Tumor-derived extracellular vesicles inhibit osteogenesis and exacerbate myeloma bone disease

Abstract

Background: As a hallmark driver of multiple myeloma (MM), MM bone disease (MBD) is unique in that it is characterized by severely impaired osteoblast activity resulting from blocked osteogenesis in bone marrow-derived mesenchymal stem cells (BM-MSCs). The mechanisms underlying this preferential blockade are incompletely understood. Methods: miRNA expression of MM cell-derived extracellular vesicles (MM-EVs) was detected by RNA sequencing. MM-EVs impaired osteogenesis and exacerbated MBD were in vitro and in vivo validated by histochemical staining, qPCR and micro-CT. We additionally examined the correlation between CD138⁺ circulating EVs (cirEVs) count and bone lesion in de novo MM patients. Results: Here, by sequencing and bioinformatics analysis, we found that MM-EVs were enriched in various molecules negatively regulating osteogenesis. We experimentally verified that MM-EVs inhibited BM-MSC osteogenesis, induced elevated expression of miR-103a-3p inhibiting osteogenesis in BM-MSCs, and increased cell viability and interleukin-6 secretion in MM cells. In a mouse model, MM-EVs that were injected into the marrow space of the left tibia led to impaired osteogenesis and exacerbated MBD and MM progression. Furthermore, the levels of CD138⁺ cirEVs in the peripheral blood were positively correlated with the number of MM bone lesions in MM patients. Conclusions: These findings suggest that MM-EVs play a pivotal role in the development of severely impaired osteoblast activity, which represents a novel biomarker for the precise diagnosis of MBD and a compelling rationale for exploring MM-EVs as a therapeutic target.

Keywords: bone lesions; extracellular vesicles; mesenchymal stem cells; multiple myeloma; osteogenesis.

Figure 1

Characteristics of the MM-EVs. (A) Transmission electron microscopy image revealing EVs (arrows) as 100-1000 nm vesicles shed from RPMI8226 cells (R-EVs). Scale bar, 500 nm (× 25600). (B) TRPS analysis of the size distribution of R-EVs and U266-EVs. (C) R-EVs were positive for Annexin-V and CD138. (D) Western blot of Alix, TSG101, CD63, MFGE8, CD9 and β-actin contained in RPMI8226, U266, R-EVs and U266-EVs (U-EVs). (E) Representative FACS analysis of PB cirEVs obtained from a de novo MM (D-MM) patient. (F) Comparison of PB and BM CD138⁺ cirEVs in D-MM patients (n = 61). n.s. Signifies P > 0.05. (G) Representative transmission electron micrograph of PB cirEVs obtained from a D-MM patient. Scale bar, 500 nm. (H) Distribution of expression levels for mRNAs, lncRNAs and miRNAs with RPKM (RPM) > 1 in R-EVs. The scale shown for piRNA is the read count. (I) Comparative expression levels of miRNAs in B cell-EVs, R-EVs and K562-EVs. miRNAs marked by “#” are associated with both osteogenesis and proliferation. (J) ELISA analysis of DKK1, IL-7 and sFRP2 in MM cells and MM-EVs.

Figure 2

MM-EVs inhibit osteogenesis in BM-MSCs. (A) ALP and Alizarin Red staining images (left, × 40) and quantification (right) of BM-MSCs treated with unfiltered MM cell medium or conditioned medium after 14 and 21 d. Conditioned medium was obtained by centrifuging MM cell medium at 16,000 ×g for 60 min and then filtering using 0.22 μm filters. (B) Confocal microscopy revealing fusion of R-EVs (PKH26, red) and BM-MSCs (DAPI, blue). Arrows: R-EVs near the cell nuclei. Imaging was performed in the x-y plane by confocal microscopy (Nikon, A1Si) with a pinhole radius of 52.9 µm (× 200). (C) FACS analyses of CD138⁺ BM-MSCs cultured with MM-EVs for 24 h. Negative control: BM-MSCs alone and isotype control. (D-E) ALP staining (D) and Alizarin Red staining (E) images (left, × 40) and quantification (right) of BM-MSCs treated with 50 ng/mL MM-EVs and K562-EVs for 14 and 21 d. (F) Real-time PCR analysis of OB marker genes (RUNX2, ALP, osteocalcin and collagen I) in BM-MSCs treated with MM-EVs or K562-EVs (50 ng/mL) for 3 or 6 d. The results were normalized to β-actin. (G) ALP activity and secreted osteocalcin (OC) protein in the BM-MSCs treated with MM-EVs (50 ng/mL) during OB differentiation. All data are shown as the mean ± SD from at least three experiments. Experiments were confirmed in eight different donors (A, D-G) or three different donors (B-C). *** P < 0.001, ** P < 0.01, * P < 0.05, via one-way ANOVA.

Figure 3

MM-EVs induce elevated expression of miR-103a-3p, inhibiting osteogenesis in BM-MSCs. (A) Expression heatmap of the 18 MM-EVs' specifically highly expressed miRNA (Figure 1I) in MM-EVs, BM-MSCs and BM-MSCs treated with MM-EVs. (B) Real-time PCR showing the expression of miR-103a-3p in MM cells (RPMI8226) and MM-EVs (R-EVs). (C) The expression of miR-103a-3p in BM-MSCs treated with MM-EVs for 24 h. (D) BM-MSCs were transfected with miR-103a-3p mimics and the miR-103a-3p level was increased by 38.3-fold in the mimics group compared with the NC group. (E) BM-MSCs were transfected with miR-103a-3p inhibitor and the miR-103a-3p level was significantly decreased in BM-MSCs. (F) Alizarin Red staining of BM-MSCs transfected with miR-103a-3p mimics or inhibitor after 21 d (× 200). (G) The expression of miR-103a-3p in MM cells treated with MM-EVs for 24 h. (H) MM cells were analyzed with CCK-8 assay after being transfected with miR-103a-3p mimics or inhibitor for 24 and 48 h. All data are shown as the mean ± SD from at least four independent experiments; ** P < 0.01, * P < 0.05 by two-tailed Student's t-test.

Figure 4

Micro-CT analysis of tibiae collected. (A) A schematic diagram illustrating the experimental design. (B-F) Micro-CT bone quantitative analysis of BMD (B), BV/TV (C), BS/TV (D), Tb.N (E), and Tb.Th (F) in tibiae collected. (G) Representative 3D reconstructive images of separated trabecular bones. (H-I) Representative 3D reconstructive images (H) and section maps (I) of the collected tibiae. All data are shown as the mean ± SD. *** P< 0.001, ** P < 0.01, * P < 0.05 by one-way ANOVA followed by Sidak's multiple comparison test.

Figure 5

Decreased osteogenesis and exacerbated MBD in mice with MM-EVs. H&E (A), anti-CD138 (B), IHC (C), and TRAP (D) staining of sections of tibiae collected from the indicated groups of mice. Scale bars for (A), 200 μm (left) and 50 μm (right). Scale bars for (B-D), 20 μm. Osteocalcin is stained yellow in (C). (E) OB and OC numbers (left) and ratios (right). (F) Survival of the MM+MM-EVs group (n = 13) and the MM group (n = 13) compared by the log rank test. HE: H&E staining; IHC: immunohistochemistry; TRAP: tartrate resistant acid phosphatase. All data are shown as the mean ± SD. *** P< 0.001, ** P < 0.01, * P < 0.05 by one-way ANOVA followed by Sidak's multiple comparison test.

Figure 6

Peripheral CD138⁺ cirEV counts indicate bone lesions in D-MM patients. CD138⁺ cirEV counts in D-MM patients (n = 61) and healthy donors (n = 29) (A) and corresponding ROC curve (n = 56) (B) CD138⁺CD38⁺ cirEV counts in D-MM patients (n = 40) and healthy donors (n = 29) (C) and corresponding ROC curve (D). (E) The correlation between CD138⁺ cirEV count and bone lesion number in D-MM patients (n = 56). (F) The levels of PB CD138⁺ cirEVs among patients with 0 (n = 19), ≤ 3 (n = 15) or > 3 bone lesions (n = 22). CD138⁺ cirEV counts in D-MM patients with ≤ 3 lesions (n = 34) and those with > 3 lesions (n = 22) (G) and corresponding ROC curve (H). (I) Comparative analysis of CD138⁺CD38⁺ cirEV counts in D-MM patients with ≤ 3 lesions (n = 32) and those with > 3 lesions (n = 8). (J) Proposed MM-EV-mediated cycles between BM-MSCs and MM cells.