Multiple myeloma (MM) drug resistance and bone disease are closely associated with the remodeling of the bone marrow microenvironment, yet the mechanisms of exosome-mediated communication remain poorly understood. In this study, we utilized a dual-model approach combining patient and mouse data through multi-omics to explore the MM-exosomal miR-21-5p/TGF-β1–CXCL12/RANKL axis and assess its potential for therapeutic intervention. We isolated CD63⁺CD81⁺ exosomes measuring 100–150 nm from 28 newly diagnosed and 17 relapsed/refractory (R/R) MM patients using ultracentrifugation-SEC, yielding a concentration of 2.3 × 10¹¹ particles per mL. miRNA sequencing and LC-MS/MS analysis revealed a greater than tenfold enrichment of miR-21-5p (p < 0.001) and identified TGF-β1 among the top 5% of most abundant proteins (iTRAQ ratio 12.4), with similar profiles confirmed in exosomes from 5TGM1 syngeneic serum. Live-cell imaging using PKH26 demonstrated that over 78% of human or murine mesenchymal stem cells (MSCs) internalized these exosomes within two hours. Dual-luciferase assays indicated that miR-21-5p binds to a specific seed sequence (nucleotides 624-631) in the 3′ untranslated region (UTR) of TGF-β1, leading to a suppression of mRNA and protein expression by 2.8-fold and 3.4-fold, respectively, after 24 hours. This resulted in a 4.1-fold increase in the nuclear accumulation of phosphorylated SMAD2/3, which directed MSCs toward a cancer-associated MSC (CA-MSC) phenotype characterized by α-SMA⁺FAP⁺ markers (GSEA FDR < 0.01). CA-MSCs were found to secrete 5.2-fold more CXCL12 and 6.7-fold more RANKL, which enhanced the chemotaxis of MM cells by 4.5-fold (with anti-CXCL12 treatment reversing 82% of this migration) and increased the number of TRAP⁺ multinucleated osteoclasts from 12 to 89 per well. Micro-CT imaging of calvariae showed a 2.1-fold increase in the bone surface to bone volume ratio compared to controls. Importantly, the administration of a TGF-β1-neutralizing antibody or miR-21-5p antagomiR encapsulated in exosome-mimetic liposomes was able to reverse this pathological condition (p < 0.01).

In bortezomib-resistant 5TGM1-BR cells, which have an IC₅₀ of 17.4 nM, the depletion of exosomes or the delivery of antagomiR significantly restored sensitivity, lowering the IC₅₀ to 3.3 nM and increasing apoptosis from 14% to 71% when treated with 5 nM for 48 hours. In a study involving NSG mice with 5TGM1-BR tumors, the combination of bortezomib administered at a dose of 0.5 mg kg⁻¹ twice weekly and MLN4924-antagomiR liposomes at 2 mg kg⁻¹ weekly resulted in a notable reduction of tumor bioluminescence imaging (BLI) flux from 1.1 × 10⁷ to 3.2 × 10⁶ photons s⁻¹, and it also extended the median survival of the mice from 34 to 71 days (p < 0.001). Additionally, there was a significant recovery of trabecular bone volume by 68% (p < 0.01). These findings suggest that multiple myeloma (MM) exosomes play a crucial role in remodeling the bone marrow niche through the miR-21-5p/TGF-β1–CXCL12/RANKL signaling axis, which drives the conversion of cancer-associated mesenchymal stem cells (CA-MSC), osteolysis, and resistance to proteasome inhibitors. Therefore, targeting this exosome-mediated pathway presents a promising strategy for overcoming drug resistance and improving conditions associated with MM-related bone disease.

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2025
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