Mir-106a-5p and Mir-146a-5p Delivered By Tumor-Derived Exosomes Promote the Induction of Myeloid-Derived Suppressor Cells from Normal Peripheral Blood Mononuclear Cells in Multiple Myeloma

Despite the great advance in treatment, including immunochemotherapy using monoclonal antibodies and immunomodulatory drugs, and cell therapies, such as chimeric-antigen receptor T cell therapies, multiple myeloma (MM) remains to be mostly incurable. One of the major mechanisms for the resistance to various immune-mediated treatment approaches for MM is the collapse of the tumor-immune surveillance system which is induced by the predominance of immunosuppressive cellular components over the surveillance system. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells which suppress T-cell-mediated immune response, and its increase has been associated with disease progression and treatment resistance in MM. We previously reported that the co-culture with some human-myeloma-derived cell lines (HMCLs), KMS-12-BM cells, and KMS-28-PE cells, but not others, such as AMO-1 cells and RPMI8226 cells, induced monocytic-MDSCs (M-MDSCs) from normal peripheral blood mononuclear cells (PBMCs), and showed that the combinatory secretion of CCL5, MIF, and MIP-1a, by HMCLs partly contributed to the induction of M-MDSCs in MM (Kuwahara-Ota S, Br J Haematol 2020). In addition to soluble factors, exosomes play a crucial role in cell-to-cell communication by transferring various molecules, including proteins, mRNAs, and microRNAs (miRNAs), and we here investigated the role of tumor-derived exosomes (TEXs) in the induction of M-MDSCs in MM. First, we isolated TEXs from the culture supernatant of various HMCLs. The presence of TEXs was confirmed by nanoparticle tracking analysis and flow cytometry. To examine whether TEXs promote the induction of M-MDSC, we treated PBMCs with TEXs from HMCLs. As the result, the exposure of TEXs from MDSC-inducible HMCLs, but not from MDSC-non-inducible HMCLs, induced M-MDSCs from PBMCs in a dose-dependent manner, indicating the involvement of TEXs in M-MDSC induction in MM. Next, we comparatively performed the comprehensive miRNA profiling by microarray analysis among TEXs from MDSC-inducible and non-inducible HMCLs and identified that miRNAs, including miR-16-5p, miR-17-5p, miR-20a-5p, miR-106a-5p, miR-107, and miR-146a-5p, were specifically upregulated in TEXs from MDSC-inducible HMCLs compared with MDSC-non-inducible HMCLs, which was validated by the quantitative RT-PCR. Interestingly, the expression levels of these 6 miRNAs were mostly equivalent in MDSC-inducible HMCLs and MDSC-non-inducible HMCLs, suggesting the crucial role of TEX-mediated delivery of those miRNAs. By the transfection experiments of these 6 miRNAs into PBMCs, we identified that PBMCs transfected by miR-106a-5p and miR-146a-5p exhibited differentiation into M-MDSCs. In addition, the combination of miRNA, i.e., miR-106a-5p or miR-146a-5p, and chemokine, namely, CCL5, MIP-1a, or MIF, further enhanced the induction of M-MDSC from PBMCs. Finally, to clarify the functional roles of miR-106-5p and miR-146a-5p in the TEX-mediated induction of M-MDSCs, we analyzed the comprehensive gene expression changes by the introduction of miR-106-5p or miR-146a-5p in PBMCs using gene expression microarray. As the result, the gene introduction of miR-106a-5p or miR-146a-5p was found to modulate various gene sets, including those associated with IFN-α signaling, IFN-γ signaling, inflammatory response, TNF-a signaling via NF-kB, or interleukin-6-JAK-STAT3 signaling, in CD33-positive myeloid cells, and these findings mirror the gene expression modulation induced by the co-culture with MDSC-inducible HMCLs in CD33-positive myeloid cells. In conclusion, this study for the first time identified the multi-layer functional involvement of TEX-mediated miR-106a-5p and miR-146a-5p transfer in the induction of M-MDSCs in MM.

Kuroda:Kyowa Kirin: Research Funding, Speakers Bureau; Chugai Phamaceutical: Research Funding, Speakers Bureau; Japan Blood Products Organization: Research Funding; Sysmex cooporation: Research Funding; Fujimoto Pharmaceutical: Research Funding, Speakers Bureau; Abbvie: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; CLS Behring: Research Funding; Janssen Pharmaceutical: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Takeda Pharmaceutical: Research Funding, Speakers Bureau; Asahikasei: Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Sumitomo Pharmaceutical: Research Funding, Speakers Bureau; Taiho Phamaceutical: Research Funding; Eisai: Research Funding, Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Ono Pharmaceutical: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding, Speakers Bureau.