Abstract
Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function.
Highlights
Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are rich in membrane proteins
Characterization of HT1376 Exosomes—Exosomes were purified from HT1376 cells, and preparations were subjected to several forms of analysis to evaluate sample quality/purity prior to analysis using proteomics workflows
The accuracy of overrepresentation analysis (ORA) can be limited by the quality and size of the gene sets queried, our analysis suggests that HT1376 exosomes express proteins strongly related to neoplastic diseases in general and to carcinomas in particular (Fig. 2, B and C)
Summary
Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. Exosomes have not yet been studied in the context of other urological malignancies such as renal cancer, and to date, only one report describes the urine-derived microparticles from bladder cancer patients [17] In that report, they examined the proteome of a highly complex mixture of microvesicles, exosomes, and other urinary constituents that can be pelleted by high speed ultracentrifugation, identifying eight proteins that may be elevated in cancer. Given the nature of the sample analyzed, it is unknown whether these proteins are exosomally expressed
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