort has been devoted to understanding the molecular mechanisms of these processes. Although the importance of the extracellular matrix and of surface properties in these mechanisms is evident, the direct impact of distinct physical and chemical surfaces characteristics on cell fate remains unclear. Here, we have addressed this question using HT1080 fibrosarcoma cells as a model. To examine the relationship between surface topography, chemistry, and cell behavior, hydrophobic poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA-EDMA) and hydrophilic poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) surfaces with three different topographies (microporous, nanoporous, and nonporous) were generated. These surfaces were then modified by photoinitiated grafting of three different methacrylate monomers to create surface chemistry gradients of either negatively (AMPS) or positively (META) charged or zwitterionic (MDSA) functionalities. Our results show that AMPS promotes cell spreading, but that META abolishes cell growth. META and MDSA grafted on microporous BMA-EDMA produced superhydrophilic surfaces with high globularity and elasticity, which modified the cell phenotype by inhibiting cell spreading, followed by loss of mesenchymal characteristics and a reduction in protein levels of the mesenchymal markers N-cadherin, beta-catenin, p120 catenin, and also of the adaptor proteins vinculin and paxillin that are associated with adhesion and cancer cell invasion. The effect was strengthened along the gradient, suggesting that the density of the functional groups plays a role in this process. On the nanoporous surface, only MDSA grafting resulted in a significant increase in cell number, a reduction in N-cadherin expression, increased beta-catenin and p120 catenin levels, as well as the appearance of the epithelial marker E-cadherin. This indicates that the cancer cells have a high plasticity that is triggered by the collaborative effect of physical and chemical surface properties.

Extracellular vesicles in ovarian cancer: applications to tumor biology, immunotherapy and biomarker discovery.

In recent years there has been tremendous interest in both the basic biology and applications of extracellular vesicles (EVs) in translational cancer research. This includes a better understanding of their biogenesis and mechanisms of selective cargo packaging, their precise roles in horizontal communication, and their application as non-invasive biomarkers. The rapid advances in next-generation omics technologies are the driving forces for these discoveries. In this review, the authors focus on recent results of EV research in ovarian cancer. A deeper understanding of ovarian cancer-derived EVs, the types of cargo molecules and their biological roles in cancer growth, metastases and drug resistance, could have significant impact on the discovery of novel biomarkers and innovative therapeutics. Insights into the role of EVs in immune regulation could lead to novel approaches built on EV-based immunotherapy.