title: Proteomic characterization of circulating extracellular vesicles identifies novel serum myeloma associated markers.
abstract: Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells in the bone marrow (BM). The microenvironment plays a key role in MM cell survival and drug resistance through release of soluble factors, expression of adhesion molecules and release of extracellular vesicles (EVs). The aim of this manuscript is to use proteomic profiling of EVs as a tool to identify circulating tumor associated markers in MM patients. First, we characterized the EV protein content obtained from different MM cell lines. Then, we established differences in protein abundance among EVs isolated from MM patient serum and BM and the serum of healthy donors. These data show that the Major Histocompatibility Complex Class I is highly enriched in EVs of MM cell lines and MM patient's serum. Next, we show that CD44 is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R. Furthermore, CD44 was found to be differentially expressed in EVs isolated from newly diagnosed MM patients. Finally through ELISA analysis, we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as an easily accessible source for MM biomarkers.
  
  
  CONCLUSIONS
  Extracellular vesicles are becoming a research focus due to their roles in cancer cell biology such as immune evasion, therapeutic resistance, proliferation and metastases. While numerous studies of vesicle characterization and biology have been conducted in many cancer models, the role of EV in MM remains relatively unstudied. Here we found that EVs isolated from MM cells are enriched in MHC-1 antigen presenting complex and its binding protein β2-MG, this observation is compatible with the enhanced proteasome activity of MM cells compared to other cancers and the ability of functional MHC-1 to bind and present peptides, generated from protein degradation by the proteasome. Additionally, our experiments show that CD44 is particularly enriched in the EV fraction of corticosteroid resistant MM.1R cells and is differentially expressed in the EV fraction of MM patients. This is of high significance due to the established role of CD44 in adhesion of MM cells to BMSC and induction of IL-6, the primary cytokine for MM cell survival, secretion by the BMSC. Furthermore, ELISA assays for CD44 content from the serum of 254 newly diagnosed MM patients enrolled in a Phase 3 randomized trial show highly variable CD44 levels and those patients with >280 ng/mL serum CD44 showing a reduced overall survival time. These results suggest the potential use of CD44 as a prognostic biomarker in MM.
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH, USA.|Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
authors.forename: Sean W
authors.initials: SW
authors.lastname: Harshman
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Canella
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Ciarlariello
authors.affiliation: The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH, USA.|Nanoscale Science and Engineering Center, The Ohio State University, Columbus, OH, USA.
authors.forename: Kitty
authors.initials: K
authors.lastname: Agarwal
authors.affiliation: The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH, USA.
authors.forename: Owen E
authors.initials: OE
authors.lastname: Branson
authors.affiliation: Department of Haematology, Manchester Royal Infirmary hospital, Oxford Road, Manchester, UK.
authors.forename: Alberto
authors.initials: A
authors.lastname: Rocci
authors.affiliation: Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA.
authors.forename: Hector
authors.initials: H
authors.lastname: Cordero
authors.affiliation: Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH, USA.
authors.forename: Mitch A
authors.initials: MA
authors.lastname: Phelps
authors.affiliation: Center for Biostatistics, Department of Biomedical Informatics, The Ohio State University, Columbus, OH, USA.
authors.forename: Erinn M
authors.initials: EM
authors.lastname: Hade
authors.affiliation: Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Dubovsky
authors.affiliation: Myeloma Unit, Division of Hematology, University of Turin, Azienda Ospedaliera Citta'della Salute e della Scienza di Torino, 10126 Torino, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Palumbo
authors.affiliation: Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA.
authors.forename: Ashley
authors.initials: A
authors.lastname: Rosko
authors.affiliation: Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Byrd
authors.affiliation: Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA.
authors.forename: Craig C
authors.initials: CC
authors.lastname: Hofmeister
authors.affiliation: Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA.
authors.forename: Don M
authors.initials: DM
authors.lastname: Benson
authors.affiliation: Nanoscale Science and Engineering Center, The Ohio State University, Columbus, OH, USA.|Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Paulaitis
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH, USA.|Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Freitas
authors.affiliation: Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA.
authors.forename: Flavia
authors.initials: F
authors.lastname: Pichiorri
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood*; D045744:Cell Line, Tumor; D018572:Disease-Free Survival; D005260:Female; D006801:Humans; D018960:Hyaluronan Receptors / Q000097:blood*; D008297:Male; D009101:Multiple Myeloma / Q000097:blood* / Q000401:mortality*; D009363:Neoplasm Proteins / Q000097:blood*; D040901:Proteomics; D015996:Survival Rate
chemical_list: D014408:Biomarkers, Tumor; C497483:CD44 protein, human; D018960:Hyaluronan Receptors; D009363:Neoplasm Proteins
keywords: Extracellular vesicles; Label-free relative quantitation; Multiple myeloma; Shotgun proteomics; cd44