Mastocytosis-derived extracellular vesicles deliver miR-23a and miR-30a into pre-osteoblasts and prevent osteoblastogenesis and bone formation

Osteoporosis and other manifestations of bone disease are frequent in patients with systemic mastocytosis (SM) in association with the presence of mast cell infiltrates in bone marrow, although the mechanisms behind bone disease remain poorly understood. We find that extracellular vesicles (EVs) released by neoplastic mast cells and present in the serum of patients with SM (SM-EVs) block osteoblast differentiation and mineralization in culture, and when injected into mice diminish the expression of osteoblast markers, and trabecular bone volume and microarchitecture. We demonstrate that miRNA-30a and miRNA-23a, increased in SM-EVs and neoplastic mast cell-derived EVs, attenuate osteoblast maturation by suppressing expression of RUNX2 and SMAD1/5, essential drivers of osteogenesis. Thus, SM-EVs carry and deliver miRNAs that epigenetically interfere with bone formation and can contribute to bone mass reduction in SM. These findings also suggest possibilities for novel approaches to the management of bone disease in mast cell proliferative disorders.

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
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Software and code
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Data analysis
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Ana Olivera
Jan 15, 2021 Western blot images were obtained using Li-Cor Odyssey infrared blot imager Extracellular vesicle number and size distribution by Nanoparticle tracking analysis (NTA) using a NanoSight NS300 system (NanoSight) Measurements of Absorbance were performed using a plate reader (Spectra max 340PC), with a SoftMax Pro 6.2 Bone scan for Micro-CT was performed using a Bruker Micro CT analyzer Skyscan1172 Quantitative PCR was performed using a BioRad CFX96 Real-time System All studies must disclose on these points even when the disclosure is negative.

Sample size
Data exclusions Replication All data supporting the findings of this study are available within the main manuscript and the supplementary files or provided upon reasonable request. Raw data and original gel images corresponding to all the main and supplementary figures are included in the Source Data file.
For each experiment with osteoblasts, five separate wells were plated for each condition (n=5 biological replicates) , and the results obtained in the biological replicates were of high fidelity. Each experiment (with the various treatments) was repeated at least two additional times, with very similar results when comparing the different conditions. This is stated in the legends to figures and in the Statistical section in Methods. No statistical methods were used to predetermine sample size. The number of biological replicates per experiment were decided based on the literature and prior experience in our laboratory, and were sufficient to provide strong statistical significance between treatments (where observed).
For isolation of extracellular vesicles from patient serum samples we used 10-11 individuals per group, 21 patients mostly with indolent SM (divided into 2 groups of 10 and 11 respectively each, depending on their tryptase levels) and 10 healthy subjects, as indicated in Supplementary Table 1, group 1. Extracellular vesicles were also isolated from 10 additional patients with ISM to confirm the effects on osteoblasts. Given that SM is a rare disease, a total of 21 to 31 samples represent a sizable cohort. No statistical methods were used to predetermine sample size. Sample size was based on patient availability and on prior experience with extracellular vesicle isolation from patients and the reproducibility of the method. Due to the complexity of the experiments, only in several instances each sample was tested individually, while in most experiments, vesicles from individuals within a group were pooled to test their effects on osteoblasts for a better representation of the effects . This is indicated in the legends to figures or the results section.
For mouse studies, no statistical methods were used to predetermine sample size. Sample sizes were decided based on the literature (see examples below) and prior experience. For microCT, due to the cost and limited availability of the imaging facility, we show data for a total of 7-8 separate mice per group. In Figure 9b (microCT), for simplification purposes, data from mice injected with PBS or with EVs isolated from control individuals were combined (although they are noted with distinct symbols in the figure) as these groups were not significantly different and were both considered control samples; similarly, data from mice injected with vesicles from SM patients with high tryptase or SM with osteoporosis were combined in a single group ( Data were generally not excluded from the analyses.
All experiments were performed independently and successfully at least 3 times with very similar results using newly isolated extracellular vesicles each time from the same patient's populations. Mice were randomly assigned to groups. All had similar weight and appearance. Cells were treated randomly with the different types of extracellular vesicles Most of the studies in vitro in osteoblasts were not blinded. Blinding of test samples was not deemed applicable because measurements were empirical and not subjective. Samples were processed identically and were reproducible. Effects of bone marrow EVs on osteoblast differentiation were blinded, and histomorphology of the bone was evaluated by a pathologist blindly. For the microCT analysis the samples were numbered without knowing the exact group they were coming from. Table 4  The antibodies are from commercial sources and validated by suppliers for the reactivity species (human and/or mouse) and application (western blot or IHC) and by previous publications. For the following antibodies, where the supplier did not clearly specify whether they were validated, validation is shown by previous publications:

Specific information about all the antibodies used in this study is presented in Supplementary
Phospho-AKT (Thr308),Cell Signaling ( Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Certificate of analysis of hFOB1.19 cells (Batch number 63791506) was provided by ATCC and was mycoplasma free. Although we didn't have the specific batch fro HEK293 to find the certificate of analysis, ATCC thoroughly tests all cell cultures produced at ATCC before distribution to ensure that the cultures are free of mycoplasma contamination. Furthermore, all cells in the lab are randomly tested from time to time for mycoplasma since cultures of primary mast cells (handled in the same biosafety cabinets) are always ongoing in the lab and they are highly sensitive to mycoplasma No commonly misidentified cell lines were used in this study.
C57/B6 mice were from Jackson laboratories. Mice were 5-week old females at the time of injection (about 7-8 weeks at the time of euthanasia). For osteoclast differentiation, naive mice were 7 weeks old. The animals were shipped the NIAID (NIH) facilities, where they were housed according with the Office of Animal Care and Use guidelines under the NIH/NIAID Animal Care and Use Committee-approved animal study proposal (LAD2E), as explained in Methods