Osteocyte-derived exosomes confer multiple myeloma resistance to chemotherapy through acquisition of cancer stem cell-like features

Osteocyte-derived exosomes confer multiple myeloma resistance to chemotherapy through acquisition of cancer stem cell-like features | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Osteocyte-derived exosomes confer multiple myeloma resistance to chemotherapy through acquisition of cancer stem cell-like features Feifei Cheng, Zhiming Wang, Gichun You, Yuhong Liu, Jin He, Jing Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2535332/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 7 You are reading this latest preprint version Abstract Therapeutic resistance is a major challenge in multiple myeloma treatment. Understanding the underlying mechanisms is required to develop effective strategies against drug resistance and improve the prognosis of myeloma patients. Here, we identify osteocytes, the major cellular component of bone tissue, as key regulators of myeloma therapeutic resistance. Osteocyte-derived exosomes can be efficiently taken up by myeloma cells and exert a protective effect against chemotherapy-induced apoptosis. Mechanistic studies further reveal that osteocyte-derived exosomal miR-483-3p and miR-513a-5p promote cancer stem cell-like features in myeloma cells by regulating HIF-1α stabilization, thus conferring myeloma cells resistance to chemotherapy. Strikingly, combination treatment of miR-483-3p and miR-513a-5p inhibitors significantly reduces tumor burden and potentiates the therapeutic efficacy of bortezomib in the myeloma mouse model. Our findings, therefore, demonstrate the functional impact of osteocytes on myeloma therapeutic resistance, and suggest that osteocyte-derived exosomal miRNAs may serve as potential therapeutic targets for overcoming drug resistance in multiple myeloma. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Multiple myeloma, characterized by a clonal proliferation of malignant plasma cells within the bone marrow, is the second most common hematological malignancy in the United States and remains largely incurable [ 1 – 3 ]. With conventional treatment, the median survival time for patients with myeloma is 3 to 4 years [ 4 ]. In the past decades, several advanced therapeutic strategies, such as proteasome inhibitors, cell-cycle specific agents, monoclonal antibodies, and immunomodulatory drugs, have been used, which increase the survival time of myeloma patients to 5 to 7 years or longer [ 3 – 5 ]. However, most patients are still prone to quickly developing relapsed or refractory disease after treatment [ 6 – 8 ]. Patients with relapsed or refractory myeloma generally have a shorter duration of response and poorer survival outcomes with each successive treatment regimen [ 6 , 8 ]. This clinical problem is thought to be caused by the bone marrow microenvironment, which provides a friendly “bed” for myeloma cell proliferation and resistance to treatment [ 9 , 10 ]. Thus, an in-depth understanding of how the bone marrow microenvironment supports myeloma growth and drug resistance is the key to improving the efficacy of therapeutic strategies and prognosis in patients with myeloma. Osteocytes, which originate from osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs), are the most abundant cell type within bone tissue [ 11 , 12 ]. Osteocytes are linked to normal bone homeostasis and benign and malignant bone diseases through communication with other cells on the bone surface or within the bone marrow [ 13 , 14 ]. For example, osteocytes have been suggested to drive normal bone remodeling through the regulation of osteoblast and osteoclast differentiation and activity [ 15 ]. There is growing evidence that osteocytes are involved in myeloma growth, angiogenesis, homing to specific bone areas, and the pathogenesis of myeloma-associated bone disease [ 16 – 19 ]. However, whether and how osteocytes affect myeloma chemoresistance remain unclear. Exosomes are nanovesicles with a diameter ranging from 30–120 nm that originate from multivesicular bodies [ 20 , 21 ]. These nanovesicles contain a wide range of miRNAs, mRNAs, and functional proteins and show unique cellular membrane markers, such as CD63 and HSP90 [ 22 , 23 ]. Growing evidence has shown that exosomes can mediate the cellular communication between stroma and tumor, thus playing a crucial role in tumor development, progression and chemoresistance [ 23 – 25 ]. Like other stromal cell types, osteocytes can produce exosomes and release them into the microenvironment [ 26 ]. However, evidence for the link between osteocyte-derived exosomes and myeloma drug resistance is limited. Here, we found that osteocyte-derived exosomes can be taken up by myeloma cells. Importantly, our data further show that osteocyte-derived exosomes exert a protective effect on myeloma cells against chemotherapy-induced apoptosis. Mechanistic studies indicate that exosomal miR-483-3p and miR-513a-5p promote cancer stem cell-like features in myeloma cells by regulating the degradation of hypoxia-inducible factor-1 α subunit (HIF-1α) via heat shock cognate 70-kDa protein (HSC70), thus conferring myeloma cells resistance to chemotherapy. Moreover, our mouse xenograft analysis suggests that combination treatment of miR-483-3p and miR-513a-5p inhibitor reduces tumor burden and potentiates the therapeutic efficacy of bortezomib in vivo . Hence, our study identifies the functional impact of osteocytes on myeloma therapeutic resistance, implicating osteocyte-derived exosomal miRNAs as therapeutic targets for overcoming tumor drug resistance in multiple myeloma. Materials And Methods Cell lines and primary cells The myeloma cell line ARP-1 was kindly provided by the University of Arkansas for Medical Sciences. RPMI8226, MM.1S, and U266 cell lines were purchased from the American Type Culture Collection (VA, USA). Primary myeloma cells were isolated from bone marrow aspirates from newly diagnosed myeloma patients using anti-CD138 antibody-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Myeloma cells were maintained in RPMI 1640 medium with 10% fetal bovine serum. In vitro generation, isolation and characterization of human MSCs were performed as previously described [ 27 ]. MSCs were maintained in the MSC medium (ScienCell Research Laboratory, CA, USA). Mature osteoblasts and osteocytes were differentiated from human MSCs. The murine osteocyte cell lines MLO-Y4 and MLO-A5 were purchased from Kerafast, Inc. (MA, USA). MLO-A5 cells were differentiated to the mature type before used. All patient samples were obtained from the Biorepository Core of Houston Methodist Research Institute. Informed consent was obtained from study participants, and the collection of patient samples was conducted by Houston Methodist Research Institute in accordance with the criteria set by the Declaration of Helsinki. This study was approved by the Institutional Review Board of Houston Methodist Research Institute. miRNA and reagents miR-483-3p mimic, miR-513a-5p mimic, anti-miR-483-3p inhibitor, anti–miR-513a-5p inhibitor, and their scrambled controls were obtained from Integrated DNA Technologies (IA, USA). Lipofectamine 3000 used for miRNA mimic transfection and red fluorescent dye Dil were purchased from Thermo Fisher Scientific (MA, USA). Bortezomib, carfilzomib, and heparin were purchased from MedChemExpress (NJ, USA). Melphalan was purchased from Sigma-Aldrich (MA, USA). HIF-1α inhibitor CAY10585 was from Cayman Chemical (MI, USA). Exosome isolation, characterization and uptake assay To isolate osteocyte-secreted exosomes, mature osteocytes were cultured for 3 days in DMEM medium with 10% exosome-free FBS, and then culture supernatants were collected. Exosomes were isolated from filtered culture supernatants using total exosome isolation reagent (Thermo Fisher Scientific). Briefly, culture supernatants were mixed with the isolation reagent in a 1:2 ratio, and was vortexed to form a homogenized solution. After overnight incubation at 4°C, the mixture was centrifuged at 10,000 g for 1 h. The pellet containing exosomes was then resuspended in PBS. As for the characterization of isolated exosomes, it was performed as previously described [ 25 ]. The expression of exosomal marker proteins, such as HSP90 and CD63, were examined. To detect exosome uptake of myeloma cells, the red fluorescent dye Dil was used to label osteocyte-secreted exosomes. After incubation of myeloma cells with labeled osteocyte-secreted exosomes for 0, 4, 8, or 24 hours, confocal microscopy (Olympus, Tokyo, Japan) was used for imaging. RT-qPCR Total cellular RNA was extracted using the RNeasy kit (QIAGEN, Venlo, Netherlands) according to the manufacturer’s protocol. Exosomal RNA was isolated from exosomes using the total exosome RNA & Protein Isolation Kit. For quantitative real-time PCR, reverse transcription was conducted as per the protocol of the SuperScript II RT-PCR kit (Life Technologies, MA, USA), and quantitative PCR was performed using SYBR Green Master Mix (Life Technologies) with the QuantStudio 3 Real-Time PCR System (Life Technologies). GAPDH mRNA was used as input control. Primers for qPCR are listed in Supplementary Table S1. Western blot Cells were harvested and lysed in lysis buffer (Cell Signaling Technology, MA, USA). To obtain exosomal proteins, exosomes were lysed with RIPA lysis and extraction buffer (Cell Signaling Technology) and isolated using the total exosome RNA & Protein Isolation Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The lysates were then subjected to SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies against CD63, HSP90, SOX2, OCT4, NANOG, HIF-1α, HSC70, VHL, MDM2, PTEN, and GAPDH. The level of GAPDH served as the protein loading control. The details of antibodies used in the study are listed in Supplementary Table S2. ELISA and immunofluorescence assay Serum M-protein levels were measured by an ELISA kit (Bethyl Laboratories, TX, USA) according to the manufacturer’s protocol. The immunofluorescence assay was performed as previously described [ 3 , 28 ]. Briefly, cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 20 minutes at room temperature. After blocking with 2% goat serum for 1hour, the cells were stained with antibodies against SOX2, OCT4, and HIF-1α at 4°C overnight. Next, the cell nuclei were stained with DAPI and mounted with ProLong Gold antifade reagent (Molecular Probes, OR, USA). Immunofluorescent images were acquired with an IX81 confocal microscope system (Olympus). Apoptotic assay Annexin V binding assay was used to detect cell apoptosis by flow cytometry. 2×10 5 cells were plated into 24-well plates, followed by treatment with bortezomib (5 nM), melphalan (25 µM), or carfilzomib (25 nM) for 24 hours. Cells were then collected and stained with APC Annexin V (Biolegend, CA, USA) and propidium iodide (Invitrogen, MA, USA). After a 20-minute incubation at room temperature, cells were measured by a BD FACS Symphony A3 flow cytometer (BD Biosciences, CA, USA). Sphere formation assay Sphere formation assay was performed as previously described [ 29 ]. Briefly, cells were plated into an ultra-low attached 96-well plate at a density of 100 per well, followed by culture in serum-free cancer stem cell growth media consisting of DMEM/F12 medium (Hyclone, UT, USA), 1% penicillin-streptomycin, 2% B27 supplements, 10 ng/ml basic FGF, 20 ng/ml EGF, and 5 µg/ml insulin. After 4 days, an appropriate amount of fresh media was added. Cells were incubated for about 12 days, and sphere diameters were measured. Colony formation assay Colony formation assay was performed as previously described [ 3 ]. Briefly, cells were incubated with osteocyte-derived exosomes. After 24 hours, cells were collected and resuspended with 0.4% agarose in the serum-free RPMI medium. Then, cells were seeded into a 6-well plate pre-coated with 0.8% agar in the complete growth medium, followed by incubation at 37°C in a humidified atmosphere containing 5% CO 2 . After a 3-week culture, colonies were photographed and counted. Side population analysis Cells were collected and resuspended in RPMI1640 supplemented with 2% FBS and 10 mM HEPES at a density of 1 × 10 6 cells/mL. Subsequently, Hoechst 33342 dye was added to the cells at a concentration of 5 µg/mL. After incubation for 2 hours at 37°C with intermittent shaking, cells were collected and resuspended in pre-cold HBSS containing 2% FBS and 10 mM HEPES. Propidium iodide solution was added to the cells to a final concentration of 5 µg/mL. Side population cells were examined by a BD FACS Symphony A3 flow cytometer (BD Biosciences). Immunohistochemistry Immunohistochemistry was performed as previously described [ 30 ]. In brief, formalin-fixed, paraffin-embedded sections of samples from the bone marrow or subcutaneous tissue of mice were deparaffinized and stained with anti-SOX2, anti-OCT4, anti-NANOG, and HIF-1α, antibodies using an EnVision System (DAKO, Glostrup, Denmark) according to the manufacturer’s protocol and counterstained with hematoxylin. In some experiments, stained slides were scanned with the Vectra 3 automated quantitative pathology imaging system and analyzed by inForm software (Perkin Elmer, MA, USA). Images were obtained using a microscope (Leica, Wetzlar, Germany). In vivo mouse experiments NOD-scid IL2Rgnull (NSG) mice were purchased from The Jackson Laboratory (ME, USA). All mice were maintained in American Association of Laboratory Animal Science-accredited facilities and all in vivo mouse studies were approved by the Institutional Animal Care and Use Committees of Houston Methodist Research Institute. Six-week-old male NSG mice were subcutaneously injected with RPMI8226 (5 × 10 5 cells) or RPMI8226 cells preincubated with osteocyte-derived exosomes (5 × 10 5 cells). For drug treatment, ARP-1 cells (5×10 5 cells/mouse) were intrafemorally injected into the NSG mice. After 2 weeks, mice were intraperitoneally injected with bortezomib (0.5 mg/kg) alone or in combination with anti-miR-483-3p or anti-miR-513a-5p inhibitor (5 mg/kg) three times a week for 2 weeks. Mice receiving an equal amount of vehicle served as controls. Tumor burden was evaluated by serum M-protein level and bioluminescence imaging. qPCR was used to measure the expression of SOX2, NANOG, OCT4, HIF-1α, and HSC70 in the bone marrow of randomly selected mice. Statistical analysis Statistical analyses were performed using GraphPad Prism 9 (CA, USA) with two tailed unpaired Student t-tests for comparison of two groups and one-way ANOVA for comparison of more than two groups. Pearson’s correlation analysis was used to examine the correlation between two variables and Kaplan-Meier analysis was used in survival analysis. The level of significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001. Results are presented as mean ± standard deviation (SD). Results Uptake of osteocyte-derived exosomes by myeloma cells To study the potential role of osteocyte-derived exosomes in myeloma, we first examined whether osteocyte-derived exosomes can be transmitted into myeloma cells. Figure 1 A shows the procedure for the isolation, Dil labeling, and myeloma cell uptake of osteocyte-derived exosomes. Osteocytes were generated by inducing the osteogenic differentiation of human-derived BMMSCs. The differentiated cells exhibited high expression of osteocyte-specific markers DMP-1 and SOST and low expression of osteoblast-specific markers RUNX2 and OSX (Fig. 1 B), suggesting the generation of osteocytes. Next, exosomes were isolated from the conditioned medium of differentiated osteocytes and characterized as described in our previous study [ 25 ]. In line with previous results, osteocyte-derived exosomes presented high expression of the exosome-specific markers HSP90 and CD63 (Fig. 1 C). To determine whether osteocyte-derived exosomes were transferred to myeloma cells, we incubated several human myeloma cell lines with fluorescent Dil-labeled exosomes (Fig. 1 A). Obvious red fluorescence signals were observed in ARP-1, RPMI 8226, and U266 cells at 24 hours after exposure to osteocyte-derived exosomes (Fig. 1 D and Supplementary Fig. 1A), while no fluorescence signals were observed in the control group, suggesting the internalization of Dil-labeled exosomes by myeloma cells. Notably, the fluorescence signals were increased in ARP-1 cells in a time-dependent manner (Fig. 1 E and F). Moreover, we found that treatment of heparin, which can block the exosome uptake of tumor cells, significantly reduced the fluorescence signals (Fig. 1 G). Taken together, these findings indicate that osteocyte-derived exosomes can be taken up by myeloma cells. Osteocyte-derived exosomes inhibit chemotherapy-induced apoptosis in myeloma cells Multiple studies have reported that stroma-derived exosomes play a crucial role in myeloma resistance to chemotherapy [ 23 , 25 ]. Therefore, we next sought to evaluate whether osteocyte-derived exosomes are involved in myeloma therapeutic response. We treated ARP-1 cells with three commonly used chemotherapeutic drugs including bortezomib, carfilzomib, and melphalan in the presence or absence of osteocyte-derived exosomes. It was observed that drug treatment markedly induced apoptosis in ARP-1 cells, while the addition of the exosomes reduced the efficacy of these drugs (Fig. 2 A). Similar results were also observed in three other myeloma cell lines: RPMI8226, MM.1S, and U266 (Fig. 2 B-D). In addition to the exosomes derived from BMMSC-differentiated human osteocytes, the addition of exosomes derived from two murine osteocyte cell lines and conditioned medium from BMMSC-differentiated human osteocytes also exhibited anti-apoptotic effects in myeloma cells (Fig. 2 E). Notably, consistent results were obtained in primary malignant plasma cells isolated from patients with newly diagnosed myeloma when treated with bortezomib (Fig. 2 F). These results indicate that osteocyte-secreted exosomes can induce therapeutic resistance in myeloma. Osteocyte-derived exosomes promote cancer stem cell-like features of myeloma cells Next, we investigated the mechanism by which osteocyte-derived exosomes protect myeloma cells against chemotherapy. It has been widely reported that cancer cell stemness contributes to chemoresistance [ 31 , 32 ]. Hence, we examined whether osteocyte-derived exosomes regulate cancer stem cell-like features of myeloma cells. CD19 + /CD27 + /CD138 − cells have been identified as a myeloma stem cell population [ 32 ]. As shown in Fig. 3 A, it was observed that osteocyte-derived exosome treatment significantly increased the percentage of CD19 + /CD27 + /CD138 − cells in ARP-1 and RPMI8226 cells. In addition, cancer side population cells represent a subpopulation of cancer cells with stem-like features and have been suggested to play a crucial role in drug resistance due to their high drug extrusion ability [ 33 ]. Therefore, we assessed the effect of osteocyte-derived exosomes on myeloma side population cells. Our data showed that incubation with osteocyte-derived exosomes increased the percentage of side population cells in RPMI8226 and MM.1S cells (Fig. 3 B). It was also demonstrated that osteocyte-derived exosome incubation promoted the sphere formation ability of myeloma cells (Fig. 3 C). Consistently, colony formation assay showed that osteocyte-derived exosome incubation increased the number and size of colonies compared with the control group (Fig. 3 D-F). However, heparin treatment attenuated the increase in colony number and size (Fig. 3 G-I). Subsequently, we sought to determine whether cultures with osteocyte-derived exosomes affect stemness-associated gene expression in myeloma. As shown by RT-qPCR assay, osteocyte-derived exosome incubation significantly increased the mRNA expression of the stemness-associated genes SOX2 , OCT4 , and NANOG in myeloma cells (Fig. 3 J). The expression of these genes was further confirmed at the protein level using western blot and immunofluorescence analysis (Fig. 3 K-L). In addition, it was demonstrated that heparin treatment abrogated the increased stemness-associated gene expression induced by incubation with osteocyte-derived exosomes (Fig. 3 M). Hence, osteocyte-derived exosomes promote cancer stem cell-like features of myeloma cells. Osteocyte-derived exosomes facilitate myeloma tumorigenesis in vivo We next assessed the effect of osteocyte-derived exosomes on myeloma tumorigenesis in vivo . We incubated RPMI8226 cells with osteocyte-derived exosomes in vitro and then subcutaneously implanted the serial dilution of the cells into the right flank of NSG mice (Fig. 4 A). The cells without exosome incubation were implanted into the left flank of mice as a control (Fig. 4 A). As shown in Fig. 4 B-D, preincubation with osteocyte-derived exosomes facilitated myeloma tumorigenesis. Notably, in the group injected with 500 myeloma cells, 50% (2/4) mice did not develop visible tumors in the left flank, whereas all mice presented a visible tumor in the right flank (Fig. 4 B-D). This finding indicated that preincubation with osteocyte-derived exosomes enhanced the tumor initiation ability of myeloma cells, which further supported the view that osteocyte-derived exosomes promote cancer stem cell-like features of myeloma cells. Indeed, immunohistochemical staining analysis showed that tumors derived from exosome-preincubated myeloma cells had a significantly higher expression of SOX2, OCT4, and NANOG compared with those in the control group (Fig. 4 E-F). These in vivo findings, along with our previous in vitro studies, suggest that osteocyte-derived exosomes promote cancer stem cell-like features of myeloma cells. Osteocyte-derived exosomal miR-483-3p and miR-513a-5p promote myeloma cell chemoresistance and cancer stem cell-like features Exosomes carry a wide range of miRNAs, mRNAs, and functional proteins [ 34 ]. Previous studies have identified exosomal miRNAs as important regulators in myeloma progression [ 23 , 34 ]. To explore whether and which kind(s) of miRNAs carried by osteocyte-derived exosomes regulate myeloma chemoresistance and cancer stem cell-like features, we first analyzed a published dataset of serum-derived exosomal miRNA expression profile changes in bortezomib-resistant and bortezomib-responsive myeloma patients (GSE71435). It was demonstrated that 9 miRNAs were significantly upregulated in the serum of bortezomib-resistant patients compared with that of bortezomib-responsive patients (Fig. 5 A). Among these 9 miRNAs, miR-483-3p, miR-513a-5p, and miR-3976 were found to be highly expressed in osteocyte-derived exosomes (Fig. 5 B). In addition, it was also observed that there was significantly higher expression of miR-483-3p and miR-513a-5p in osteocyte-derived exosomes compared with those in exosomes from BMMSCs, osteoblasts and myeloma cells (Fig. 5 C). Next, we examined the effect of miR-483-3p and miR-513a-5p in myeloma. Our data showed that overexpression of miR-483-3p or miR-513a-5p significantly attenuated the increased apoptosis of RPMI 8226 cells induced by bortezomib and carfilzomib treatment (Fig. 5 D). Conversely, inhibition of miR-483-3p or miR-513a-5p by the specific inhibitor blocked the protective effect of osteocyte-derived exosomes in RPMI8226 cells (Fig. 5 E). In addition, overexpression of miR-483-3p or miR-513a-5p significantly increased the percentage of side population cells in myeloma cells (Fig. 5 F). However, inhibition of miR-483-3p and miR-513a-5p abrogated the increased side population cell percentage induced by osteocyte-derived exosome incubation (Fig. 5 G). These results indicate that osteocyte-derived exosomal miR-483-3p and miR-513a-5p promote myeloma cell chemoresistance and cancer stem cell-like features. miR-483-3p and miR-513a-5p promote cancer stem cell-like features of myeloma cells through regulation of HIF-1α stabilization We further investigated the molecular pathway by which osteocyte-derived exosomal miR-483-3p and miR-513a-5p regulate myeloma cell stemness. Previous studies have demonstrated that HIF-1α signaling plays a critical role in the interactions between bone marrow cells and myeloma cells, as well as myeloma cell stemness and drug resistance [ 35 – 37 ]. Thus, we sought to evaluate whether HIF-1α signaling is involved in the regulation of exosomal miR-483-3p and miR-513a-5p in myeloma cell stemness. As shown in Fig. 6 A-B, incubation with osteocyte-derived exosomes increased the protein expression of HIF-1α in myeloma cells. This finding was also supported by immunohistochemical analysis of HIF-1α protein in tumors generated in the osteocyte exosome-pretreated myeloma cell xenograft model (Fig. 4 A and 6 C). Survival analysis also revealed that high HIF-1α expression was significantly linked to the decreased overall survival of myeloma patients (Fig. 6 D). In addition, HIF-1α inhibitor treatment significantly attenuated the increased expression of stemness-associated genes induced by incubation with osteocyte-derived exosomes (Fig. 6 E). These findings indicate that osteocyte-derived exosomes promote cancer stem cell-like features of myeloma cells through HIF-1α signaling. Notably, qPCR assay showed that osteocyte-derived exosome incubation did not affect the mRNA expression of HIF1A (Fig. 6 F), implying that osteocyte-derived exosomes might regulate HIF-1α protein expression by modulating its stabilization. Therefore, we evaluated whether incubation with osteocyte-derived exosomes affect the protein levels of the genes HSC70 , VHL , MDM2 , and PTEN , which are closely associated with HIF-1α stabilization [ 38 , 39 ]. As shown by the Western blot analysis, osteocyte-derived exosome incubation reduced the protein expression of HSC70 (Fig. 6 A), which has been shown to participate in lysosomal degradation of HIF-1α through chaperone-mediated autophagic pathway [ 39 ]. Consistently, it was also observed that overexpression of miR-483-3p or miR-513a-5p significantly increased the expression of HIF-1α and Sox2, but reduced HSC70 expression in myeloma cells (Fig. 6 G). Moreover, inhibition of miR-483-3p and miR-513a-5p reverted the protein expression of HIF-1α, SOX2, and HSC70 (Fig. 6 H). Taken together, these findings suggest that osteocyte-derived exosomal miR-483-3p and miR-513a-5p promote cancer stem cell-like features of myeloma cells by downregulating HSC70-mediated HIF-1α protein degradation (Fig. 6 I). Combination treatment of miR-483-3p and miR-513a-5p inhibitors potentiate the therapeutic efficacy of bortezomib in the myeloma mouse model Having verified the role of osteocyte-derived exosomal miR-483-3p and miR-513a-5p in myeloma cancer cell stemness and chemoresistance in vitro , we further investigated whether combination treatment of miR-483-3p and miR-513a-5p inhibitors can potentiate the efficacy of chemotherapy in a myeloma xenograft mouse model by intravenously injecting the luciferase-labeled myeloma cells into NSG mice (Fig. 7 A). On the 14th day after myeloma cell injection, myeloma-bearing mice were randomized into 7 groups and given intraperitoneal injections of vehicle, bortezomib, miR-483-3p inhibitor, miR-513a-5p inhibitor single or in a combination. Tumor burden was monitored by bioluminescence imaging. After 2-week treatment, single miR-483-3p or miR-513a-5p inhibitor treatment significantly decreased tumor burden compared with the vehicle group (Fig. 7 B-C). Furthermore, combination of bortezomib with miR-483-3p or miR-513a-5p inhibitor showed a greater reduction in tumor burden than the vehicle or single-drug group (Fig. 7 B-C). The triple combination of miR-483-3p inhibitor, miR-513a-5p inhibitor, and bortezomib resulted in the highest effect on tumor burden (Fig. 7 B-C). These results were also confirmed by measuring serum levels of M-proteins, which are secreted from myeloma cells (Fig. 7 D). Hence, combination treatment of miR-483-3p and miR-513a-5p inhibitors potentiates the anti-myeloma therapeutic efficacy of bortezomib in vivo . In addition, it was also demonstrated that single miR-483-3p or miR-513a-5p inhibitor treatment significantly reduced the expression level of stemness-associated genes and increased HSC70 expression compared with the vehicle group, similar to in vitro results (Fig. 7 E-G). These findings further support the view that osteocyte-derived exosomal miR-483-3p and miR-513a-5p promote cancer stem cell-like features of myeloma cells by downregulating HSC70-mediated HIF-1α protein degradation. Discussion Although advanced therapies have improved the prognosis of patients with myeloma, myeloma remains largely incurable [ 3 , 40 ]. In parallel with the development and clinical use of these advanced therapies is the emergence of novel mechanisms of drug resistance, many of which remain elusive. Herein, we demonstrate for the first time that osteocytes, the most abundant cell type within bone tissue, are new regulators of myeloma therapeutic resistance. We identified two osteocyte-derived miRNAs, miR-483-3p and miR-513a-5p, both of which promoted cancer stem cell-like features in myeloma cells by regulating HIF-1α stabilization, thus conferring myeloma cells resistance to chemotherapy. Moreover, combination treatment of miR-483-3p and miR-513a-5p inhibitors significantly reduced myeloma tumor burden and potentiated the therapeutic efficacy of bortezomib. Thus, our study provides insights into the molecular mechanisms underlying myeloma chemoresistance, implicating osteocyte-derived exosomal miRNAs as novel therapeutic targets for overcoming chemoresistance in myeloma. During the past several decades, our understanding of the biological function of osteocytes has been changing. Originally, osteocytes were considered as an inactive placeholder [ 41 ]. Then, they were linked to the process of normal bone remodeling by regulating the differentiation and activity of osteoclasts and osteoblasts [ 15 ]. Currently, there is growing evidence that osteocytes play a crucial role in myeloma-associated bone disease, which is a major problem in myeloma patients [ 19 , 42 , 43 ]. By producing osteolytic cytokines, osteocytes can enhance myeloma-induced bone lesions. Using genetically engineered mice, Delgado-Calle J et al. reported that osteocyte-secreted sclerostin contributes to myeloma-induced bone loss [ 42 ]. A preclinical study by McDonald MM et al. showed that pharmaceutical inhibition of sclerostin reduced lytic lesions and enhanced new bone formation [ 43 ], suggesting a potential application of the inhibitor against osteocyte-secreted sclerostin to treat myeloma patients with lytic lesions. Previous studies also found that osteocyte production of osteolytic cytokines can be enhanced by myeloma cells [ 16 , 19 ]. These results indicate the important role of osteocytes in the development and pathogenesis of myeloma-associated bone disease. However, the biological function of osteocytes in myeloma growth and survival is still vague. In this study, we explored the role of osteocytes in myeloma cell response to chemotherapy, another major problem in most myeloma patients, and found the protective effect of osteocytes on myeloma cells against all tested chemotherapeutic drugs. Our findings unveil a novel unexplored role of osteocytes in myeloma pathogenesis. To the best of our knowledge, this is the first study to investigate the role of osteocyte-derived exosomes in myeloma therapeutic response. Exosomes are nanovesicles and have been shown to be a common mediator for the cellular communication between tumor cells and microenvironmental stromal cells [ 23 ]. Though buried within lacunae, osteocytes can secrete exosomes, which circulate in the blood [ 26 ]. Accumulating evidence shows that osteocyte-secreted exosomes are involved in bone homeostasis and benign diseases [ 44 – 46 ], but few studies have investigated their role in tumors, including myeloma. Here, we observed reduced chemotherapy-induced myeloma cell apoptosis once myeloma cells take up osteocyte-derived exosomes, indicating that osteocyte-derived exosomes are required for chemoresistance, at least partly. We also examined the effect of osteocyte-derived exosomes on myeloma cancer cell stemness, a common mechanism for inducing therapeutic resistance. Previous studies have shown that the population with CD19 + /CD27 + /CD138 − is the myeloma stem cell population [ 32 ]. We measured the percentage change of this population in myeloma cells after incubation with osteocyte-derived exosomes and found a significantly increased percentage. In addition, we also used several well-established approaches, including in vitro sphere formation, colony formation, side population, stemness-associated gene expression, and in vivo tumorigenesis, to confirm our finding that osteocyte-secreted exosomes enhance myeloma cancer cell stemness. We identified two osteocyte-derived exosomal miRNAs that are responsible for myeloma drug resistance using a dataset containing thousands of circulating exosomal miRNAs in a cohort of myeloma patients with bortezomib-based therapy. miRNAs, composed of 18 to 25 nucleotides, can be transferred from stromal cells to tumor cells through exosomes and then transmit a signal for supporting tumor development [ 47 , 48 ]. In this study, we observed that miR-483-3p and miR-513a-5p are enriched in osteocyte-derived exosomes, while their levels are much lower in the exosomes from the precursor MSCs, mature osteoblasts, and myeloma cells. Previous studies show that the level of miR-483-3p is upregulated during MSC differentiation to mature bone cells [ 49 , 50 ]. These findings indicate that osteocytes express unique miRNAs. We further found that these two miRNAs have an inhibitory effect on the expression of HSC70, which contributes to lysosomal degradation of HIF-1α through the chaperone-mediated autophagic pathway [ 39 ], thus enhancing HIF-1α stabilization, cancer cell stemness, and chemoresistance in myeloma cells. Our study originally links osteocytes to myeloma therapeutic resistance by exosomal miRNA-induced myeloma cancer cell stemness. Our next goal is to identify other osteocyte-derived unique factors, which may be used as biomarkers to evaluate myeloma therapeutic efficacy and even used as therapeutic targets. In summary, we have systematically elucidated the role of osteocyte-derived exosomal miRNAs in myeloma chemoresistance herein. Considering the paucity of knowledge of the specific determinants conferring myeloma cells resistance to chemotherapy, our study provides mechanistic insights into myeloma chemoresistance, and suggests that targeting osteocyte-derived exosomal miRNAs may overcome chemoresistance in multiple myeloma. Declarations Competing Interests statement: The authors declare no competing interests. Acknowledgments This work was supported by the National Institutes of Health/National Cancer Institute (R01 awards CA190863 and CA193362), the American Cancer Society (Research Scholar Grant 127337-RSG-15-069-01-TBG), and the Cancer Prevention & Research Institute of Texas (RP220639). We thank the Myeloma Tissue Bank at Houston Methodist Research Institute. Supports also came from Research Pathology Core, Flow Cytometry Core, and Translational Imaging Core at Houston Methodist Research Institute. We would like to thank Dr. Shaefali P. Rodgers, Houston Methodist Hospital, who edited the manuscript. Author contributions J.Y. and F.C. designed the study and wrote the manuscript; F.C., Z.W., and G.Y. performed the experiments; F.C., Z.W., Y.L., and J.H. analyzed the data and conducted statistical analysis. All authors reviewed the final manuscript. Data availability The authors declare that all data supporting the findings of this study are available within the article or its Supplementary Materials and from the corresponding author upon reasonable request. References Van de Donk NWCJ, Pawlyn C, Yong KL. Multiple myeloma. Lancet. 2021;397(10272):410-427. https://doi.org/10.1016/s0140-6736(21)00135-5. Liu H, He J, Koh SP, Zhong Y, Liu Z, Wang Z, et al. Reprogrammed marrow adipocytes contribute to myeloma-induced bone disease. Sci Transl Med. 2019;11(494):eaau9087. https://doi.org/10.1126/scitranslmed.aau9087. Li Z, Liu H, He J, Wang Z, Yin Z, You G, et al. Acetyl-CoA Synthetase 2: A Critical Linkage in Obesity-Induced Tumorigenesis in Myeloma. 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Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 16 Feb, 2023 Review # 1 received at journal 13 Feb, 2023 Reviewer # 1 agreed at journal 03 Feb, 2023 Reviewers invited by journal 02 Feb, 2023 Editor assigned by journal 01 Feb, 2023 Submission checks completed at journal 01 Feb, 2023 First submitted to journal 31 Jan, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2535332","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":172674519,"identity":"132cdb12-61c5-4df4-aadf-aba1cfec3047","order_by":0,"name":"Feifei Cheng","email":"","orcid":"https://orcid.org/0000-0002-3234-513X","institution":"Houston Methodist Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Feifei","middleName":"","lastName":"Cheng","suffix":""},{"id":172674520,"identity":"e83d545f-4348-4807-a973-ca533a8ea7c4","order_by":1,"name":"Zhiming Wang","email":"","orcid":"","institution":"Houston Methodist Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhiming","middleName":"","lastName":"Wang","suffix":""},{"id":172674521,"identity":"bcdff9af-3512-4388-aac9-164d95d9962d","order_by":2,"name":"Gichun You","email":"","orcid":"","institution":"Houston Methodist Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gichun","middleName":"","lastName":"You","suffix":""},{"id":172674522,"identity":"63c23c6b-0603-48ad-8519-e57b088544e6","order_by":3,"name":"Yuhong Liu","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuhong","middleName":"","lastName":"Liu","suffix":""},{"id":172674523,"identity":"4a3b0716-b3d3-4e63-8aef-7e03a925cfe4","order_by":4,"name":"Jin He","email":"","orcid":"","institution":"Houston Methodist Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"He","suffix":""},{"id":172674524,"identity":"7c6f0f02-6252-4239-a720-e8fa37cea1aa","order_by":5,"name":"Jing Yang","email":"data:image/png;base64,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","orcid":"","institution":"Cancer Center for Hematological Malignancies, Houston Methodist Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2023-01-31 19:26:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2535332/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2535332/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":32443244,"identity":"961b7d47-614e-4bff-87aa-d0ad2f604818","added_by":"auto","created_at":"2023-02-03 15:54:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":854217,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic workflow of the incubation of osteocyte-derived exosomes with myeloma cells. (B) qPCR analysis for mRNA expression levels of osteocyte- and osteoblast- specific markers in osteocytes generated from the osteogenic differentiation of human-derived BMMSCs. (C) Western blot analysis for protein expression levels of HSP90 and CD63 in purified exosomes from two different human BMMSC-differentiated osteocytes (OS exo 1 and 2). (D) Confocal microscopy images of ARP-1 and RPMI8226 cells that were exposed to Dil pre‐stained osteocyte‐derived exosomes for 24 hours. Nuclei were counterstained with DAPI. Scale bar, 50 μm. (E) Confocal microscopy images of ARP-1 cells that were exposed to Dil pre‐stained osteocyte‐derived exosomes for 0, 4, 8, or 24 hours. Nuclei were counterstained with DAPI. (F) Time course of relative fluorescence intensity (AU) of ARP-1 cells after exposure to Dil pre‐stained osteocyte‐derived exosomes. (G) Confocal microscopy images of RPMI8226 cells that were exposed to Dil pre‐stained osteocyte‐derived exosomes with or without heparin treatment.\u003c/p\u003e\n\u003cp\u003eError bars are SD of biological replicates and p values: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2535332/v1/6db4da70f0300b36a247d638.png"},{"id":32444614,"identity":"887f40fa-f44b-4298-960e-9f5087dfbbc1","added_by":"auto","created_at":"2023-02-03 16:02:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":334173,"visible":true,"origin":"","legend":"\u003cp\u003e(A-D) Annexin V‐binding assay shows the percentages of apoptotic myeloma cells treated with 5 nM bortezomib (BTZ), 25 μM melphalan (Mel), or 25 nM carfilzomib (CFZ) in the presence or absence of osteocyte-derived exosomes. (E) Annexin V‐binding assay shows the percentages of apoptotic ARP-1 cells treated with 5 nM bortezomib (BTZ) in cultures with PBS control, the exosomes isolated from culture medium of osteocytes differentiated from BMMSCs or two murine osteocyte cell lines MLO-Y4 and MLO-A5, or the conditioned medium (CM) of osteocytes differentiated from BMMSCs. (F) Annexin V‐binding assay shows the percentages of apoptotic primary malignant plasma cells from three myeloma patients (Pt) after 5 nM bortezomib (BTZ) treatment in the presence or absence of osteocyte-derived exosomes.\u003c/p\u003e\n\u003cp\u003eError bars are SD of biological replicates and p values: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2535332/v1/72438d37ebb03f2beeeb4099.png"},{"id":32443240,"identity":"3aec4ec3-f513-4717-8a08-c306daeade20","added_by":"auto","created_at":"2023-02-03 15:54:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1053203,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The percentages of CD19\u003csup\u003e+\u003c/sup\u003e/CD27\u003csup\u003e+\u003c/sup\u003e/CD138\u003csup\u003e−\u003c/sup\u003e populations in ARP-1 and RPMI8226 cells in presence or absence of osteocyte-derived exosomes. (B) The percentages of side population cells in RPMI8226 and MM.1s cells in the presence or absence of osteocyte-derived exosomes. (C) Sphere formation ability of ARP-1 and RPMI8226 cells in the presence or absence of osteocyte-derived exosomes. (D) Representative images of colonies generated from ARP-1 and RPMI8226 cells in the presence or absence of osteocyte-derived exosomes. (E, F) Numbers (E) and diameters (F) of colonies generated from ARP-1 and RPMI8226 cells in the presence or absence of osteocyte-derived exosomes. (G) Representative images of colonies generated from ARP-1 and RPMI8226 cells after incubation with osteocyte-derived exosomes, or in combination with heparin. (H, I) Numbers (H) and diameters (I) of colonies generated from ARP-1 and RPMI8226 cells after incubation with osteocyte-derived exosomes, or in combination with heparin. (J) qPCR analysis for mRNA level changes of \u003cem\u003eSOX2\u003c/em\u003e, \u003cem\u003eOCT4\u003c/em\u003e, and \u003cem\u003eNANOG\u003c/em\u003e in ARP-1 and RPMI8226 cells after incubation with osteocyte-derived exosomes. (K) Western blot analysis for protein level changes of SOX2, OCT4, and NANOG in ARP-1 and RPMI8226 cells after incubation with osteocyte-derived exosomes. (L) Immunofluorescence analysis for protein level changes of SOX2 and OCT4 in ARP-1 cells after incubation with osteocyte-derived exosomes. (M) qPCR analysis for mRNA level changes of \u003cem\u003eSOX2\u003c/em\u003e, \u003cem\u003eOCT4\u003c/em\u003e, and \u003cem\u003eNANOG\u003c/em\u003e in ARP-1 and RPMI8226 cells after incubation with osteocyte-derived exosomes, or in combination with heparin.\u003c/p\u003e\n\u003cp\u003eError bars are SD of biological replicates and p values: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2535332/v1/e95286ab2e833d4f068d7e7e.png"},{"id":32444617,"identity":"23bcc5ae-54b8-4f3b-815c-99efd6f1c1c6","added_by":"auto","created_at":"2023-02-03 16:02:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1594819,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Illustrated injection strategies of NSG mice in the myeloma mouse model. Myeloma cells with exosome incubation (MM+ OS exo) were implanted into the right flank of mice, and myeloma cells without exosome incubation (MM) were implanted into the left flank of mice as a control. (B) Representative bioluminescent images of mice 4 weeks after subcutaneous injection of RPMI8226 cells with or without incubation of osteocyte-derived exosomes. (C) Quantitative bioluminescent signals of mice 4 weeks after subcutaneous injection of RPMI8226 cells with or without incubation of osteocyte-derived exosomes. n = 4 mice/group. (D) Tumor volume of mice 4 weeks after subcutaneous injection of RPMI8226 cells with or without incubation of osteocyte-derived exosomes. (E) Representative micrographs of immunohistochemical staining for SOX2, OCT4 and NANOG in the indicated tumor tissues. Scale bars, 20 μm. (F) H-scores for SOX2, OCT4, and NANOG in the indicated tumor tissues.\u003c/p\u003e\n\u003cp\u003eError bars are SD of biological replicates and p values: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2535332/v1/b8bc648f4842dc9f3a0279bb.png"},{"id":32443246,"identity":"279614f8-ec72-40d7-956f-b424f8bbeed8","added_by":"auto","created_at":"2023-02-03 15:54:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":423347,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Upregulated miRNAs in the serum of bortezomib-resistant patients compared with that in patients who responded to bortezomib treatment based on a public database (GSE71435) (B) Levels of indicated miRNAs in osteocyte-derived exosomes. (C) Levels of indicated miRNAs in exosomes isolated from BMMSCs, osteoblasts (OB), osteocytes (OS) and myeloma cells. (D) Annexin V‐binding assay shows the percentages of apoptotic ARP-1 cells treated with 5 nM bortezomib (BTZ) or 25 nM carfilzomib (CFZ) in the presence or absence of miR-483-3p or miR-513a-5p. (E) Annexin V‐binding assay shows the percentages of apoptotic ARP-1 cells treated with 5 nM bortezomib (BTZ) or 25 nM carfilzomib (CFZ) in the presence or absence of osteocyte-derived exosomes, miR-483-3p inhibitor or miR-513a-5p inhibitor. (F) The side population cell percentages in ARP-1 cells in the presence or absence of miR-483-3p or miR-513a-5p. (G) The side population cell percentages in ARP-1 cells in the presence or absence of osteocyte-derived exosomes, miR-483-3p inhibitor or miR-513a-5p inhibitor.\u003c/p\u003e\n\u003cp\u003eError bars are SD of biological replicates and p values: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2535332/v1/4303ea7c19804190f0b5fe8c.png"},{"id":32444825,"identity":"f2df4e25-ccfb-4211-890d-367547f4b871","added_by":"auto","created_at":"2023-02-03 16:10:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1088627,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Western blot analysis for protein level changes of HIF-1α, HSC70, VHL MDM2, and PTEN in ARP-1 and RPMI8226 cells after incubation with osteocyte-derived exosomes. (B) Immunofluorescence analysis for protein level changes of HIF-1α in ARP-1 cells after incubation with osteocyte-derived exosomes. (C) Representative micrographs and H-scores of IHC staining for HIF-1α in the tumor tissues derived from myeloma cells or exosome-pretreated myeloma cells. Scale bar, 20 μm. (D) Overall survival of 542 myeloma patients with high HIF-1α (red) and low HIF-1α (blue) expression levels, calculated with the ‘‘R2: Tumor Myeloma - Hanamura - 542 - MAS5.0 - u133p2’’ dataset (GSE2658; http://r2.amc.nl). (E) qPCR analysis for mRNA level changes of \u003cem\u003eSOX2\u003c/em\u003e, \u003cem\u003eOCT4\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;NANOG\u003c/em\u003e in ARP-1 and RPMI8226 cells after incubation with osteocyte-derived exosomes or HIF-1α inhibitor. (F) qPCR analysis for mRNA level changes of \u003cem\u003eHIF1A\u003c/em\u003e in ARP-1 and RPMI8226 cells after incubation with osteocyte-derived exosomes. (G) Western blot analysis for protein level changes of SOX2, HIF-1α, and HSC70 in ARP-1 and RPMI8226 cells after addition of miR-483-3p or miR-513a-5p. (H) Western blot analysis for protein level changes of SOX2, HIF-1α, and HSC70 in ARP-1 and RPMI8226 cells after addition of osteocyte-derived exosomes, miR-483-3p inhibitor or miR-513a-5p inhibitor. (I) Depiction of exosomal miR-483-3p and miR-513a-5p–mediated drug resistance in myeloma. Osteocyte-derived exosomal miR-483-3p and miR-513a-5p promote cancer stem cell-like features of myeloma cells by downregulating the HSC70-mediated HIF-1α protein degradation, conferring myeloma cells resistance to chemotherapy.\u003c/p\u003e\n\u003cp\u003eError bars are SD of biological replicates and p values: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2535332/v1/79ec7bf38fcc70ad8795df1e.png"},{"id":32443247,"identity":"06df7a73-4e1c-4b4c-a56b-ab0c9d451f10","added_by":"auto","created_at":"2023-02-03 15:54:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1064119,"visible":true,"origin":"","legend":"\u003cp\u003e(A)\u003cstrong\u003e \u003c/strong\u003eSchematic workflow of drug treatment in the myeloma mouse model. (B) Representative bioluminescent images of mice at different time points in the indicated group. (C) Quantitative bioluminescent signals of mice in the indicated group. n = 4 or 5 mice/group. (D) Serum M-protein levels of mice at different time points in the indicated group. n = 4 or 5 mice/group. (E) qPCR analysis for mRNA levels of \u003cem\u003eSOX2\u003c/em\u003e, \u003cem\u003eOCT4\u003c/em\u003e, and \u003cem\u003eNANOG\u003c/em\u003e in tumor tissues from the indicated group. (F) qPCR analysis for mRNA level of \u003cem\u003eHSC70\u003c/em\u003e in tumor tissues from the indicated group.\u003c/p\u003e\n\u003cp\u003eError bars are SD of biological replicates and p values: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2535332/v1/9a1c2aeebcb2409a9b259fc2.png"},{"id":32444826,"identity":"0e79f0f4-b8a0-4325-aa4b-30661bd6547d","added_by":"auto","created_at":"2023-02-03 16:11:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2741135,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2535332/v1/255d15ea-5e9b-4abc-815f-67f1d53508bc.pdf"},{"id":32444615,"identity":"2db3a1c6-a860-4acc-8725-239344c0239d","added_by":"auto","created_at":"2023-02-03 16:02:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":44539,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-2535332/v1/be0f716b678c7958c9b36601.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Osteocyte-derived exosomes confer multiple myeloma resistance to chemotherapy through acquisition of cancer stem cell-like features","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMultiple myeloma, characterized by a clonal proliferation of malignant plasma cells within the bone marrow, is the second most common hematological malignancy in the United States and remains largely incurable [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. With conventional treatment, the median survival time for patients with myeloma is 3 to 4 years [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the past decades, several advanced therapeutic strategies, such as proteasome inhibitors, cell-cycle specific agents, monoclonal antibodies, and immunomodulatory drugs, have been used, which increase the survival time of myeloma patients to 5 to 7 years or longer [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, most patients are still prone to quickly developing relapsed or refractory disease after treatment [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Patients with relapsed or refractory myeloma generally have a shorter duration of response and poorer survival outcomes with each successive treatment regimen [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This clinical problem is thought to be caused by the bone marrow microenvironment, which provides a friendly \u0026ldquo;bed\u0026rdquo; for myeloma cell proliferation and resistance to treatment [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Thus, an in-depth understanding of how the bone marrow microenvironment supports myeloma growth and drug resistance is the key to improving the efficacy of therapeutic strategies and prognosis in patients with myeloma.\u003c/p\u003e \u003cp\u003eOsteocytes, which originate from osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs), are the most abundant cell type within bone tissue [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Osteocytes are linked to normal bone homeostasis and benign and malignant bone diseases through communication with other cells on the bone surface or within the bone marrow [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For example, osteocytes have been suggested to drive normal bone remodeling through the regulation of osteoblast and osteoclast differentiation and activity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. There is growing evidence that osteocytes are involved in myeloma growth, angiogenesis, homing to specific bone areas, and the pathogenesis of myeloma-associated bone disease [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, whether and how osteocytes affect myeloma chemoresistance remain unclear.\u003c/p\u003e \u003cp\u003eExosomes are nanovesicles with a diameter ranging from 30\u0026ndash;120 nm that originate from multivesicular bodies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These nanovesicles contain a wide range of miRNAs, mRNAs, and functional proteins and show unique cellular membrane markers, such as CD63 and HSP90 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Growing evidence has shown that exosomes can mediate the cellular communication between stroma and tumor, thus playing a crucial role in tumor development, progression and chemoresistance [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Like other stromal cell types, osteocytes can produce exosomes and release them into the microenvironment [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, evidence for the link between osteocyte-derived exosomes and myeloma drug resistance is limited. Here, we found that osteocyte-derived exosomes can be taken up by myeloma cells. Importantly, our data further show that osteocyte-derived exosomes exert a protective effect on myeloma cells against chemotherapy-induced apoptosis. Mechanistic studies indicate that exosomal miR-483-3p and miR-513a-5p promote cancer stem cell-like features in myeloma cells by regulating the degradation of hypoxia-inducible factor-1 α subunit (HIF-1α) via heat shock cognate 70-kDa protein (HSC70), thus conferring myeloma cells resistance to chemotherapy. Moreover, our mouse xenograft analysis suggests that combination treatment of miR-483-3p and miR-513a-5p inhibitor reduces tumor burden and potentiates the therapeutic efficacy of bortezomib \u003cem\u003ein vivo\u003c/em\u003e. Hence, our study identifies the functional impact of osteocytes on myeloma therapeutic resistance, implicating osteocyte-derived exosomal miRNAs as therapeutic targets for overcoming tumor drug resistance in multiple myeloma.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and primary cells\u003c/h2\u003e \u003cp\u003eThe myeloma cell line ARP-1 was kindly provided by the University of Arkansas for Medical Sciences. RPMI8226, MM.1S, and U266 cell lines were purchased from the American Type Culture Collection (VA, USA). Primary myeloma cells were isolated from bone marrow aspirates from newly diagnosed myeloma patients using anti-CD138 antibody-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Myeloma cells were maintained in RPMI 1640 medium with 10% fetal bovine serum.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e generation, isolation and characterization of human MSCs were performed as previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. MSCs were maintained in the MSC medium (ScienCell Research Laboratory, CA, USA). Mature osteoblasts and osteocytes were differentiated from human MSCs. The murine osteocyte cell lines MLO-Y4 and MLO-A5 were purchased from Kerafast, Inc. (MA, USA). MLO-A5 cells were differentiated to the mature type before used.\u003c/p\u003e \u003cp\u003eAll patient samples were obtained from the Biorepository Core of Houston Methodist Research Institute. Informed consent was obtained from study participants, and the collection of patient samples was conducted by Houston Methodist Research Institute in accordance with the criteria set by the Declaration of Helsinki. This study was approved by the Institutional Review Board of Houston Methodist Research Institute.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003emiRNA and reagents\u003c/h2\u003e \u003cp\u003emiR-483-3p mimic, miR-513a-5p mimic, anti-miR-483-3p inhibitor, anti\u0026ndash;miR-513a-5p inhibitor, and their scrambled controls were obtained from Integrated DNA Technologies (IA, USA). Lipofectamine 3000 used for miRNA mimic transfection and red fluorescent dye Dil were purchased from Thermo Fisher Scientific (MA, USA). Bortezomib, carfilzomib, and heparin were purchased from MedChemExpress (NJ, USA). Melphalan was purchased from Sigma-Aldrich (MA, USA). HIF-1α inhibitor CAY10585 was from Cayman Chemical (MI, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExosome isolation, characterization and uptake assay\u003c/h2\u003e \u003cp\u003eTo isolate osteocyte-secreted exosomes, mature osteocytes were cultured for 3 days in DMEM medium with 10% exosome-free FBS, and then culture supernatants were collected. Exosomes were isolated from filtered culture supernatants using total exosome isolation reagent (Thermo Fisher Scientific). Briefly, culture supernatants were mixed with the isolation reagent in a 1:2 ratio, and was vortexed to form a homogenized solution. After overnight incubation at 4\u0026deg;C, the mixture was centrifuged at 10,000 g for 1 h. The pellet containing exosomes was then resuspended in PBS. As for the characterization of isolated exosomes, it was performed as previously described [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The expression of exosomal marker proteins, such as HSP90 and CD63, were examined. To detect exosome uptake of myeloma cells, the red fluorescent dye Dil was used to label osteocyte-secreted exosomes. After incubation of myeloma cells with labeled osteocyte-secreted exosomes for 0, 4, 8, or 24 hours, confocal microscopy (Olympus, Tokyo, Japan) was used for imaging.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRT-qPCR\u003c/h2\u003e \u003cp\u003eTotal cellular RNA was extracted using the RNeasy kit (QIAGEN, Venlo, Netherlands) according to the manufacturer\u0026rsquo;s protocol. Exosomal RNA was isolated from exosomes using the total exosome RNA \u0026amp; Protein Isolation Kit. For quantitative real-time PCR, reverse transcription was conducted as per the protocol of the SuperScript II RT-PCR kit (Life Technologies, MA, USA), and quantitative PCR was performed using SYBR Green Master Mix (Life Technologies) with the QuantStudio 3 Real-Time PCR System (Life Technologies). GAPDH mRNA was used as input control. Primers for qPCR are listed in Supplementary Table S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eCells were harvested and lysed in lysis buffer (Cell Signaling Technology, MA, USA). To obtain exosomal proteins, exosomes were lysed with RIPA lysis and extraction buffer (Cell Signaling Technology) and isolated using the total exosome RNA \u0026amp; Protein Isolation Kit (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s protocol. The lysates were then subjected to SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies against CD63, HSP90, SOX2, OCT4, NANOG, HIF-1α, HSC70, VHL, MDM2, PTEN, and GAPDH. The level of GAPDH served as the protein loading control. The details of antibodies used in the study are listed in Supplementary Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eELISA and immunofluorescence assay\u003c/h2\u003e \u003cp\u003eSerum M-protein levels were measured by an ELISA kit (Bethyl Laboratories, TX, USA) according to the manufacturer\u0026rsquo;s protocol. The immunofluorescence assay was performed as previously described [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Briefly, cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 20 minutes at room temperature. After blocking with 2% goat serum for 1hour, the cells were stained with antibodies against SOX2, OCT4, and HIF-1α at 4\u0026deg;C overnight. Next, the cell nuclei were stained with DAPI and mounted with ProLong Gold antifade reagent (Molecular Probes, OR, USA). Immunofluorescent images were acquired with an IX81 confocal microscope system (Olympus).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eApoptotic assay\u003c/h2\u003e \u003cp\u003eAnnexin V binding assay was used to detect cell apoptosis by flow cytometry. 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were plated into 24-well plates, followed by treatment with bortezomib (5 nM), melphalan (25 \u0026micro;M), or carfilzomib (25 nM) for 24 hours. Cells were then collected and stained with APC Annexin V (Biolegend, CA, USA) and propidium iodide (Invitrogen, MA, USA). After a 20-minute incubation at room temperature, cells were measured by a BD FACS Symphony A3 flow cytometer (BD Biosciences, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSphere formation assay\u003c/h2\u003e \u003cp\u003eSphere formation assay was performed as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Briefly, cells were plated into an ultra-low attached 96-well plate at a density of 100 per well, followed by culture in serum-free cancer stem cell growth media consisting of DMEM/F12 medium (Hyclone, UT, USA), 1% penicillin-streptomycin, 2% B27 supplements, 10 ng/ml basic FGF, 20 ng/ml EGF, and 5 \u0026micro;g/ml insulin. After 4 days, an appropriate amount of fresh media was added. Cells were incubated for about 12 days, and sphere diameters were measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay\u003c/h2\u003e \u003cp\u003eColony formation assay was performed as previously described [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Briefly, cells were incubated with osteocyte-derived exosomes. After 24 hours, cells were collected and resuspended with 0.4% agarose in the serum-free RPMI medium. Then, cells were seeded into a 6-well plate pre-coated with 0.8% agar in the complete growth medium, followed by incubation at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. After a 3-week culture, colonies were photographed and counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSide population analysis\u003c/h2\u003e \u003cp\u003eCells were collected and resuspended in RPMI1640 supplemented with 2% FBS and 10 mM HEPES at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL. Subsequently, Hoechst 33342 dye was added to the cells at a concentration of 5 \u0026micro;g/mL. After incubation for 2 hours at 37\u0026deg;C with intermittent shaking, cells were collected and resuspended in pre-cold HBSS containing 2% FBS and 10 mM HEPES. Propidium iodide solution was added to the cells to a final concentration of 5 \u0026micro;g/mL. Side population cells were examined by a BD FACS Symphony A3 flow cytometer (BD Biosciences).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eImmunohistochemistry was performed as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In brief, formalin-fixed, paraffin-embedded sections of samples from the bone marrow or subcutaneous tissue of mice were deparaffinized and stained with anti-SOX2, anti-OCT4, anti-NANOG, and HIF-1α, antibodies using an EnVision System (DAKO, Glostrup, Denmark) according to the manufacturer\u0026rsquo;s protocol and counterstained with hematoxylin. In some experiments, stained slides were scanned with the Vectra 3 automated quantitative pathology imaging system and analyzed by inForm software (Perkin Elmer, MA, USA). Images were obtained using a microscope (Leica, Wetzlar, Germany).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vivo\u003c/span\u003e \u003cb\u003emouse experiments\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNOD-scid IL2Rgnull (NSG) mice were purchased from The Jackson Laboratory (ME, USA). All mice were maintained in American Association of Laboratory Animal Science-accredited facilities and all \u003cem\u003ein vivo\u003c/em\u003e mouse studies were approved by the Institutional Animal Care and Use Committees of Houston Methodist Research Institute. Six-week-old male NSG mice were subcutaneously injected with RPMI8226 (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) or RPMI8226 cells preincubated with osteocyte-derived exosomes (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells). For drug treatment, ARP-1 cells (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mouse) were intrafemorally injected into the NSG mice. After 2 weeks, mice were intraperitoneally injected with bortezomib (0.5 mg/kg) alone or in combination with anti-miR-483-3p or anti-miR-513a-5p inhibitor (5 mg/kg) three times a week for 2 weeks. Mice receiving an equal amount of vehicle served as controls. Tumor burden was evaluated by serum M-protein level and bioluminescence imaging. qPCR was used to measure the expression of \u003cem\u003eSOX2, NANOG, OCT4, HIF-1α, and HSC70\u003c/em\u003e in the bone marrow of randomly selected mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 9 (CA, USA) with two tailed unpaired Student t-tests for comparison of two groups and one-way ANOVA for comparison of more than two groups. Pearson\u0026rsquo;s correlation analysis was used to examine the correlation between two variables and Kaplan-Meier analysis was used in survival analysis. The level of significance was defined as *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eUptake of osteocyte-derived exosomes by myeloma cells\u003c/h2\u003e \u003cp\u003eTo study the potential role of osteocyte-derived exosomes in myeloma, we first examined whether osteocyte-derived exosomes can be transmitted into myeloma cells. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows the procedure for the isolation, Dil labeling, and myeloma cell uptake of osteocyte-derived exosomes. Osteocytes were generated by inducing the osteogenic differentiation of human-derived BMMSCs. The differentiated cells exhibited high expression of osteocyte-specific markers \u003cem\u003eDMP-1\u003c/em\u003e and \u003cem\u003eSOST\u003c/em\u003e and low expression of osteoblast-specific markers \u003cem\u003eRUNX2\u003c/em\u003e and \u003cem\u003eOSX\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), suggesting the generation of osteocytes. Next, exosomes were isolated from the conditioned medium of differentiated osteocytes and characterized as described in our previous study [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In line with previous results, osteocyte-derived exosomes presented high expression of the exosome-specific markers HSP90 and CD63 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To determine whether osteocyte-derived exosomes were transferred to myeloma cells, we incubated several human myeloma cell lines with fluorescent Dil-labeled exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Obvious red fluorescence signals were observed in ARP-1, RPMI 8226, and U266 cells at 24 hours after exposure to osteocyte-derived exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Supplementary Fig.\u0026nbsp;1A), while no fluorescence signals were observed in the control group, suggesting the internalization of Dil-labeled exosomes by myeloma cells. Notably, the fluorescence signals were increased in ARP-1 cells in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F). Moreover, we found that treatment of heparin, which can block the exosome uptake of tumor cells, significantly reduced the fluorescence signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Taken together, these findings indicate that osteocyte-derived exosomes can be taken up by myeloma cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOsteocyte-derived exosomes inhibit chemotherapy-induced apoptosis in myeloma cells\u003c/h2\u003e \u003cp\u003eMultiple studies have reported that stroma-derived exosomes play a crucial role in myeloma resistance to chemotherapy [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, we next sought to evaluate whether osteocyte-derived exosomes are involved in myeloma therapeutic response. We treated ARP-1 cells with three commonly used chemotherapeutic drugs including bortezomib, carfilzomib, and melphalan in the presence or absence of osteocyte-derived exosomes. It was observed that drug treatment markedly induced apoptosis in ARP-1 cells, while the addition of the exosomes reduced the efficacy of these drugs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Similar results were also observed in three other myeloma cell lines: RPMI8226, MM.1S, and U266 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). In addition to the exosomes derived from BMMSC-differentiated human osteocytes, the addition of exosomes derived from two murine osteocyte cell lines and conditioned medium from BMMSC-differentiated human osteocytes also exhibited anti-apoptotic effects in myeloma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Notably, consistent results were obtained in primary malignant plasma cells isolated from patients with newly diagnosed myeloma when treated with bortezomib (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results indicate that osteocyte-secreted exosomes can induce therapeutic resistance in myeloma.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOsteocyte-derived exosomes promote cancer stem cell-like features of myeloma cells\u003c/h2\u003e \u003cp\u003eNext, we investigated the mechanism by which osteocyte-derived exosomes protect myeloma cells against chemotherapy. It has been widely reported that cancer cell stemness contributes to chemoresistance [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Hence, we examined whether osteocyte-derived exosomes regulate cancer stem cell-like features of myeloma cells. CD19\u003csup\u003e+\u003c/sup\u003e/CD27\u003csup\u003e+\u003c/sup\u003e/CD138\u003csup\u003e\u0026minus;\u003c/sup\u003e cells have been identified as a myeloma stem cell population [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, it was observed that osteocyte-derived exosome treatment significantly increased the percentage of CD19\u003csup\u003e+\u003c/sup\u003e/CD27\u003csup\u003e+\u003c/sup\u003e/CD138\u003csup\u003e\u0026minus;\u003c/sup\u003e cells in ARP-1 and RPMI8226 cells. In addition, cancer side population cells represent a subpopulation of cancer cells with stem-like features and have been suggested to play a crucial role in drug resistance due to their high drug extrusion ability [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, we assessed the effect of osteocyte-derived exosomes on myeloma side population cells. Our data showed that incubation with osteocyte-derived exosomes increased the percentage of side population cells in RPMI8226 and MM.1S cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). It was also demonstrated that osteocyte-derived exosome incubation promoted the sphere formation ability of myeloma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Consistently, colony formation assay showed that osteocyte-derived exosome incubation increased the number and size of colonies compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). However, heparin treatment attenuated the increase in colony number and size (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we sought to determine whether cultures with osteocyte-derived exosomes affect stemness-associated gene expression in myeloma. As shown by RT-qPCR assay, osteocyte-derived exosome incubation significantly increased the mRNA expression of the stemness-associated genes \u003cem\u003eSOX2\u003c/em\u003e, \u003cem\u003eOCT4\u003c/em\u003e, and \u003cem\u003eNANOG\u003c/em\u003e in myeloma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). The expression of these genes was further confirmed at the protein level using western blot and immunofluorescence analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-L). In addition, it was demonstrated that heparin treatment abrogated the increased stemness-associated gene expression induced by incubation with osteocyte-derived exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). Hence, osteocyte-derived exosomes promote cancer stem cell-like features of myeloma cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOsteocyte-derived exosomes facilitate myeloma tumorigenesis\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e\u003c/p\u003e \u003cp\u003eWe next assessed the effect of osteocyte-derived exosomes on myeloma tumorigenesis \u003cem\u003ein vivo\u003c/em\u003e. We incubated RPMI8226 cells with osteocyte-derived exosomes \u003cem\u003ein vitro\u003c/em\u003e and then subcutaneously implanted the serial dilution of the cells into the right flank of NSG mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The cells without exosome incubation were implanted into the left flank of mice as a control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D, preincubation with osteocyte-derived exosomes facilitated myeloma tumorigenesis. Notably, in the group injected with 500 myeloma cells, 50% (2/4) mice did not develop visible tumors in the left flank, whereas all mice presented a visible tumor in the right flank (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). This finding indicated that preincubation with osteocyte-derived exosomes enhanced the tumor initiation ability of myeloma cells, which further supported the view that osteocyte-derived exosomes promote cancer stem cell-like features of myeloma cells. Indeed, immunohistochemical staining analysis showed that tumors derived from exosome-preincubated myeloma cells had a significantly higher expression of SOX2, OCT4, and NANOG compared with those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F). These \u003cem\u003ein vivo\u003c/em\u003e findings, along with our previous \u003cem\u003ein vitro\u003c/em\u003e studies, suggest that osteocyte-derived exosomes promote cancer stem cell-like features of myeloma cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eOsteocyte-derived exosomal miR-483-3p and miR-513a-5p promote myeloma cell chemoresistance and cancer stem cell-like features\u003c/h2\u003e \u003cp\u003eExosomes carry a wide range of miRNAs, mRNAs, and functional proteins [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Previous studies have identified exosomal miRNAs as important regulators in myeloma progression [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To explore whether and which kind(s) of miRNAs carried by osteocyte-derived exosomes regulate myeloma chemoresistance and cancer stem cell-like features, we first analyzed a published dataset of serum-derived exosomal miRNA expression profile changes in bortezomib-resistant and bortezomib-responsive myeloma patients (GSE71435). It was demonstrated that 9 miRNAs were significantly upregulated in the serum of bortezomib-resistant patients compared with that of bortezomib-responsive patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Among these 9 miRNAs, miR-483-3p, miR-513a-5p, and miR-3976 were found to be highly expressed in osteocyte-derived exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In addition, it was also observed that there was significantly higher expression of miR-483-3p and miR-513a-5p in osteocyte-derived exosomes compared with those in exosomes from BMMSCs, osteoblasts and myeloma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we examined the effect of miR-483-3p and miR-513a-5p in myeloma. Our data showed that overexpression of miR-483-3p or miR-513a-5p significantly attenuated the increased apoptosis of RPMI 8226 cells induced by bortezomib and carfilzomib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Conversely, inhibition of miR-483-3p or miR-513a-5p by the specific inhibitor blocked the protective effect of osteocyte-derived exosomes in RPMI8226 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In addition, overexpression of miR-483-3p or miR-513a-5p significantly increased the percentage of side population cells in myeloma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). However, inhibition of miR-483-3p and miR-513a-5p abrogated the increased side population cell percentage induced by osteocyte-derived exosome incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). These results indicate that osteocyte-derived exosomal miR-483-3p and miR-513a-5p promote myeloma cell chemoresistance and cancer stem cell-like features.\u003c/p\u003e \u003cp\u003e \u003cb\u003emiR-483-3p and miR-513a-5p promote cancer stem cell-like features of myeloma cells through regulation of HIF-1α stabilization\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe further investigated the molecular pathway by which osteocyte-derived exosomal miR-483-3p and miR-513a-5p regulate myeloma cell stemness. Previous studies have demonstrated that HIF-1α signaling plays a critical role in the interactions between bone marrow cells and myeloma cells, as well as myeloma cell stemness and drug resistance [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Thus, we sought to evaluate whether HIF-1α signaling is involved in the regulation of exosomal miR-483-3p and miR-513a-5p in myeloma cell stemness. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B, incubation with osteocyte-derived exosomes increased the protein expression of HIF-1α in myeloma cells. This finding was also supported by immunohistochemical analysis of HIF-1α protein in tumors generated in the osteocyte exosome-pretreated myeloma cell xenograft model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Survival analysis also revealed that high HIF-1α expression was significantly linked to the decreased overall survival of myeloma patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In addition, HIF-1α inhibitor treatment significantly attenuated the increased expression of stemness-associated genes induced by incubation with osteocyte-derived exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These findings indicate that osteocyte-derived exosomes promote cancer stem cell-like features of myeloma cells through HIF-1α signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, qPCR assay showed that osteocyte-derived exosome incubation did not affect the mRNA expression of \u003cem\u003eHIF1A\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), implying that osteocyte-derived exosomes might regulate HIF-1α protein expression by modulating its stabilization. Therefore, we evaluated whether incubation with osteocyte-derived exosomes affect the protein levels of the genes \u003cem\u003eHSC70\u003c/em\u003e, \u003cem\u003eVHL\u003c/em\u003e, \u003cem\u003eMDM2\u003c/em\u003e, and \u003cem\u003ePTEN\u003c/em\u003e, which are closely associated with HIF-1α stabilization [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. As shown by the Western blot analysis, osteocyte-derived exosome incubation reduced the protein expression of HSC70 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), which has been shown to participate in lysosomal degradation of HIF-1α through chaperone-mediated autophagic pathway [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Consistently, it was also observed that overexpression of miR-483-3p or miR-513a-5p significantly increased the expression of HIF-1α and Sox2, but reduced HSC70 expression in myeloma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Moreover, inhibition of miR-483-3p and miR-513a-5p reverted the protein expression of HIF-1α, SOX2, and HSC70 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Taken together, these findings suggest that osteocyte-derived exosomal miR-483-3p and miR-513a-5p promote cancer stem cell-like features of myeloma cells by downregulating HSC70-mediated HIF-1α protein degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCombination treatment of miR-483-3p and miR-513a-5p inhibitors potentiate the therapeutic efficacy of bortezomib in the myeloma mouse model\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHaving verified the role of osteocyte-derived exosomal miR-483-3p and miR-513a-5p in myeloma cancer cell stemness and chemoresistance \u003cem\u003ein vitro\u003c/em\u003e, we further investigated whether combination treatment of miR-483-3p and miR-513a-5p inhibitors can potentiate the efficacy of chemotherapy in a myeloma xenograft mouse model by intravenously injecting the luciferase-labeled myeloma cells into NSG mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). On the 14th day after myeloma cell injection, myeloma-bearing mice were randomized into 7 groups and given intraperitoneal injections of vehicle, bortezomib, miR-483-3p inhibitor, miR-513a-5p inhibitor single or in a combination. Tumor burden was monitored by bioluminescence imaging. After 2-week treatment, single miR-483-3p or miR-513a-5p inhibitor treatment significantly decreased tumor burden compared with the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). Furthermore, combination of bortezomib with miR-483-3p or miR-513a-5p inhibitor showed a greater reduction in tumor burden than the vehicle or single-drug group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). The triple combination of miR-483-3p inhibitor, miR-513a-5p inhibitor, and bortezomib resulted in the highest effect on tumor burden (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-C). These results were also confirmed by measuring serum levels of M-proteins, which are secreted from myeloma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Hence, combination treatment of miR-483-3p and miR-513a-5p inhibitors potentiates the anti-myeloma therapeutic efficacy of bortezomib \u003cem\u003ein vivo\u003c/em\u003e. In addition, it was also demonstrated that single miR-483-3p or miR-513a-5p inhibitor treatment significantly reduced the expression level of stemness-associated genes and increased \u003cem\u003eHSC70\u003c/em\u003e expression compared with the vehicle group, similar to \u003cem\u003ein vitro\u003c/em\u003e results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-G). These findings further support the view that osteocyte-derived exosomal miR-483-3p and miR-513a-5p promote cancer stem cell-like features of myeloma cells by downregulating HSC70-mediated HIF-1α protein degradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough advanced therapies have improved the prognosis of patients with myeloma, myeloma remains largely incurable [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In parallel with the development and clinical use of these advanced therapies is the emergence of novel mechanisms of drug resistance, many of which remain elusive. Herein, we demonstrate for the first time that osteocytes, the most abundant cell type within bone tissue, are new regulators of myeloma therapeutic resistance. We identified two osteocyte-derived miRNAs, miR-483-3p and miR-513a-5p, both of which promoted cancer stem cell-like features in myeloma cells by regulating HIF-1α stabilization, thus conferring myeloma cells resistance to chemotherapy. Moreover, combination treatment of miR-483-3p and miR-513a-5p inhibitors significantly reduced myeloma tumor burden and potentiated the therapeutic efficacy of bortezomib. Thus, our study provides insights into the molecular mechanisms underlying myeloma chemoresistance, implicating osteocyte-derived exosomal miRNAs as novel therapeutic targets for overcoming chemoresistance in myeloma.\u003c/p\u003e \u003cp\u003eDuring the past several decades, our understanding of the biological function of osteocytes has been changing. Originally, osteocytes were considered as an inactive placeholder [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Then, they were linked to the process of normal bone remodeling by regulating the differentiation and activity of osteoclasts and osteoblasts [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Currently, there is growing evidence that osteocytes play a crucial role in myeloma-associated bone disease, which is a major problem in myeloma patients [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. By producing osteolytic cytokines, osteocytes can enhance myeloma-induced bone lesions. Using genetically engineered mice, Delgado-Calle J et al. reported that osteocyte-secreted sclerostin contributes to myeloma-induced bone loss [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. A preclinical study by McDonald MM et al. showed that pharmaceutical inhibition of sclerostin reduced lytic lesions and enhanced new bone formation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], suggesting a potential application of the inhibitor against osteocyte-secreted sclerostin to treat myeloma patients with lytic lesions. Previous studies also found that osteocyte production of osteolytic cytokines can be enhanced by myeloma cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These results indicate the important role of osteocytes in the development and pathogenesis of myeloma-associated bone disease. However, the biological function of osteocytes in myeloma growth and survival is still vague. In this study, we explored the role of osteocytes in myeloma cell response to chemotherapy, another major problem in most myeloma patients, and found the protective effect of osteocytes on myeloma cells against all tested chemotherapeutic drugs. Our findings unveil a novel unexplored role of osteocytes in myeloma pathogenesis.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, this is the first study to investigate the role of osteocyte-derived exosomes in myeloma therapeutic response. Exosomes are nanovesicles and have been shown to be a common mediator for the cellular communication between tumor cells and microenvironmental stromal cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Though buried within lacunae, osteocytes can secrete exosomes, which circulate in the blood [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Accumulating evidence shows that osteocyte-secreted exosomes are involved in bone homeostasis and benign diseases [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], but few studies have investigated their role in tumors, including myeloma. Here, we observed reduced chemotherapy-induced myeloma cell apoptosis once myeloma cells take up osteocyte-derived exosomes, indicating that osteocyte-derived exosomes are required for chemoresistance, at least partly. We also examined the effect of osteocyte-derived exosomes on myeloma cancer cell stemness, a common mechanism for inducing therapeutic resistance. Previous studies have shown that the population with CD19\u003csup\u003e+\u003c/sup\u003e/CD27\u003csup\u003e+\u003c/sup\u003e/CD138\u003csup\u003e\u0026minus;\u003c/sup\u003e is the myeloma stem cell population [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We measured the percentage change of this population in myeloma cells after incubation with osteocyte-derived exosomes and found a significantly increased percentage. In addition, we also used several well-established approaches, including \u003cem\u003ein vitro\u003c/em\u003e sphere formation, colony formation, side population, stemness-associated gene expression, and \u003cem\u003ein vivo\u003c/em\u003e tumorigenesis, to confirm our finding that osteocyte-secreted exosomes enhance myeloma cancer cell stemness.\u003c/p\u003e \u003cp\u003eWe identified two osteocyte-derived exosomal miRNAs that are responsible for myeloma drug resistance using a dataset containing thousands of circulating exosomal miRNAs in a cohort of myeloma patients with bortezomib-based therapy. miRNAs, composed of 18 to 25 nucleotides, can be transferred from stromal cells to tumor cells through exosomes and then transmit a signal for supporting tumor development [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In this study, we observed that miR-483-3p and miR-513a-5p are enriched in osteocyte-derived exosomes, while their levels are much lower in the exosomes from the precursor MSCs, mature osteoblasts, and myeloma cells. Previous studies show that the level of miR-483-3p is upregulated during MSC differentiation to mature bone cells [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. These findings indicate that osteocytes express unique miRNAs. We further found that these two miRNAs have an inhibitory effect on the expression of HSC70, which contributes to lysosomal degradation of HIF-1α through the chaperone-mediated autophagic pathway [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], thus enhancing HIF-1α stabilization, cancer cell stemness, and chemoresistance in myeloma cells. Our study originally links osteocytes to myeloma therapeutic resistance by exosomal miRNA-induced myeloma cancer cell stemness. Our next goal is to identify other osteocyte-derived unique factors, which may be used as biomarkers to evaluate myeloma therapeutic efficacy and even used as therapeutic targets.\u003c/p\u003e \u003cp\u003eIn summary, we have systematically elucidated the role of osteocyte-derived exosomal miRNAs in myeloma chemoresistance herein. Considering the paucity of knowledge of the specific determinants conferring myeloma cells resistance to chemotherapy, our study provides mechanistic insights into myeloma chemoresistance, and suggests that targeting osteocyte-derived exosomal miRNAs may overcome chemoresistance in multiple myeloma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests statement:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was\u0026nbsp;supported by the National Institutes of Health/National Cancer Institute (R01 awards CA190863 and CA193362), the American Cancer Society (Research Scholar Grant 127337-RSG-15-069-01-TBG), and the Cancer Prevention \u0026amp; Research Institute of Texas (RP220639).\u0026nbsp;We thank the Myeloma Tissue Bank at Houston Methodist Research Institute. Supports also came from Research Pathology Core, Flow Cytometry Core, and Translational Imaging Core at Houston Methodist Research Institute. We would like to thank\u0026nbsp;Dr. Shaefali P. Rodgers,\u0026nbsp;Houston Methodist Hospital, who edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.Y. and F.C. designed the study and wrote the manuscript;\u0026nbsp;F.C., Z.W., and G.Y. performed the experiments; F.C., Z.W., Y.L., and J.H. analyzed the data and conducted statistical analysis. All authors reviewed the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that all data supporting the findings of this study are available within the article or its Supplementary Materials and from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVan de Donk NWCJ, Pawlyn C, Yong KL. Multiple myeloma. Lancet. 2021;397(10272):410-427. https://doi.org/10.1016/s0140-6736(21)00135-5.\u003c/li\u003e\n\u003cli\u003eLiu H, He J, Koh SP, Zhong Y, Liu Z, Wang Z, et al. Reprogrammed marrow adipocytes contribute to myeloma-induced bone disease. Sci Transl Med. 2019;11(494):eaau9087. https://doi.org/10.1126/scitranslmed.aau9087.\u003c/li\u003e\n\u003cli\u003eLi Z, Liu H, He J, Wang Z, Yin Z, You G, et al. Acetyl-CoA Synthetase 2: A Critical Linkage in Obesity-Induced Tumorigenesis in Myeloma. 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Int J Mol Med. 2020;46(4):1571-1581. https://doi.org/10.3892/ijmm.2020.4694.\u003c/li\u003e\n\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2535332/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2535332/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTherapeutic resistance is a major challenge in multiple myeloma treatment. Understanding the underlying mechanisms is required to develop effective strategies against drug resistance and improve the prognosis of myeloma patients. Here, we identify osteocytes, the major cellular component of bone tissue, as key regulators of myeloma therapeutic resistance. Osteocyte-derived exosomes can be efficiently taken up by myeloma cells and exert a protective effect against chemotherapy-induced apoptosis. Mechanistic studies further reveal that osteocyte-derived exosomal miR-483-3p and miR-513a-5p promote cancer stem cell-like features in myeloma cells by regulating HIF-1α stabilization, thus conferring myeloma cells resistance to chemotherapy. Strikingly, combination treatment of miR-483-3p and miR-513a-5p inhibitors significantly reduces tumor burden and potentiates the therapeutic efficacy of bortezomib in the myeloma mouse model. Our findings, therefore, demonstrate the functional impact of osteocytes on myeloma therapeutic resistance, and suggest that osteocyte-derived exosomal miRNAs may serve as potential therapeutic targets for overcoming drug resistance in multiple myeloma.\u003c/p\u003e","manuscriptTitle":"Osteocyte-derived exosomes confer multiple myeloma resistance to chemotherapy through acquisition of cancer stem cell-like features","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-03 15:54:50","doi":"10.21203/rs.3.rs-2535332/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2023-02-16T12:10:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-02-13T16:43:06+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-02-03T07:44:58+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-02-02T06:33:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-02-01T11:44:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-02-01T11:44:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Leukemia","date":"2023-01-31T19:23:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"37d3fa76-5f75-4b61-a451-3d0850933b57","owner":[],"postedDate":"February 3rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2023-03-27T09:22:15+00:00","versionOfRecord":[],"versionCreatedAt":"2023-02-03 15:54:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2535332","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2535332","identity":"rs-2535332","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}