Bone Marrow Stromal Cell-Derived Exosomes Facilitate Multiple Myeloma Cell Survival Through Inhibition Of The JNK Pathway

Interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells plays a crucial role in MM pathogenesis by secreting growth factors, cytokines, and other functional components. Exosomes are 30-100nm diameter membranous vesicles constitutively released by several cell types including reticulocytes, cytotoxic T lymphocytes, B lymphocytes, epithelial and endothelial cells. Exosomes mediate local cell-cell communication by transferring mRNAs, miRNAs and proteins. Due to their ability to transfer functional components, exosomes play multiple roles by stimulating target cells, transferring membrane receptors, delivering proteins, and inducing epigenetic changes in recipient cells. Although the promotion of MM growth and survival induced by BMSCs has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here, we investigated the effect and mechanisms of BMSC-derived exosomes on the proliferation and survival of MM cells using the murine 5T33MM model. This model mimics the human disease closely and of this model two lines exist: the 5T33MMvv model which is propagated in vivo and the 5T33MMvt line which is derived from 5T33MMvv cells but which can grow stroma-independently.

Exosomes were isolated from conditioned medium using the ExoQuick-TC Exosome Precipitation Solution (System Biosciences) after culture of primary BMSCs obtained from naïve C57BL/KaLwRij mice or 5T33MM diseased mice. The size of exosomes derived from naïve BMSCs, 5T33 BMSCs and 5T33MMvt cells were confirmed using a NanoSight LM10. Several exosomal markers such as CD63, Flotillin-1, heat shock protein 90 (HSP90), and HSP70 were detected using Western blot. We co-cultured the BMSCs or MM cells with fluorescent dye-labeled exosomes to examine whether exosomes could be transferred into cells. The results showed that both naïve and 5T33 BMSC-derived exosomes could fuse with 5T33MMvt cells and that the uptake of 5T33MMvt cell-derived exosomes by BMSCs was also observed. As several cytokines were found to be present in BMSC- and MMvt cell-derived exosomes, this suggests that BMSCs and MM cells could exchange cytokines with each other through exosomes secretion and uptake. Furthermore, the cytokine composition of 5T33BMSC-derived exosomes compared to naïve BMSC-derived exosomes was different.

We next performed luminescent cell viability assays, BrdU cell proliferation assays and 7-AAD/annexin-V stainings to examine the effects of BMSC-derived exosomes on MM cell viability, proliferation and survival, respectively. Both naïve and 5T33 BMSC-derived exosomes increased 5T33MMvt and MMvv cell viability in a dose- and time-dependently manner. BrdU uptake in 5T33MMvt and MMvv cells was also increased after treatment with BMSC-derived exosomes. Significantly reduced apoptosis of 5T33 MMvt and MMvv cells was observed when they were treated with BMSC-derived exosomes as judged by 7-AAD/annexin-V staining. 5T33MMvt and MMvv cells were treated with different amounts of BMSC-derived exosomes and apoptosis-related proteins Bcl-2, Bax, and caspase-3 were determined using western blot. Bcl-2 was increased slightly and activated (cleaved) caspase-3 was reduced after co-culture with exosomes, coinciding with the results of 7-AAD/annexin-V staining. To elucidate the mechanisms responsible for exosome-induced MM cell survival, we examined the activation of several proteins involved. Reduced phosphorylation of p53, p38MAPK and JNK were detected when 5T33MMvt were treated with naïve-BMSC-derived exosomes for 24h, whereas phosphorylated Erk1/2, Akt, and IGF1Rβ were not changed. Surprisingly, activation of p53 and p38MAPK were not changed after the treatment with 5T33 BMSC-derived exosomes. 5T33 BMSC-derived exosomes further decreased the activation of JNK, Bim expression and phosphorylated Bim compared to naïve BMSC-derived exosomes. As Bim is a pro-apoptosis protein, mainly regulated by the JNK pathway; promotion of MM cell survival likely results from the inhibition of the JNK pathway by BMSC-derived exosomes.

In summary, our results demonstrate a positive role for BMSC-derived exosomes in induction of MM cell proliferation and survival. BMSC-derived exosomes could inhibit the JNK pathway, thereby reducing caspase-3 activation and protecting MM cells from apoptosis.