Abstract B62: Stroma-derived exosomes mediate progression in multiple meyloma
Authors: Roccaro Aldo M. , Sacco Antonio , Maiso Patricia , Moschetta Michele , Manier Salomon , Mishima Yuji , Reagan Michaela , Aljawai Yosra , Ghobrial Irene M. 
Published: May 2015

Background. Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) support multiple myeloma (MM) cell growth, but little is known about the putative mechanisms that may regulate the interaction between clonal MM plasma cells and the surrounding BM milieu. We characterized the role BM-MSC-derived exosomes as key regulators of MM pathogenesis.

Purpose. 1) To determine the ability of BM-MSCs to release and transfer exosomes to MM cells. 2) To determine the role of BM-MSC-derived exosomes in inducing MM tumor growth and MM cell dissemination

Methods. MSCs were collected from BM of healthy subjects and MM patients, showing a multipotent MSC phenotype (CD34-; CD14-; CD45-; CD19-; CD138-; CD73+; CD90+; CD105+; CD106+). Exosomes were collected from conditioned-medium of normal-BM-MSCs and MM-BM-MSCs, or HS-5 cells; and studied using electron microscopy, immunogold-labeling, and western-blot for CD63 and CD81 detection. Transfer of PKH67-fluorescently-labeled-exosomes to MM cells was evaluated by time-lapse confocal microscopy. Transfer of murine-derived miRNA-containing exosomes into human MM cells was evaluated by qRT-PCR (exosomes were collected from BM-MSCs of C57BL/6 miRNA-15a/16-1-/- or C57BL/6 mice). miRNA expression profiling was obtained from normal (n=4) and MM (n=9) BM-MSCs-derived exosomes (TaqMan-human-miRNA-profiling). Normal and MM BM-MSCs-derived exosomes were loaded into tissue-engineered bones (TEB) with MM.1S-GFP+/Luc+ cells: MM cell homing and MM tumor growth have been tested in vivo by using confocal-microscopy and bioluminescence-imaging (BLI), respectively. Loss- and gain-of-function studies were performed using normal-BM-MSCs, MM-BM-MSCs and HS-5 cells transfected with either pre- or anti-miRNA-15a.

Results. Normal-BM-MSCs and MM-BM-MSCs released CD63+/CD81+ exosomes, as confirmed by electron microscopy, immunogold labeling, and western blot. BM-MSCs exosomes are transferred into MM cells, as shown by confocal microscopy; and further validated by qRT-PCR in human MM cell lines incubated with murine (C57BL/6 miRNA-15a/16-1-/- and wild-type) BM-MSCs-derived exosomes. The impact of normal-BM-MSC- and MM-BM-MSC-derived exosomes on MM cell behavior in vivo was next evaluated. MM cells co-cultured with MM BM-MSC-derived exosomes induced rapid tumor growth at the site of the TEB scaffold, as well as rapid dissemination to distant BM niches, as compared to MM cells co-cultured with exosomes derived from normal BM-MSCs. We next performed miRNA expression profiling on exosomes isolated from MSCs, and found increased expression of 24 miRNAs and reduced expression of 3 miRNAs in MM-BM-MSCs-derived exosomes versus normal (1.5 fold-change; P&lt;0.05). Specifically, miRNA15a was significantly lower in MM-BM-MSC-derived exosomes, similarly to primary MM cells that present with reduced miRNA-15a expression. We therefore sought to examine whether lack of transfer of the tumor suppressor miRNA15a can lead to significant change in tumor growth and dissemination in MM, and found that by over-expressing miRNA-15a in normal BM-MSCs and HS-5 cells inhibited MM cell proliferation and adhesion to fibronectin. Next MM cells were cultured in presence of BM-MSCs isolated from either C57BL/6 mice or C57BL/6 miRNA15a/16-1-/-: miRNA15a-deficient BM-MSCs significantly induced MM cell proliferation (P&lt;0.05). Moreover, exosomes isolated from HS-5 pre-miRNA15a-transfected cells both inhibited MM cell proliferation and reduced their adhesion properties.

Conclusion. These findings demonstrate the existence of exosome-driven interactions between the BM milieu and MM cells, suggesting that exosomes might constitute a novel mechanism for intercellular transfer of miRNAs to MM cells.

Citation Format: Aldo M. Roccaro, Antonio Sacco, Patricia Maiso, Michele Moschetta, Salomon Manier, Yuji Mishima, Michaela Reagan, Yosra Aljawai, Irene M. Ghobrial. Stroma-derived exosomes mediate progression in multiple meyloma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B62. doi:10.1158/1538-7445.CHTME14-B62