Mirna Expression Profiling and Proteomic Analysis Of Circulating Exosomes From Multiple Myeloma Patients

Exosomes are small vesicles (50-100 nm) of endocytic origin, which are released in the extra-cellular milieu by several cell types. It is known that cell-to-cell communication is partially mediated by exosomes. Exosomes play a role in tumor progression where they have been shown to carry and transfer microRNAs (miRNAs) and proteins to the recipient cells. In this study, we sought to characterize circulating exosomes in terms of their ability to modulate the microenvironment, leading to Multiple Myeloma (MM) progression.

Exosomes were collected from peripheral blood obtained from healthy individuals (n=5), MGUS patients (n=5) and MM patients (n=10), using ultracentrifugation. Further characterization was carried out using electron microscopy and immunogold labeling for the detection of CD63 and CD81 and for the size using Nanosight® analysis. MiRNA were isolated using miRNeasy mini kit (Qiagen®) and profiling has been performed using nCounter miRNA expression assay (Nanostring® Technologies, Seattle WA). Bioinformatic software tools (TargetScan, MIRDB) were used to predict the target genes of identified miRNA to define their function. Proteins were isolated from exosomes following lysis and precipitated by acetone before in-solution trypsin digestion and ZipTip® purification. Proteomic analysis was performed using mass spectrometry (BIDMC Mass Spectrometry, ObiTrap Elite®). Spectral count numbers were determined with a false discovery rate (FDR) less than 0.5%.

Circulating exosomes were studied at ultrastructural level showing positivity for CD81 and CD63, as demonstrated by immunogold labeling and electron microscopy. Exosome number and size did not differ based on clinical stage on Nanosight® analysis. We identified 16 miRNAs differentially expressed in circulating exosomes obtained from MGUS patients compared to healthy subjects (FC >2 or <-2; p<0.05): specifically, higher expression of miR-450a, -30e, -125a, -300 and lower expression of miR-185, -150, -98 were observed in MGUS- compared to healthy individual-derived circulating exosomes. Interestingly, miR-30e and -150 modulate NK cell activity by targeting perforin and c-Myb, respectively. We found 96 miRNAs differentially expressed in circulating exosomes from MM patients as compared to healthy donors: specifically lower expression of Let-7 family members, miR-150, -15a and higher expression of miR-125b, -144 and -363 were observed. Interestingly, miR-15a is involved in angiogenesis regulating VEGFA and FGF2. Let-7 family members are tumor suppressors targeting k-Ras and c-Myc and miR-150 regulates CXCR-4 expression. Moreover, these patterns have been described in MM cells suggesting that circulating exosomes in MM are mainly released from MM cells and could play a role in modulating the tumor micro-environment. The mass spectrometry analysis was performed on protein derived from circulating exosomes from 5 healthy donors, 5 MGUS and 10 MM patients. 272 proteins were identified in circulating exosomes including proteins highly associated with exosomes such as CD9, HSP70, Rab proteins (Rab7a; Rab5; Rab27b) and annexins. Comparing MM exosomal proteins to healthy donor exosomal proteins, we found significantly distinctive peptide counts for fibronectin (FC=3.5; p=0.002), AMBP protein (FC=3; p=0.001) and Ig gamma-1 chain C region (FC=2.5; p=0.006). Interestingly, fibronectin expression level in the microenvironment has been reported to be associated with tumor proliferation and drug resistance in MM.

These findings indicate that circulating exosomes differ between normal, MGUS and MM patients in terms of miRNA and protein content. Circulating exosomes could potentially be involved in modulating the host microenvironment for specific homing of clonal plasma cells to the bone marrow; thus providing a better understanding of the epigenetic changes responsible for the transition to MM stage.

Leleu:CELGENE: Honoraria; JANSSEN: Honoraria. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.