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.

Biological significance: 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.

Keywords: Extracellular vesicles; Label-free relative quantitation; Multiple myeloma; Shotgun proteomics; cd44.

Figure 1. MM cell line EVs are enriched for MHCI and β₂-MG

A) Cryo-TEM images of vesicles derived from MM.1R and RPMI-8226 cells. B) Mean size distributions for vesicles derived from U266, MM.1S, MM.1R and RPMI-8226 cell lines by Nanosight analysis (bars indicate 95 % CI). C) 4-way Venn diagram of vesicle protein identifications from MM cell lines. D) Expanded cluster map of extracellular vesicle and cellular lysate spectral counts for MHCI, β2-MG and GAPDH from MM cell lines E) Immunoblot of extracellular vesicle and cellular lysates for MHCI, β2-MG and GAPDH from MM cell lines. Ponceau S is provided for loading comparison. Collectively, these data show MM cell line derived vesicles are enriched in MHCI and β₂-MG.

Figure 2. MM Patient EVs are enriched for MHCI and β₂-MG

A) Mean serum vesicle size distributions from healthy donors (n=3) and MM patients of different stages (MGUS n=3, Smoldering n=8, Active n=3, error bars indicate 95 % CI). B) Venn diagram of overlapping protein identifications from MM serum, MM bone marrow aspirates and healthy donor peripheral blood samples (whole blood & leukopaks). C) Immunoblot for β₂-MG, MHCI and GAPDH from serum of representative random healthy donors (lanes 1-4) and MM patients (lanes 5-8). Ponceau S is provided for loading comparison. Collectively, these results illustrate an enrichment of MHCI and β₂-MG in the MM ex vivo EV samples when compared to healthy donor EVs.

Figure 3. Corticosteroid resistant cell lines harbor EVs with proteins associated drug resistance

A) A cluster map of 403 extracellular vesicle and cellular lysate protein spectral counts from the resistant (MM.1R) and parent (MM.1S) cell lines with at least one observation in each replicate of greater than five spectral counts. B) Expanded cluster maps of CD44, β₂-MG and GAPDH from the MM.1R and MM.1S data. C) Immunoblot for CD44, β₂-MG, Lamin B1 and GAPDH from extracellular vesicle and cellular lysates. D) Flow cytometry for surface expression of CD44 from the MM cell lines. Collectively, these data show an enrichment of CD44 in the corticosteroid resistant cell line MM.1R EVs compared the parent MM.1S cell line.

Figure 4. Identification of the extracellular vesicles as the primary location of serum CD44 in MM patients

A) Immunoblot for CD44 from whole serum of healthy individuals and MM patients. B) Immunoblot for CD44 from whole serum (+) and vesicle depleted serum (−) from those samples labeled 1 & 2 in Figure 4A. C) ELISA for CD44 from whole serum and vesicle depleted serum from those samples labeled 1 & 2 in Figure 4A. Collectively, these results show CD44 is primarily located in the serum vesicles of MM patients.

Figure 5. Identification of serum CD44 as a predictor of overall survival in MM patients

A) ELISA for CD44 from 13 non-cancer donor age matched serum. B) ELISA for CD44 from MM patient serum in 233 newly diagnosed MM patients. C) Multivariable model for overall survival, HR: hazard ratio, 95% CI: 95% confidence interval. D) Kaplan Meier survival curve for those patients with serum CD44 concentrations greater than 280 ng/mL. Collectively, these data show serum CD44 levels predict overall survival in multiple myeloma patients.