BackgroundHuman hyaluronic acid (HA) molecules are synthesized by three membrane spanning Hyaluronic Acid Synthases (HAS1, HAS2 and HAS3). Of the three, HAS1 is found to be localized more into the cytoplasmic space where it synthesizes intracellular HA. HA is a ubiquitous glycosaminoglycan, mainly present in the extracellular matrix (ECM) and on the cell surface, but are also detected intracellularly. Accumulation of HA in cancer cells, the cancer-surrounding stroma, and ECM is generally considered an independent prognostic factors for patients. Higher HA production also correlates with higher tumor grade and more genetic heterogeneity in multiple cancer types which is known to contribute to drug resistance and results in treatment failure. Tumor heterogeneity and intra-tumor clonal diversity are major challenges for diagnosis and treatment. Identification of the driver pathway(s) that initiate genomic instability, tumor heterogeneity and subsequent phenotypic/clinical manifestations, are fundamental for the diagnosis and treatment of cancer. Thus far, no evidence was shown to correlate intracellular HA status (produced by HAS1) and the generation of genetic diversity in tumors.MethodsWe tested different cell lines engineered to induce HAS1 expression. We measured the epithelial traits, centrosomal abnormalities, micronucleation and polynucleation of those HAS1-expressing cells. We performed real-time PCR, 3D cell culture assay, confocal microscopy, immunoblots and HA-capture methods.ResultsOur results demonstrate that overexpression of HAS1 induces loss of epithelial traits, increases centrosomal abnormalities, micronucleation and polynucleation, which together indicate manifestation of malignant transformation, intratumoral genetic heterogeneity, and possibly create suitable niche for cancer stem cells generation.ConclusionsThe intracellular HA produced by HAS1 can aggravate genomic instability and intratumor heterogeneity, pointing to a fundamental role of intracellular HA in cancer initiation and progression.Electronic supplementary materialThe online version of this article (10.1186/s12964-017-0204-z) contains supplementary material, which is available to authorized users.
Several studies have demonstrated the potential uses of extracellular vesicles (EVs) for liquid biopsy‐based diagnostic tests and therapeutic applications; however, clinical use of EVs presents a challenge as many currently‐available EV isolation methods have limitations related to efficiency, purity, and complexity of the methods. Moreover, many EV isolation methods do not perform efficiently in all biofluids due to their differential physicochemical properties. Thus, there continues to be a need for novel EV isolation methods that are simple, robust, non‐toxic, and/or clinically‐amenable. Here we demonstrate a rapid and efficient method for small extracellular vesicle (sEV) isolation that uses chitosan, a linear cationic polyelectrolyte polysaccharide that exhibits biocompatibility, non‐immunogenicity, biodegradability, and low toxicity. Chitosan‐precipitated material was characterized using Western blotting, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and relevant proteomic‐based gene ontology analyses. We find that chitosan facilitates the isolation of sEVs from multiple biofluids, including cell culture‐conditioned media, human urine, plasma and saliva. Overall, our data support the potential for chitosan to isolate a population of sEVs from a variety of biofluids and may have the potential to be a clinically amenable sEV isolation method.
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