Hyaluronic Acid and Large Extracellular Vesicles (EVs) in Synovial Fluid and Plasma of Patients With End-Stage Arthritis: Positive Association of EVs to Joint Pain.

OBJECTIVE: Hyaluronic acid (HA) in synovial fluid (SF) contributes to boundary lubrication with altered levels in osteoarthritis (OA) and rheumatoid arthritis (RA). SF extracellular vesicles (EVs) may participate in arthritis by affecting inflammation and cartilage degradation. It remains unknown whether HA and EVs display joint-specific alterations in arthritic SFs. DESIGN: We investigated the numbers and characteristics of HA-particles and large EVs in SF from knees and shoulders of 8 OA and 8 RA patients and 8 trauma controls, and in plasma from 10 healthy controls and 11 knee OA patients. The plasma and SF HA concentrations were determined with a sandwich-type enzyme-linked sorbent assay, and EVs and HA-particles were characterized from plasma and unprocessed and centrifuged SFs with confocal microscopy. The data were compared according to diagnosis, location, and preanalytical processing. RESULTS: The main findings were: (1) OA and RA SFs can be distinguished from trauma joints based on the distinctive profiles of HA-particles and large EVs, (2) there are differences in the SF HA and EV characteristics between shoulder and knee joints that could reflect their dissimilar mobility, weight-bearing, and shock absorption properties, (3) EV counts in SF and plasma can positively associate with pain parameters independent of age and body adiposity, and (4) low-speed centrifugation causes alterations in the features of HA-particles and EVs, complicating their examination in the original state. CONCLUSIONS: Arthritis and anatomical location can affect the characteristics of HA-particles and large EVs that may have potential as biomarkers and effectors in joint degradation and pain.

DPYSL5 is highly expressed in treatment-induced neuroendocrine prostate cancer and promotes lineage plasticity via EZH2/PRC2.

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is a lethal subtype of castration-resistant prostate cancer resistant to androgen receptor (AR) inhibitors. Our study unveils that AR suppresses the neuronal development protein dihydropyrimidinase-related protein 5 (DPYSL5), providing a mechanism for neuroendocrine transformation under androgen deprivation therapy. Our unique CRPC-NEPC cohort, comprising 135 patient tumor samples, including 55 t-NEPC patient samples, exhibits a high expression of DPYSL5 in t-NEPC patient tumors. DPYSL5 correlates with neuroendocrine-related markers and inversely with AR and PSA. DPYSL5 overexpression in prostate cancer cells induces a neuron-like phenotype, enhances invasion, proliferation, and upregulates stemness and neuroendocrine-related markers. Mechanistically, DPYSL5 promotes prostate cancer cell plasticity via EZH2-mediated PRC2 activation. Depletion of DPYSL5 decreases proliferation, induces G1 phase cell cycle arrest, reverses neuroendocrine phenotype, and upregulates luminal genes. In conclusion, DPYSL5 plays a critical role in regulating prostate cancer cell plasticity, and we propose the AR/DPYSL5/EZH2/PRC2 axis as a driver of t-NEPC progression.

Counts of hyaluronic acid-containing extracellular vesicles decrease in naturally occurring equine osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease with inadequately understood pathogenesis leading to pain and functional limitations. Extracellular vesicles (EVs) released by synovial joint cells can induce both pro- and anti-OA effects. Hyaluronic acid (HA) lubricates the surfaces of articular cartilage and is one of the bioactive molecules transported by EVs. In humans, altered EV counts and composition can be observed in OA synovial fluid (SF), while EV research is in early stages in the horse-a well-recognized OA model. The aim was to characterize SF EVs and their HA cargo in 19 horses. SF was collected after euthanasia from control, OA, and contralateral metacarpophalangeal joints. The SF HA concentrations and size distribution were determined with a sandwich-type enzyme-linked sorbent assay and size-exclusion chromatography. Ultracentrifugation followed by nanoparticle tracking analysis (NTA) were utilized to quantify small EVs, while confocal laser scanning microscopy (CLSM) and image analysis characterized larger EVs. The number and size distribution of small EVs measured by NTA were unaffected by OA, but these results may be limited by the lack of hyaluronidase pre-treatment of the samples. When visualized by CLSM, the number and proportion of larger HA-containing EVs (HA-EVs) decreased in OA SF (generalized linear model, count: p = 0.024, %: p = 0.028). There was an inverse association between the OA grade and total EV count, HA-EV count, and HA-EV % (rs = - 0.264 to - 0.327, p = 0.012-0.045). The total HA concentrations were also lower in OA (generalized linear model, p = 0.002). To conclude, the present study discovered a potential SF biomarker (HA-EVs) for naturally occurring equine OA. The roles of HA-EVs in the pathogenesis of OA and their potential as a joint disease biomarker and therapeutic target warrant future studies.

A quick pipeline for the isolation of 3D cell culture-derived extracellular vesicles.

Recent advances in cell biology research regarding extracellular vesicles have highlighted an increasing demand to obtain 3D cell culture-derived EVs, because they are considered to more accurately represent EVs obtained in vivo. However, there is still a grave need for efficient and tunable methodologies to isolate EVs from 3D cell cultures. Using nanofibrillar cellulose (NFC) scaffold as a 3D cell culture matrix, we developed a pipeline of two different approaches for EV isolation from cancer spheroids. A batch method was created for delivering high EV yield at the end of the culture period, and a harvesting method was created to enable time-dependent collection of EVs to combine EV profiling with spheroid development. Both these methods were easy to set up, quick to perform, and they provided a high EV yield. When compared to scaffold-free 3D spheroid cultures on ultra-low affinity plates, the NFC method resulted in similar EV production/cell, but the NFC method was scalable and easier to perform resulting in high EV yields. In summary, we introduce here an NFC-based, innovative pipeline for acquiring EVs from 3D cancer spheroids, which can be tailored to support the needs of variable EV research objectives.

MCF10CA Breast Cancer Cells Utilize Hyaluronan-Coated EV-Rich Trails for Coordinated Migration.

Invasion of tumor cells through the stroma is coordinated in response to migratory cues provided by the extracellular environment. One of the most abundant molecules in the tumor microenvironment is hyaluronan, a glycosaminoglycan known to promote many hallmarks of tumor progression, including the migratory potential of tumor cells. Strikingly, hyaluronan is also often found to coat extracellular vesicles (EVs) that originate from plasma membrane tentacles of tumor cells crucial for migration, such as filopodia, and are abundant in tumor niches. Thus, it is possible that hyaluronan and hyaluronan-coated EVs have a cooperative role in promoting migration. In this work, we compared the hyaluronan synthesis, EV secretion and migratory behavior of normal and aggressive breast cell lines from MCF10 series. Single live cell confocal imaging, electron microscopy and correlative light and electron microscopy experiments revealed that migrating tumor cells form EV-rich and hyaluronan -coated trails. These trails promote the pathfinding behavior of follower cells, which is dependent on hyaluronan. Specifically, we demonstrated that plasma membrane protrusions and EVs left behind by tumor cells during migration are strongly positive for CD9. Single cell tracking demonstrated a leader-follower behavior, which was significantly decreased upon removal of pericellular hyaluronan, indicating that hyaluronan promotes the pathfinding behavior of follower cells. Chick chorioallantoic membrane assays in ovo suggest that tumor cells behave similarly in 3D conditions. This study strengthens the important role of extracellular matrix production and architecture in coordinated tumor cell movements and validates the role of EVs as important components and regulators of tumor matrix. The results suggest that tumor cells can modify the extracellular niche by forming trails, which they subsequently follow coordinatively. Future studies will clarify in more detail the orchestrated role of hyaluronan, EVs and other extracellular cues in coordinated migration and pathfinding behavior of follower cells.

Fatty Acid Fingerprints and Hyaluronic Acid in Extracellular Vesicles from Proliferating Human Fibroblast-like Synoviocytes.

Extracellular vesicles (EVs) function as conveyors of fatty acids (FAs) and other bioactive lipids and can modulate the gene expression and behavior of target cells. EV lipid composition influences the fluidity and stability of EV membranes and reflects the availability of lipid mediator precursors. Fibroblast-like synoviocytes (FLSs) secrete EVs that transport hyaluronic acid (HA). FLSs play a central role in inflammation, pannus formation, and cartilage degradation in joint diseases, and EVs have recently emerged as potential mediators of these effects. The aim of the present study was to follow temporal changes in HA and EV secretion by normal FLSs, and to characterize the FA profiles of FLSs and EVs during proliferation. The methods used included nanoparticle tracking analysis, confocal laser scanning microscopy, sandwich-type enzyme-linked sorbent assay, quantitative PCR, and gas chromatography. The expression of hyaluronan synthases 1-3 in FLSs and HA concentrations in conditioned media decreased during cell proliferation. This was associated with elevated proportions of 20:4n-6 and total n-6 polyunsaturated FAs (PUFAs) in high-density cells, reductions in n-3/n-6 PUFA ratios, and up-regulation of cluster of differentiation 44, tumor necrosis factor α, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Compared to the parent FLSs, 16:0, 18:0, and 18:1n-9 were enriched in the EV fraction. EV counts decreased during cell growth, and 18:2n-6 in EVs correlated with the cell count. To conclude, FLS proliferation was featured by increased 20:4n-6 proportions and reduced n-3/n-6 PUFA ratios, and FAs with a low degree of unsaturation were selectively transferred from FLSs into EVs. These FA modifications have the potential to affect membrane fluidity, biosynthesis of lipid mediators, and inflammatory processes in joints, and could eventually provide tools for translational studies to counteract cartilage degradation in inflammatory joint diseases.

Microscopic characterization reveals the diversity of EVs secreted by GFP-HAS3 expressing MCF7 cells.

We have shown the connection of hyaluronan synthesis activity with the enhanced shedding of extracellular vesicles, but detailed morphological analysis of those hyaluronan-induced EVs is still missing. In this study we utilized a comprehensive set of high-resolution imaging techniques to characterize in high detail the size and morphology of EVs originating from stable MCF7 breast cancer cell line and transiently transfected cells expressing GFP-HAS3. To avoid possible artefacts or loss of EVs resulting from the isolation process, special attention was paid to analysis of EVs in situ in monolayer and in 3D cultures. The results of this study show that GFP-HAS3 expressing MCF7 cells produce morphologically diverse EVs but also demonstrates the variation in results obtained with different experimental setup, which emphasizes the importance of comparison between different methods when interpreting the observations.

Characterization of hyaluronan-coated extracellular vesicles in synovial fluid of patients with osteoarthritis and rheumatoid arthritis.

BACKGROUND: Hyaluronic acid (HA) is the major extracellular matrix glycosaminoglycan with a reduced synovial fluid (SF) concentration in arthropathies. Cell-derived extracellular vesicles (EV) have also been proposed to contribute to pathogenesis in joint diseases. It has recently been shown that human SF contains HA-coated EV (HA-EV), but their concentration and function in joint pathologies remain unknown. METHODS: The aim of the present study was to develop an applicable method based on confocal laser scanning microscopy (CLSM) and image analysis for the quantification of EV, HA-particles, and HA-EV in the SF of the human knee joint. Samples were collected during total knee replacement surgery from patients with end-stage rheumatoid arthritis (RA, n = 8) and osteoarthritis (OA, n = 8), or during diagnostic/therapeutic arthroscopy unrelated to OA/RA (control, n = 7). To characterize and quantify EV, HA-particles, and HA-EV, SF was double-stained with plasma membrane and HA probes and visualized by CLSM. Comparisons between the patient groups were performed with the Kruskal-Wallis analysis of variance. RESULTS: The size distribution of EV and HA-particles was mostly similar in the study groups. Approximately 66% of EV fluorescence was co-localized with HA verifying that a significant proportion of EV carry HA. The study groups were clearly separated by the discriminant analysis based on the CLSM data. The intensities of EV and HA-particle fluorescences were lower in the RA than in the control and OA groups. CONCLUSIONS: CLSM analysis offers a useful tool to assess HA-EV in SF samples. The altered EV and HA intensities in the RA SF could have possible implications for diagnostics and therapy.

The Density and Length of Filopodia Associate with the Activity of Hyaluronan Synthesis in Tumor Cells.

Filopodia are multifunctional finger-like plasma membrane protrusions with bundles of actin filaments that exist in virtually all cell types. It has been known for some time that hyaluronan synthesis activity induces filopodial growth. However, because of technical challenges in the studies of these slender and fragile structures, no quantitative analyses have been performed so far to indicate their association with hyaluronan synthesis. In this work we comprehensively address the direct quantification of filopodial traits, covering for the first time length and density measurements in a series of human cancer cell lines with variable levels of hyaluronan synthesis. The synthesis and plasma membrane binding of hyaluronan were manipulated with hyaluronan synthase 3 (HAS3) and hyaluronan receptor CD44 overexpression, and treatments with mannose, 4-methylumbelliferone (4-MU), and glucosamine. The results of this work show that the growth of filopodia was associated with the levels of hyaluronan synthesis but was not dependent on CD44 expression. The results confirm the hypothesis that abundance and length of filopodia in cancer cells is associated with the activity of hyaluronan synthesis.

CD44s Assembles Hyaluronan Coat on Filopodia and Extracellular Vesicles and Induces Tumorigenicity of MKN74 Gastric Carcinoma Cells.

CD44 is a multifunctional adhesion molecule typically upregulated in malignant, inflamed and injured tissues. Due to its ability to bind multiple ligands present in the tumor microenvironment, it promotes multiple cellular functions related to tumorigenesis. Recent data has shown that CD44 and its principal ligand hyaluronan (HA) are carried by extracellular vesicles (EV) derived from stem and tumor cells, but the role of CD44 in EV shedding has not been studied so far. To answer this question, we utilized CD44-negative human gastric carcinoma cell line MKN74 manipulated to stably express CD44 standard form (CD44s). The effect of CD44s expression on HA metabolism, EV secretion, morphology and growth of these cells was studied. Interestingly, HAS2 and HYAL2 expression levels were significantly upregulated in CD44s-expressing cells. Cell-associated HA levels were significantly increased, while HA levels in the culture medium of CD44s-positive cells was lower compared to CD44s-negative MOCK cells. CD44s expression had no significant effect on the proliferation capacity of cells, but cells showed diminished contact inhibition. Superresolution imaging revealed that CD44s and HA were accumulated on filopodia and EVs secreted from CD44s-positive cells, but no differences in total numbers of secreted EV between CD44s-negative and -positive cells was detected. In 3D cultures, CD44s-expressing cells had an enhanced invasion capacity in BME gel and increased spheroidal growth when cultured in collagen I gel. No significant differences in mitotic activity, tumor size or morphology were detected in CAM assays. However, a significant increase in HA staining coverage was detected in CD44s-positive tumors. Interestingly, CD44s-positive EVs embedded in HA-rich matrix were detected in the stromal areas of tumors. The results indicate that CD44s expression significantly increases the HA binding capacity of gastric cancer cells, while the secreted HA is downregulated. CD44s is also carried by EVs secreted by CD44s-expressing cells. These findings highlight the potential usefulness of CD44s and its ligands as multipurpose EV biomarkers, because they are upregulated in inflammatory, injured, and cancer cells and accumulate on the surface of EVs secreted in these situations.

Correlation between E-cadherin interactions, survivin expression, and apoptosis in MDCK and ts-Src MDCK cell culture models.

Survivin, a member of inhibitor of apoptosis (IAP) protein family, is a multifunctional protein expressed in most cancers. In addition to inhibition of apoptosis, it regulates proliferation and promotes migration. Its presence and function in cells is strongly regulated via transcription factors, intracellular localization, and degradation. We analyzed the presence of survivin at protein level in various culture environments and under activation of Src tyrosine kinase in epithelial canine kidney MDCK cells in order to elucidate factors controlling survivin 'lifespan'. We used untransformed and temperature sensitive ts-Src MDCK cells as a model and forced them to grow in suspension (1D), in 2D on hard and soft surfaces and in soft 3D Matrigel environment with or without EGTA. In addition, we tested the effect of stressful conditions by cultivating the cells in the presence of an anti-cancer drug and a generator of reactive oxygen species (ROS), piperlongumine (PL) with or without an antioxidant, N-acetylcysteine (NAC). We could confirm that inhibition of apoptosis and simultaneous downregulation of survivin in MDCK cells required both intact cell-cell junctions, trans-interactions of E-cadherin and soft 3D matrix environment. In ts-Src-transformed MDCK cells, survivin was upregulated as soon as the cell-cell junctions were disintegrated. ROS generation with PL-induced cell death of ts-Src MDCK cells concomitantly with survivin downregulation. NAC rescued the ts-Src MDCK cells from ROS-induced apoptosis without upregulation of survivin resulting in a situation resembling untransformed MDCK cells in 3D environment and E-cadherin delineating the lateral cell walls.

MDCK cells are capable of water secretion and reabsorption in response to changes in the ionic environment.

A prerequisite for tissue electrolyte homeostasis is highly regulated ion and water transport through kidney or intestinal epithelia. In the present work, we monitored changes in the cell and luminal volumes of type II Madin-Darby canine kidney (MDCK) cells grown in a 3D environment in response to drugs, or to changes in the composition of the basal extracellular fluid. Using fluorescent markers and high-resolution spinning disc confocal microscopy, we could show that lack of sodium and potassium ions in the basal fluid (tetramethylammonium chloride (TMACl) buffer) induces a rapid increase in the cell and luminal volumes. This transepithelial water flow could be regulated by inhibitors and agonists of chloride channels. Hence, the driving force for the transepithelial water flow is chloride secretion, stimulated by hyperpolarization. Chloride ion depletion of the basal fluid (using sodium gluconate buffer) induces a strong reduction in the lumen size, indicating reabsorption of water from the lumen to the basal side. Lumen size also decreased following depolarization of the cell interior by rendering the membrane permeable to potassium. Hence, MDCK cells are capable of both absorption and secretion of chloride ions and water; negative potential within the lumen supports secretion, while depolarizing conditions promote reabsorption.

Different responses in transformation of MDCK cells in 2D and 3D culture by v-Src as revealed by microarray techniques, RT-PCR and functional assays.

Differentiation and transformation of untransformed and ts-Src-transformed canine kidney MDCK cells in 2D and 3D environment were investigated using microarray technique, RT-PCR, confocal microscopy and functional assays. Activated Src induced epithelial-mesenchymal transition (EMT) in 2D environment followed by translocation of junctional proteins to the cytoplasm, without significant changes in protein expression. In 3D environment untransformed MDCK cells formed cell cysts with apical domain facing a lumen, E-cadherin delineating the lateral membranes, ZO-1 at tight junctions and caspase-3 in apoptotic cells captured within the lumen. This was accompanied by reduced expression of an apoptosis inhibitor, survivin and vesicle transport effectors, rab 7 and 8, whereas rab 5 expression increased. In 3D environment activated Src induced changes in expression of over 100 genes as revealed by microarray analysis, mostly involved in cell signaling, division and energy metabolism. Only response in cytoskeletal components was decreased expression of actin and Arp2/3 by v-Src, whereas two p120catenin binding proteins Kaiso and Nanos increased their expression. Concomitantly, apoptosis was inhibited by v-Src resulting in formation of a sphere with epitheloid cells facing extracellular matrix and undifferentiated cells captured within the cluster. This was accompanied by increased expression of apoptosis inhibitor survivin, as revealed by western blotting. Mitochondrial membrane potential in untransformed MDCK cells was lower than in ts-Src-MDCK cells in early days of cluster formation correlating with the induction of apoptosis. Hence, v-Src activation in 3D environment did not induce EMT, but brought about inhibition of apoptosis and increased proliferation where increased expression of survivin and inhibition of the mitochondrial permeability have a role.