Deregulation of Stemness and Senescence Genes in Bone Marrow Mesenchymal Stem Cells of Multiple Myeloma: Implications for Therapeutic Approaches

Deregulation of Stemness and Senescence Genes in Bone Marrow Mesenchymal Stem Cells of Multiple Myeloma: Implications for Therapeutic Approaches | 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 Research Article Deregulation of Stemness and Senescence Genes in Bone Marrow Mesenchymal Stem Cells of Multiple Myeloma: Implications for Therapeutic Approaches Fatemeh Soleymani, Saeideh Kavousi, Nastaran Khodakarim, Mohammad Ahmadvand This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7802296/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Purpose: Multiple myeloma (MM) is a hematologic malignancy with a poor prognosis. MM-derived MSCs (MM-MSCs) contribute to disease progression by creating a supportive stromal microenvironment for malignant cells. Therefore, elucidating transcriptomic alterations in MSCs may facilitate the development of novel therapeutic strategies for treatment resistant MM patients. Methods: Total RNA was extracted from cultured MSCs isolated from bone marrow aspirates of MM patients and normal donors (ND-MSCs). Gene expression of stemness markers ( NANOG , OCT4 ) and senescence-related genes ( P16 , P21 , IL-6 , IL-8 ) were analyzed using Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), while senescence status was assessed using SA-β-gal staining. Results: Compared to ND-MSCs, MM-MSCs demonstrated a higher percentage of SA-β-gal-positive cells. Gene expression analysis revealed upregulation of P21 and IL-6 in MM-MSCs, whereas NANOG and OCT4 were significantly downregulated. Notably, this downregulation was corroborated by analyzing a publicly available RNA-seq dataset, reinforcing our findings Conclusions: While confirming previous reports on increased senescence, our study provides novel evidence for the significant downregulation of stemness-related genes ( NANOG , OCT4 ) in MSCs from newly diagnosed, untreated MM patients. This concurrent dysregulation of stemness and increased senescent phenotype may be a key contributor to the pathogenicity of the tumor microenvironment, highlighting a potential axis for therapeutic intervention. Multiple Myeloma Tumor Microenvironment Bone Marrow Mesenchymal Stem Cells Stemness Senescence-Related Genes Figures Figure 1 Figure 2 Figure 3 Introduction Multiple myeloma (MM) is a prevalent hematologic cancer, with an estimated 36,110 new cases (representing 1.8% of all new cancer cases) and 12,030 associated deaths (accounting for 1.9% of all cancer deaths) in the United States for the year 2025[ 1 ]. Despite therapeutic advances, MM remains largely incurable with a poor prognosis. Multiple myeloma is defined by the clonal expansion of abnormal plasma cells (PCs) in the bone marrow [ 2 ]. Numerous studies have explored the role of the bone marrow microenvironment and its intricate interactions with PCs [ 3 ]. The bone marrow microenvironment includes both cellular and non-cellular components .The cellular compartments include hematopoietic and stromal cells, whereas the non-cellular compartments consist of cytokines, growth factors, chemokines, and extracellular matrix [ 4 ]. Among these, mesenchymal stem cells (MSCs) are essential elements of the bone marrow, playing critical roles in its physiological and pathological states. Several studies indicate that MM patients have abnormal MSCs with multiple reproducible differentially expressed genes (DEGs) compared to normal mesenchymal stem cells [ 5 , 6 ]. In particular, these cells can overproduce growth factors such as IL-6, which stimulates the growth of MM cells [ 7 ]. Comparative gene expression analyses of MM-MSCs and MSCs from normal donors (ND-MSCs) have revealed downregulation and disruption of genes involved in cell cycle progression, providing further evidence of dysregulated gene expression in MM-MSCs [ 5 ]. MM-MSCs are believed to contribute to disease progression by supporting MM cells, promoting treatment resistance, and establishing a favorable stromal microenvironment [ 8 ]. Compared to bone marrow-derived ND-MSCs, MM-MSCs exhibit pronounced cellular signs of senescence[ 9 , 10 ], which have been significantly associated with MM development triggered by myeloma cell stimulation through LPA receptor 1 (LPA1) and 3 (LPA3) [ 8 ]. This is important, as previous studies have shown that the secretory phenotype and metabolic activity of senescent MSCs exhibit pro-tumorigenic effects, promoting tumor growth and facilitating processes that drive MM progression [ 11 ]. Senescence is characterized by the arrest of the cell cycle in the G0-G1 phase, primarily mediated by cyclin-dependent kinase inhibitors such as P21 and P16. This state is commonly associated with increased β-galactosidase activity, overexpression of cytokines, and abnormal cellular morphology [ 12 ]. While the precise mechanisms driving MSC senescence in MM remain unclear, understanding these pathways is vital, as it may provide novel therapeutic targets for improving treatment outcomes in MM patients. As multipotent adult stem cells, MSCs are defined by their dual capacity for self-renewal and multilineage differentiation into various tissue types[ 13 ]. However, evidence supports the notion that these essential properties, differentiation and proliferation, may be disrupted in MM patients. Key transcription factors such as OCT4 and NANOG regulate these functions in MSCs [ 14 ]. Studies have shown that MSCs from MM patients exhibit a diminished differentiation capacity [ 15 , 16 ], and significantly reduced proliferative potential. Additionally, MM-MSCs display severely impaired osteoblastic differentiation compared to ND-MSCs [ 6 ]. The high prevalence of senescent cells in MM-MSCs likely contributes to these functional impairments, as senescence is well known to disrupt normal cellular function. Interestingly, previous studies provide preliminary evidence that targeting mesenchymal stem cell (MSC) senescence may shift MM-MSCs from promoting tumor growth to inhibiting it [ 8 ]. In other words, modifying the altered cellular characteristics of MM-MSCs could offer a promising strategy to improve treatment outcomes. Although previous studies have highlighted an increased senescent phenotype in MM-MSCs, the concurrent status of core stemness pathways in these cells remains poorly understood. Based on this knowledge gap, we hypothesized that the MSCs of newly diagnosed MM patients are characterized by a dual dysregulation involving both increased senescence and a loss of stemness. To address this hypothesis, we compared the expression of key senescence ( P16 , P21 , IL-6 , IL-8 ) and stemness ( NANOG , OCT4 ) genes between MM-MSCs and ND-MSCs. A deeper understanding of this potential dysregulation is crucial for identifying pathological mechanisms and could ultimately inform targeted strategies to disrupt these interactions, slow disease progression, and improve treatment outcomes. Materials and Methods Patients This study included patients referred to Rasoul Hospital, Iran University of Medical Sciences, Tehran, Iran. Biopsies were performed on an initial cohort of 10 patients for the diagnosis of multiple myeloma (MM). The final study cohort consisted of six patients (three males and three females; mean age 61.7) who met the inclusion and exclusion criteria. Additionally, three healthy bone marrow biopsies (two males and one female; mean age 55.3) were collected for comparison. These samples were used to examine the molecular characteristics of MSCs from MM patients and healthy donors. Inclusion and Exclusion Criteria Patients with a confirmed diagnosis of multiple myeloma (MM) at intermediate to advanced disease stages (Stage II/III) were enrolled in this study. The patients were aged 48–77 years. Exclusion criteria encompassed the presence of systemic disorders, such as inflammation, genetic disorders, and multiple sclerosis (MS), prior treatment for MM, a history of other malignancies, or treatment for other systemic conditions, including hypothyroidism or diabetes mellitus (DM). Additionally, healthy individuals without a history of systemic diseases, inflammation, or ongoing systemic treatment were selected as healthy bone marrow donors for transplantation. MSCs Isolation and Culture A bone marrow biopsy was performed following the standard clinical procedure. Approximately 3–5 mL of bone marrow aspirate was obtained from the posterior iliac crest. MSCs were isolated using the plastic adhesion method and cultured in a low-glucose medium ( bioIDEA, Tehran, Iran) supplemented with 10% fetal bovine serum (bioIDEA) and 1% penicillin/streptomycin (bioIDEA) until they reached 80% confluency at 37°C in a 5% CO2 atmosphere. The cells were then detached using 0.25% trypsin/EDTA (bioIDEA), centrifuged at 1200 rpm for 5 minutes, and subsequently passaged. This culturing procedure was applied to both MM-MSCs and ND-MSCs in the study. All isolated MSC populations successfully reached passage 4 for subsequent analyses. MSCs Validation To confirm the validation of MSCs, several well-established criteria must be met, including their spindle-shaped morphology, adherence to plastic surfaces, differentiation potential into at least two cell lineages, and the expression or absence of specific clusters of differentiation (CD) markers. MSCs were cultured in differentiation media (bioIDEA) for osteogenic and adipogenic differentiation, with differentiation confirmed according to the bioIDEA protocol. Briefly, MSCs were seeded in 12-well plates and cultured in osteogenic and adipogenic differentiation media. The media were replaced every three days. After three weeks, the cells were fixed with 4% formaldehyde and stained with 4% Alizarin Red for osteogenic differentiation. For adipogenic evaluation, cells were fixed with 10% formalin and stained with 1% Oil Red (bioIDEA). In addition to their differentiation potential, the harvested cells were evaluated by flow cytometry to confirm the presence of specific CD markers, including CD105, and CD90, and the absence of CD45 and CD34 and HLA-DR, to confirm their identity as MSCs. Briefly, cells were detached using 0.25% trypsin (bioIDEA), and the cell pellet was resuspended in 5% FBS. The cells were then incubated with monoclonal antibodies in the dark for 30 minutes. The presence of positive and negative CD markers was evaluated using a FACSCalibur™ flow cytometer (Becton Dickinson, USA). FlowJo 10 software was employed to analyze the data and validate the MSC nature of the cells. RNA Extraction and cDNA Synthesis Total RNA was extracted from MSCs (10^6 cells) using TRIzol reagent (Sigma, Germany) according to the manufacturer's instructions. The extracted RNA was verified for purity and concentration using a NanoDrop (IMPLEN GmbH, Munich, Germany), which Confirmed absorbance ratios of approximately 2.0 at 260/280 nm and ≥ 1.8 at 260/230 nm. For cDNA synthesis, 10 µl of the extracted RNA was used for first-strand cDNA synthesis with the PrimeScript cDNA Synthesis Kit (Takara Bio, Japan), following the manufacturer's instructions. Quantitative RT-PCR for Gene Expression Level Evaluations The gene expression levels of the targeted genes were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) on a Step One, ABI system. Each reaction was performed using a high ROX RealQ Plus 2x Master Mix (Amplicon, Denmark), as specified by the manufacturer. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) was used as the internal control gene for normalization. All primers were synthesized by Sinaclon Co. (Tehran, Iran). The sequences of all primers are provided in Online Resource 1. Statistical Analysis The Mann-Whitney U test, a nonparametric statistical method, was applied to compare the expression levels of the studied genes between MM-MSCs and ND-MSCs. Each sample was analyzed in triplicate and the average CT value was used for quantification. Gene expression was quantified using the comparative ΔΔCT method. Briefly, the CT value of each target gene was normalized to the endogenous control, GAPDH , to generate a ΔCT value (ΔCT = CT target gene − CT GAPDH ​). The average ΔCT from the Normal Donor (ND-MSC) group served as the reference. Final statistical comparisons were performed on the ΔCT values between the MM-MSC and ND-MSC groups. Data analysis was performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). A two-tailed p-value of < 0.05 was considered statistically significant. Public Dataset Analysis Publicly available RNA-seq data from the Gene Expression Omnibus (GEO) dataset GSE196297 [ 17 ] were analyzed to validate the stemness gene expression trends observed in our study. This dataset includes bulk RNA-seq counts from bone marrow-derived mesenchymal stem cells (BM-MSCs) obtained from multiple myeloma (MM) patients and control donors. In the original study, RNA-seq data were processed using DESeq2 for normalization and differential expression analysis. Gene Set Enrichment Analysis (GSEA) was conducted based on MSigDB hallmark gene sets. Our analysis utilized the DESeq2-normalized count data from the study. To maintain consistency with our patient group, only MM patients at stages II/III were included and compared with control donors. Log2-transformed counts for POU5F1 ( OCT4 ) and NANOG were compared between MM patients (n = 4) and control donors (n = 5) using the Mann-Whitney U test. SA-β-gal Staining (X-GAL) SA-β-gal staining was employed to assess the percentage of senescent cells in MM-MSCs and ND-MSCs. The cells were seeded in 12-well plates to achieve approximately 80% confluence. The staining process followed the protocol established by Debacq-Chainiaux et al. [ 18 ]. Briefly, the cells were washed with PBS, fixed with a freshly prepared solution containing 2% formaldehyde (vol/vol) and 0.2% glutaraldehyde (vol/vol) in PBS, and then washed twice with PBS. The cells were incubated overnight at 37°C without CO2 in a fresh staining solution. The staining solution consisted of citric acid/sodium phosphate buffer (40 mM, pH 6), potassium ferricyanide (5 mM), potassium ferrocyanide (5 mM), magnesium chloride (2 mM), sodium chloride (150 mM), and 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-gal, 1 mg/mL) in distilled water. After 16 hours of incubation, the cells were washed three times with PBS and once with methanol before being air-dried. To determine the percentage of senescent cells, a total of at least 500 cells were counted across 5 to 10 random, non-overlapping fields per sample. The percentage was calculated as the ratio of SA-β-gal-positive (blue) cells to the total number of cells counted.. Results Identification and Differentiation of MSCs MSCs isolated from bone marrow exhibited a spindle-shaped morphology (Fig. 1a). Flow cytometry analysis confirmed the expression of CD105 and CD90 and the absence of CD45. Specifically, ND-MSCs were negative for CD34 (Fig. 1d), and MM-MSCs were negative for HLA-DR (Fig. 1e). Additionally, the MSCs demonstrated multipotent differentiation capacity. Osteogenic differentiation was confirmed by detecting mineralized deposits in the extracellular matrix after Alizarin Red staining (Fig. 1b). Adipogenic differentiation was confirmed by the presence of lipid droplet formation after Oil Red O staining (Fig. 1c). Senescence-related Genes Demonstrating Abnormal Expression Patterns in MM-MSCs IL-6 , a pro-inflammatory cytokine gene, was significantly overexpressed in MM-MSCs compared to ND-MSCs (p ≤ 0.05). Additionally, the cell cycle-regulating gene P21 was upregulated in MM-MSCs (p ≤ 0.05), indicating a potential link between senescence and the MM-MSC phenotype. While IL-6 and P21 exhibited distinct expression patterns between MM-MSCs and ND-MSCs, no statistically significant differences were observed in the expression levels of P16 and IL-8 between the two groups (Fig. 2a). Genes Involved in Stemness are Suppressed in MM-MSCs Dysregulation of stemness genes was observed in MM-MSCs relative to ND-MSCs, with NANOG and OCT4 transcripts ( OCT4A , OCT4B , OCT4B1 ) being significantly differentially expressed in MM patient-derived MSCs. All transcripts of OCT4A (p < 0.05), OCT4B (p < 0.05), OCT4B1 (p < 0.05), and NANOG (p < 0.05) exhibited downregulation in MM-MSCs (Fig. 2b). Concordance of OCT4 and NANOG Expression in RNA-seq Data RNA sequencing data showed concordance for OCT4 and NANOG expression. Log2-transformed, normalized counts revealed significant downregulation of POU5F1 ( OCT4 ) in MM patients (p = 0.0317) and marginally significant downregulation of NANOG (p = 0.0635) compared to healthy controls. These findings align with our in-house observations of suppressed stemness gene expression in MM-MSCs (Fig. 2c). MM-MSCs Demonstrating a Higher Percentage of the SA-β-Gal-Positive Cells MM-MSCs and ND-MSCs were stained with SA-β-gal, and the results revealed a significant difference between the two groups. As shown in Fig. 3 b-c, MM-MSCs exhibited a statistically significant (P ≤ 0.0169) increase in the percentage of SA-β-gal-positive cells (Fig. 3a). Discussion This study provides the first direct evidence that MSCs from newly diagnosed, untreated multiple myeloma patients exhibit a dual dysregulation characterized by both accelerated senescence and a significant loss of stemness. This finding supports the growing hypothesis that a concurrent disruption of these pathways contributes to MM pathogenesis. The reciprocal interaction between plasma cells and MM-MSCs has emerged as a key focus, with several studies suggesting that PCs play a crucial role in inducing senescence in surrounding MSCs. Interestingly, senescent MSCs, in turn, appear to contribute to treatment resistance in multiple myeloma. Understanding the underlying mechanisms of this interaction could provide valuable insights for developing novel therapeutic approaches, including precision medicine strategies tailored to target senescence-driven treatment resistance in multiple myeloma patients. Our findings substantiate this hypothesis of dual dysregulation. We observed a significant upregulation of the pro-senescent factors IL-6 and P21 in MM-MSCs, which are hallmarks of cellular aging and cell cycle arrest. Concurrently, we detected a significant downregulation of the core stemness transcription factors OCT4 and NANOG . This concurrent phenotype suggests a profound functional shift in MM-MSCs, where the cells not only enter a senescent state but also lose their fundamental self-renewal capacity. Delving into the senescence aspect, our study found that IL-6 , a key growth factor and a marker of senescence, exhibited a significant increase in MM-MSCs, consistent with previous findings [ 7 , 9 ]. IL-6 plays a pivotal role in sustaining myeloma cell survival and proliferation by activating critical signaling pathways such as JAK/STAT, PI3K/AKT, and MAPK. Previous research has demonstrated that IL-6 overexpression in senescent cells not only supports but also perpetuates the proliferation of neighboring malignant cells [ 19 ]. These findings further support the notion that MM-MSCs exist in a senescent state, aligning with prior studies that underscore their role in promoting tumorigenesis, conferring drug resistance, and sustaining the survival of malignant plasma cells. P16 and P21 are commonly used senescence biomarkers, as their overexpression results in cell cycle arrest. Our findings of elevated P21 but unchanged P16 levels align with some previous reports [ 9 ]. This suggests that the role of P16 in senescence may be context-dependent, as P16 regulates specific senescent states, while P53 governs others based on the cellular response to different stressors [ 20 ]. Additionally, endogenous P16 has been shown to inhibit IL-6 expression [ 21 ], and IL-6-dependent inhibition of G0-G1 progression is mediated through the P21 pathway [ 22 ]. This creates a coherent molecular picture that aligns with our observation regarding the expression levels of P21 and IL-6 . Although IL-8 is recognized as a senescence-associated cytokine [ 23 ], our study only detected a trend towards upregulation that did not achieve statistical significance, likely attributable to the small sample size. The transcription factors OCT4 and NANOG are critical for the survival and self-renewal of MSCs [ 24 ]. OCT4 establishes a regulatory network that inhibits differentiation-associated genes, thereby preserving pluripotency [ 25 ]. Similarly, NANOG is crucial for maintaining self-renewal and facilitating pluripotent or multipotent differentiation while also suppressing spontaneous senescence [ 26 ]. Consistent with these findings, the current study reveals a significant downregulation of OCT4 and NANOG expression in MM-MSCs compared to ND-MSCs. Although these genes have been identified as key components of the regulatory network that represses differentiation and maintains MSC pluripotency [ 27 ], their reduced expression in MM-MSCs is, to our knowledge, a novel finding. Interestingly, when using publicly available RNA sequencing data from the GEO dataset [ 17 ], the findings were concordant with our RT-qPCR results. This concordance further supports the downregulation of critical stemness genes in MM-MSCs. However, a recent study reported no significant differences in the expression of stemness-related genes between HD-MSCs and MM-MSCs [ 6 ]. Notably, Lu et al. investigated primarily treated multiple myeloma patients, whereas our study focused on newly diagnosed, untreated MM patients at intermediate to advanced disease stages. This fundamental difference in patient characterization may account for the gene expression discrepancy and suggests that disease stage and treatment status are critical determinants of MSC gene expression profiles in MM. It further emphasizes the potential significance of OCT4 and NANOG dysregulation in the pathogenesis of newly diagnosed, untreated multiple myeloma. Crucially, the observed loss of stemness and increased senescence in MM-MSCs are not isolated events but likely represent two faces of the same coin, forming the basis for our "dual dysregulation" hypothesis. Our study strongly suggests this link by demonstrating a concurrent downregulation of OCT4 and NANOG , alongside an upregulation of the cell cycle inhibitor P21. This finding is well supported by established mechanistic connections; for instance, NANOG has been shown to counteract senescence [ 28 ] and delay its onset through specific signaling pathway [ 29 ]. Similarly, OCT4 maintains stem cell self-renewal and reverses senescence by inhibiting P21 expression [ 30 ]. Therefore, our observation of OCT4 downregulation and P21 upregulation corroborate previous reports of a negative regulatory axis between these two factors. These internally consistent findings reinforce the notion that targeting dysregulated transcription factors could present a novel therapeutic approach to overcoming treatment resistance in MM patients. Conclusion This study provides further evidence for the involvement of senescence-related genes in MM-MSCs and confirms that these cells exhibit a more pronounced senescent phenotype compared to ND-MSCs. Furthermore, we present, for the first time, distinct expression patterns of stemness-related genes in MM-MSCs in comparison to ND-MSCs. This dysregulation may contribute to the senescent state of MM-MSCs and potentially underlie the treatment resistance observed in multiple myeloma patients. These findings highlight the potential for novel therapeutic strategies aimed at targeting senescence in MM-MSCs. Specifically, regulating stemness transcription factors may offer a promising avenue for improving treatment outcomes in multiple myeloma. Our study has limitations, primarily a small sample size requiring confirmation in larger cohorts. Additionally, our findings are based on gene expression; future studies should incorporate functional assays to directly assess the consequences of stemness loss and increased senescence. Exploring the signaling pathways driving this phenotype could reveal novel therapeutic targets. Abbreviations CD markers: Clusters of Differentiation Markers DEGs: Differentially Expressed Genes DM: Diabetes Mellitus GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase GEO: Gene Expression Omnibus GSEA: Gene Set Enrichment Analysis MM: Multiple Myeloma MS: Multiple Sclerosis MSCs: Mesenchymal Stem Cells ND : Normal Donors PCs: Plasma Cells RT-qPCR: Reverse Transcription-Quantitative Polymerase Chain Reaction Declarations Author Contributions : M.A. and F.S. designed the study. N.K. was responsible for patient diagnosis, recruitment, and clinical sample acquisition. F.S. and S.K. performed the experiments, data collection, and formal analysis. F.S. wrote the first draft of the manuscript. M.A., and S.K. reviewed and edited the manuscript. All authors read and approved the final manuscript. Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Ethics Approval : This study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board and the Ethics Committee of the Tarbiat Modares University, Tehran, Iran (IR.MODARES.REC.1397.105). Consent to P articipate : Written Informed consent was obtained from all human donors for the isolation of their stem cells and their subsequent use in this research study. Consent for Publication : Not applicable. (This study does not contain any individual person’s data in any form, such as individual details, images, or videos). Clinical Trial Registration : Not applicable. (This research did not involve a clinical trial). 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Supplementary Files ESM1.docx graphicalabstract.jpg Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 Dec, 2025 Reviews received at journal 23 Nov, 2025 Reviewers agreed at journal 20 Oct, 2025 Reviewers invited by journal 15 Oct, 2025 Editor assigned by journal 14 Oct, 2025 Submission checks completed at journal 13 Oct, 2025 First submitted to journal 07 Oct, 2025 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-7802296","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":534858537,"identity":"b0e295b0-0291-4207-b345-58359b9bb563","order_by":0,"name":"Fatemeh Soleymani","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Soleymani","suffix":""},{"id":534858538,"identity":"b2eb015d-bc90-47bc-8aa0-4a93a53fb95e","order_by":1,"name":"Saeideh Kavousi","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Saeideh","middleName":"","lastName":"Kavousi","suffix":""},{"id":534858539,"identity":"7d694312-7356-4b11-bfaf-c0d23ddc4bea","order_by":2,"name":"Nastaran Khodakarim","email":"","orcid":"","institution":"Iran University","correspondingAuthor":false,"prefix":"","firstName":"Nastaran","middleName":"","lastName":"Khodakarim","suffix":""},{"id":534858540,"identity":"fa1e5c33-b377-47e3-8107-c2542343c8b9","order_by":3,"name":"Mohammad Ahmadvand","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYFACHiBmAzGSDzAkIEuwEdaSlgDWwkOClhwDOB8v0G0/e3TDj7LD8ubsOZ8/PNxhx2AvkZ38gaHGjoFP+gBWLWZn8tJu9pw7bLiz5+02icQzyQw8ErnbJBiOJTOw8SVg13Igx+wGb1sa44YbudsYEtuYwVqATj3AwIbDiWbn35jd/NuWZr/hRs7jD4lt9SAtmz8w/MOj5UaO2W3eNptEoBYGicS2wyAtGyQY2/BpeWN2W+acTfKGM8/MgFqO8/CcAXmqL5kHt8NyzG6+KZOw3XA8+fHHn23VcuztQId9+GYnJ9+DXQsGgBidQET8jIJRMApGwSjADQDT8FwmgEVdqQAAAABJRU5ErkJggg==","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Ahmadvand","suffix":""}],"badges":[],"createdAt":"2025-10-07 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13:41:25","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91853,"visible":true,"origin":"","legend":"","description":"","filename":"173464d26e7545ffa1180e71ccc1beb31structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7802296/v1/e414248796c8302d20325cf9.xml"},{"id":94673411,"identity":"e1431b8f-812d-4042-8bbf-fd9358d59b0b","added_by":"auto","created_at":"2025-10-29 13:41:23","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103618,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7802296/v1/f259994477a4d6c7e6d5d06f.html"},{"id":94672869,"identity":"86fe1a32-f584-4552-8514-f63289172d2b","added_by":"auto","created_at":"2025-10-29 13:41:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":986940,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation and characterization of MSCs from ND and\u003c/strong\u003e \u003cstrong\u003eMM patients\u003c/strong\u003e. (a) Representative images showing microscopic spindle-shaped morphology of MM-MSCs (left) and ND-MSCs(right); (b) osteogenic differentiation was confirmed by Alizarin Red staining of Calcium deposits. (c) Adipogenic differentiation was demonstrated by Oil Red O staining of intracellular lipid droplets. (d-e) Flow cytometry analysis confirmed the immunophenotype of MSCs. (d) ND-MSCs were positive for CD105 and CD90, and negative for CD45 and CD34. (e) MM-MSCs were positive for CD105, CD90, and negative for CD45 and HLA-DR.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7802296/v1/5ca16575555dec84cc777de4.png"},{"id":94664230,"identity":"07848b71-ae14-4de7-aa8d-e2c568664548","added_by":"auto","created_at":"2025-10-29 12:18:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":155584,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDysregulation of senescence and stemness gene expression in MM-MSCs. \u003c/strong\u003e(a) Relative expression of senescence-related genes (\u003cem\u003eP16\u003c/em\u003e, \u003cem\u003eP21\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eIL-8\u003c/em\u003e) in MM-MSCs compared to ND-MSCs. (b) Relative expression of stemness-related genes \u003cem\u003e(NANOG, OCT4)\u003c/em\u003e between MM-MSCs (n=6) and ND-MSCs (n=3); (c) Validation using public RNA-seq data (GSE196297), showing Log2-transformed normalized counts for \u003cem\u003ePOU5F1\u003c/em\u003e (\u003cem\u003eOCT4\u003c/em\u003e) and \u003cem\u003eNANOG\u003c/em\u003e in MM patients (n=4) and control donors (n=5). All data are presented as mean ± SEM. The significance level is indicated as follows: ns (not significant), \u003csup\u003e\u003cstrong\u003e∗\u003c/strong\u003e\u003c/sup\u003e: p ≤ 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7802296/v1/76ee71fe1023088baee47f42.png"},{"id":94664237,"identity":"e8e58040-9820-4cae-a70d-55ddc898bb81","added_by":"auto","created_at":"2025-10-29 12:18:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":501372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSA-β-Gal staining for detection of cellular senescence. \u003c/strong\u003e(a) The percentage was calculated by counting blue-stained cells and total cells across multiple random fields for each sample. MM-MSCs show a significantly higher percentage of senescent cells compared to ND-MSCs. (b) Representative image of SA-β-gal staining in MM-MSCs. (c) Representative image of SA-β-gal staining in ND-MSCs. Data in) a (are presented as mean ± SEM. ∗: p ≤ 0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7802296/v1/aa0f662dff0e6530d9a50b4e.png"},{"id":94674104,"identity":"6f9271ed-efe9-4e81-90af-7a5e34f34c13","added_by":"auto","created_at":"2025-10-29 13:42:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2325650,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7802296/v1/7419c8ab-d54e-4ca2-80d5-fe7048d3a1e0.pdf"},{"id":94664229,"identity":"d38b2ed1-21a8-4e6b-9cb1-0953dd132808","added_by":"auto","created_at":"2025-10-29 12:18:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18566,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7802296/v1/d3e6246e1d65fb01607be53b.docx"},{"id":94664233,"identity":"32c8910a-ed16-49cc-ab2f-1ef24cf0875c","added_by":"auto","created_at":"2025-10-29 12:18:01","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":895328,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7802296/v1/c6c253af9067962b468c34b3.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Deregulation of Stemness and Senescence Genes in Bone Marrow Mesenchymal Stem Cells of Multiple Myeloma: Implications for Therapeutic Approaches","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMultiple myeloma (MM) is a prevalent hematologic cancer, with an estimated 36,110 new cases (representing 1.8% of all new cancer cases) and 12,030 associated deaths (accounting for 1.9% of all cancer deaths) in the United States for the year 2025[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite therapeutic advances, MM remains largely incurable with a poor prognosis.\u003c/p\u003e\u003cp\u003eMultiple myeloma is defined by the clonal expansion of abnormal plasma cells (PCs) in the bone marrow [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Numerous studies have explored the role of the bone marrow microenvironment and its intricate interactions with PCs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The bone marrow microenvironment includes both cellular and non-cellular components .The cellular compartments include hematopoietic and stromal cells, whereas the non-cellular compartments consist of cytokines, growth factors, chemokines, and extracellular matrix [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among these, mesenchymal stem cells (MSCs) are essential elements of the bone marrow, playing critical roles in its physiological and pathological states.\u003c/p\u003e\u003cp\u003eSeveral studies indicate that MM patients have abnormal MSCs with multiple reproducible differentially expressed genes (DEGs) compared to normal mesenchymal stem cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In particular, these cells can overproduce growth factors such as IL-6, which stimulates the growth of MM cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Comparative gene expression analyses of MM-MSCs and MSCs from normal donors (ND-MSCs) have revealed downregulation and disruption of genes involved in cell cycle progression, providing further evidence of dysregulated gene expression in MM-MSCs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. MM-MSCs are believed to contribute to disease progression by supporting MM cells, promoting treatment resistance, and establishing a favorable stromal microenvironment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCompared to bone marrow-derived ND-MSCs, MM-MSCs exhibit pronounced cellular signs of senescence[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], which have been significantly associated with MM development triggered by myeloma cell stimulation through LPA receptor 1 (LPA1) and 3 (LPA3) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This is important, as previous studies have shown that the secretory phenotype and metabolic activity of senescent MSCs exhibit pro-tumorigenic effects, promoting tumor growth and facilitating processes that drive MM progression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSenescence is characterized by the arrest of the cell cycle in the G0-G1 phase, primarily mediated by cyclin-dependent kinase inhibitors such as P21 and P16. This state is commonly associated with increased β-galactosidase activity, overexpression of cytokines, and abnormal cellular morphology [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While the precise mechanisms driving MSC senescence in MM remain unclear, understanding these pathways is vital, as it may provide novel therapeutic targets for improving treatment outcomes in MM patients.\u003c/p\u003e\u003cp\u003eAs multipotent adult stem cells, MSCs are defined by their dual capacity for self-renewal and multilineage differentiation into various tissue types[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, evidence supports the notion that these essential properties, differentiation and proliferation, may be disrupted in MM patients. Key transcription factors such as \u003cem\u003eOCT4\u003c/em\u003e and \u003cem\u003eNANOG\u003c/em\u003e regulate these functions in MSCs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Studies have shown that MSCs from MM patients exhibit a diminished differentiation capacity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and significantly reduced proliferative potential. Additionally, MM-MSCs display severely impaired osteoblastic differentiation compared to ND-MSCs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The high prevalence of senescent cells in MM-MSCs likely contributes to these functional impairments, as senescence is well known to disrupt normal cellular function.\u003c/p\u003e\u003cp\u003eInterestingly, previous studies provide preliminary evidence that targeting mesenchymal stem cell (MSC) senescence may shift MM-MSCs from promoting tumor growth to inhibiting it [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In other words, modifying the altered cellular characteristics of MM-MSCs could offer a promising strategy to improve treatment outcomes.\u003c/p\u003e\u003cp\u003eAlthough previous studies have highlighted an increased senescent phenotype in MM-MSCs, the concurrent status of core stemness pathways in these cells remains poorly understood. Based on this knowledge gap, we hypothesized that the MSCs of newly diagnosed MM patients are characterized by a dual dysregulation involving both increased senescence and a loss of stemness. To address this hypothesis, we compared the expression of key senescence (\u003cem\u003eP16\u003c/em\u003e, \u003cem\u003eP21\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eIL-8\u003c/em\u003e) and stemness (\u003cem\u003eNANOG\u003c/em\u003e, \u003cem\u003eOCT4\u003c/em\u003e) genes between MM-MSCs and ND-MSCs. A deeper understanding of this potential dysregulation is crucial for identifying pathological mechanisms and could ultimately inform targeted strategies to disrupt these interactions, slow disease progression, and improve treatment outcomes.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatients\u003c/h2\u003e\u003cp\u003eThis study included patients referred to Rasoul Hospital, Iran University of Medical Sciences, Tehran, Iran. Biopsies were performed on an initial cohort of 10 patients for the diagnosis of multiple myeloma (MM). The final study cohort consisted of six patients (three males and three females; mean age 61.7) who met the inclusion and exclusion criteria. Additionally, three healthy bone marrow biopsies (two males and one female; mean age 55.3) were collected for comparison. These samples were used to examine the molecular characteristics of MSCs from MM patients and healthy donors.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInclusion and Exclusion Criteria\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePatients with a confirmed diagnosis of multiple myeloma (MM) at intermediate to advanced disease stages (Stage II/III) were enrolled in this study. The patients were aged 48\u0026ndash;77 years. Exclusion criteria encompassed the presence of systemic disorders, such as inflammation, genetic disorders, and multiple sclerosis (MS), prior treatment for MM, a history of other malignancies, or treatment for other systemic conditions, including hypothyroidism or diabetes mellitus (DM). Additionally, healthy individuals without a history of systemic diseases, inflammation, or ongoing systemic treatment were selected as healthy bone marrow donors for transplantation.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eMSCs Isolation and Culture\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eA bone marrow biopsy was performed following the standard clinical procedure. Approximately 3\u0026ndash;5 mL of bone marrow aspirate was obtained from the posterior iliac crest. MSCs were isolated using the plastic adhesion method and cultured in a low-glucose medium \u003cem\u003e(\u003c/em\u003ebioIDEA, Tehran, Iran) supplemented with 10% fetal bovine serum (bioIDEA) and 1% penicillin/streptomycin (bioIDEA) until they reached 80% confluency at 37\u0026deg;C in a 5% CO2 atmosphere. The cells were then detached using 0.25% trypsin/EDTA (bioIDEA), centrifuged at 1200 rpm for 5 minutes, and subsequently passaged. This culturing procedure was applied to both MM-MSCs and ND-MSCs in the study. All isolated MSC populations successfully reached passage 4 for subsequent analyses.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eMSCs Validation\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo confirm the validation of MSCs, several well-established criteria must be met, including their spindle-shaped morphology, adherence to plastic surfaces, differentiation potential into at least two cell lineages, and the expression or absence of specific clusters of differentiation (CD) markers. MSCs were cultured in differentiation media (bioIDEA) for osteogenic and adipogenic differentiation, with differentiation confirmed according to the bioIDEA protocol. Briefly, MSCs were seeded in 12-well plates and cultured in osteogenic and adipogenic differentiation media. The media were replaced every three days. After three weeks, the cells were fixed with 4% formaldehyde and stained with 4% Alizarin Red for osteogenic differentiation. For adipogenic evaluation, cells were fixed with 10% formalin and stained with 1% Oil Red (bioIDEA).\u003c/p\u003e\u003cp\u003eIn addition to their differentiation potential, the harvested cells were evaluated by flow cytometry to confirm the presence of specific CD markers, including CD105, and CD90, and the absence of CD45 and CD34 and HLA-DR, to confirm their identity as MSCs. Briefly, cells were detached using 0.25% trypsin (bioIDEA), and the cell pellet was resuspended in 5% FBS. The cells were then incubated with monoclonal antibodies in the dark for 30 minutes. The presence of positive and negative CD markers was evaluated using a FACSCalibur\u0026trade; flow cytometer (Becton Dickinson, USA). FlowJo 10 software was employed to analyze the data and validate the MSC nature of the cells.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eRNA Extraction and cDNA Synthesis\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTotal RNA was extracted from MSCs (10^6 cells) using TRIzol reagent (Sigma, Germany) according to the manufacturer's instructions. The extracted RNA was verified for purity and concentration using a NanoDrop (IMPLEN GmbH, Munich, Germany), which Confirmed absorbance ratios of approximately 2.0 at 260/280 nm and \u0026ge;\u0026thinsp;1.8 at 260/230 nm. For cDNA synthesis, 10 \u0026micro;l of the extracted RNA was used for first-strand cDNA synthesis with the PrimeScript cDNA Synthesis Kit (Takara Bio, Japan), following the manufacturer's instructions.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative RT-PCR for Gene Expression Level Evaluations\u003c/h2\u003e\u003cp\u003eThe gene expression levels of the targeted genes were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) on a Step One, ABI system. Each reaction was performed using a high ROX RealQ Plus 2x Master Mix (Amplicon, Denmark), as specified by the manufacturer. \u003cem\u003eGAPDH\u003c/em\u003e (Glyceraldehyde-3-phosphate dehydrogenase) was used as the internal control gene for normalization. All primers were synthesized by Sinaclon Co. (Tehran, Iran). The sequences of all primers are provided in Online Resource 1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe Mann-Whitney U test, a nonparametric statistical method, was applied to compare the expression levels of the studied genes between MM-MSCs and ND-MSCs. Each sample was analyzed in triplicate and the average CT value was used for quantification. Gene expression was quantified using the comparative ΔΔCT method. Briefly, the CT value of each target gene was normalized to the endogenous control, \u003cem\u003eGAPDH\u003c/em\u003e, to generate a ΔCT value (ΔCT\u0026thinsp;=\u0026thinsp;CT\u003cem\u003etarget gene\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;CT\u003cem\u003eGAPDH\u003c/em\u003e ​). The average ΔCT from the Normal Donor (ND-MSC) group served as the reference. Final statistical comparisons were performed on the ΔCT values between the MM-MSC and ND-MSC groups. Data analysis was performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). A two-tailed p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePublic Dataset Analysis\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePublicly available RNA-seq data from the Gene Expression Omnibus (GEO) dataset GSE196297 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] were analyzed to validate the stemness gene expression trends observed in our study. This dataset includes bulk RNA-seq counts from bone marrow-derived mesenchymal stem cells (BM-MSCs) obtained from multiple myeloma (MM) patients and control donors. In the original study, RNA-seq data were processed using DESeq2 for normalization and differential expression analysis. Gene Set Enrichment Analysis (GSEA) was conducted based on MSigDB hallmark gene sets. Our analysis utilized the DESeq2-normalized count data from the study. To maintain consistency with our patient group, only MM patients at stages II/III were included and compared with control donors. Log2-transformed counts for \u003cem\u003ePOU5F1\u003c/em\u003e (\u003cem\u003eOCT4\u003c/em\u003e) and \u003cem\u003eNANOG\u003c/em\u003e were compared between MM patients (n\u0026thinsp;=\u0026thinsp;4) and control donors (n\u0026thinsp;=\u0026thinsp;5) using the Mann-Whitney U test.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSA-β-gal Staining (X-GAL)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSA-β-gal staining was employed to assess the percentage of senescent cells in MM-MSCs and ND-MSCs. The cells were seeded in 12-well plates to achieve approximately 80% confluence. The staining process followed the protocol established by Debacq-Chainiaux et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBriefly, the cells were washed with PBS, fixed with a freshly prepared solution containing 2% formaldehyde (vol/vol) and 0.2% glutaraldehyde (vol/vol) in PBS, and then washed twice with PBS. The cells were incubated overnight at 37\u0026deg;C without CO2 in a fresh staining solution. The staining solution consisted of citric acid/sodium phosphate buffer (40 mM, pH 6), potassium ferricyanide (5 mM), potassium ferrocyanide (5 mM), magnesium chloride (2 mM), sodium chloride (150 mM), and 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-gal, 1 mg/mL) in distilled water. After 16 hours of incubation, the cells were washed three times with PBS and once with methanol before being air-dried. To determine the percentage of senescent cells, a total of at least 500 cells were counted across 5 to 10 random, non-overlapping fields per sample. The percentage was calculated as the ratio of SA-β-gal-positive (blue) cells to the total number of cells counted..\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eIdentification and Differentiation of MSCs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMSCs isolated from bone marrow exhibited a spindle-shaped morphology (Fig. 1a). Flow cytometry analysis confirmed the expression of CD105 and CD90 and the absence of CD45. Specifically, ND-MSCs were negative for CD34 (Fig. 1d), and MM-MSCs were negative for HLA-DR (Fig. 1e). Additionally, the MSCs demonstrated multipotent differentiation capacity. Osteogenic differentiation was confirmed by detecting mineralized deposits in the extracellular matrix after Alizarin Red staining (Fig. 1b). Adipogenic differentiation was confirmed by the presence of lipid droplet formation after Oil Red O staining (Fig. 1c).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSenescence-related Genes Demonstrating Abnormal Expression Patterns in MM-MSCs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIL-6\u003c/em\u003e, a pro-inflammatory cytokine gene, was significantly overexpressed in MM-MSCs compared to ND-MSCs (p \u0026le; 0.05). Additionally, the cell cycle-regulating gene \u003cem\u003eP21\u003c/em\u003e was upregulated in MM-MSCs (p \u0026le; 0.05), indicating a potential link between senescence and the MM-MSC phenotype. While \u003cem\u003eIL-6\u003c/em\u003e and \u003cem\u003eP21\u003c/em\u003e exhibited distinct expression patterns between MM-MSCs and ND-MSCs, no statistically significant differences were observed in the expression levels of \u003cem\u003eP16\u003c/em\u003e and \u003cem\u003eIL-8\u003c/em\u003e between the two groups (Fig. 2a).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGenes Involved in Stemness are Suppressed in MM-MSCs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDysregulation of stemness genes was observed in MM-MSCs relative to ND-MSCs, with \u003cem\u003eNANOG\u003c/em\u003e and \u003cem\u003eOCT4\u0026nbsp;\u003c/em\u003etranscripts (\u003cem\u003eOCT4A\u003c/em\u003e, \u003cem\u003eOCT4B\u003c/em\u003e, \u003cem\u003eOCT4B1\u003c/em\u003e) being significantly differentially expressed in MM patient-derived MSCs. All transcripts of \u003cem\u003eOCT4A\u003c/em\u003e (p \u0026lt; 0.05), \u003cem\u003eOCT4B\u003c/em\u003e (p \u0026lt; 0.05), \u003cem\u003eOCT4B1\u003c/em\u003e (p \u0026lt; 0.05), and \u003cem\u003eNANOG\u003c/em\u003e (p \u0026lt; 0.05) exhibited downregulation in MM-MSCs (Fig. 2b).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConcordance of OCT4 and NANOG Expression in RNA-seq Data\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRNA sequencing data showed concordance for \u003cem\u003eOCT4\u003c/em\u003e and \u003cem\u003eNANOG\u003c/em\u003e expression. Log2-transformed, normalized counts revealed significant downregulation of \u003cem\u003ePOU5F1\u003c/em\u003e\u003cem\u003e\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e\u003c/em\u003e(\u003cem\u003eOCT4\u003c/em\u003e) in MM patients (p = 0.0317) and marginally significant downregulation of \u003cem\u003eNANOG\u003c/em\u003e (p = 0.0635) compared to healthy controls. These findings align with our in-house observations of suppressed stemness gene expression in MM-MSCs (Fig. 2c).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMM-MSCs Demonstrating a Higher Percentage of the SA-\u0026beta;-Gal-Positive Cells\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMM-MSCs and ND-MSCs were stained with SA-\u0026beta;-gal, and the results revealed a significant difference between the two groups. As shown in Fig. 3 b-c, MM-MSCs exhibited a statistically significant (P \u0026le; 0.0169) increase in the percentage of SA-\u0026beta;-gal-positive cells (Fig. 3a).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study provides the first direct evidence that MSCs from newly diagnosed, untreated multiple myeloma patients exhibit a dual dysregulation characterized by both accelerated senescence and a significant loss of stemness. This finding supports the growing hypothesis that a concurrent disruption of these pathways contributes to MM pathogenesis. The reciprocal interaction between plasma cells and MM-MSCs has emerged as a key focus, with several studies suggesting that PCs play a crucial role in inducing senescence in surrounding MSCs. Interestingly, senescent MSCs, in turn, appear to contribute to treatment resistance in multiple myeloma. Understanding the underlying mechanisms of this interaction could provide valuable insights for developing novel therapeutic approaches, including precision medicine strategies tailored to target senescence-driven treatment resistance in multiple myeloma patients.\u003c/p\u003e\u003cp\u003eOur findings substantiate this hypothesis of dual dysregulation. We observed a significant upregulation of the pro-senescent factors \u003cem\u003eIL-6\u003c/em\u003e and \u003cem\u003eP21\u003c/em\u003e in MM-MSCs, which are hallmarks of cellular aging and cell cycle arrest. Concurrently, we detected a significant downregulation of the core stemness transcription factors \u003cem\u003eOCT4\u003c/em\u003e and \u003cem\u003eNANOG\u003c/em\u003e. This concurrent phenotype suggests a profound functional shift in MM-MSCs, where the cells not only enter a senescent state but also lose their fundamental self-renewal capacity.\u003c/p\u003e\u003cp\u003eDelving into the senescence aspect, our study found that \u003cem\u003eIL-6\u003c/em\u003e, a key growth factor and a marker of senescence, exhibited a significant increase in MM-MSCs, consistent with previous findings [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. IL-6 plays a pivotal role in sustaining myeloma cell survival and proliferation by activating critical signaling pathways such as JAK/STAT, PI3K/AKT, and MAPK. Previous research has demonstrated that \u003cem\u003eIL-6\u003c/em\u003e overexpression in senescent cells not only supports but also perpetuates the proliferation of neighboring malignant cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These findings further support the notion that MM-MSCs exist in a senescent state, aligning with prior studies that underscore their role in promoting tumorigenesis, conferring drug resistance, and sustaining the survival of malignant plasma cells.\u003c/p\u003e\u003cp\u003e\u003cem\u003eP16\u003c/em\u003e and \u003cem\u003eP21\u003c/em\u003e are commonly used senescence biomarkers, as their overexpression results in cell cycle arrest. Our findings of elevated P21 but unchanged P16 levels align with some previous reports [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This suggests that the role of \u003cem\u003eP16\u003c/em\u003e in senescence may be context-dependent, as \u003cem\u003eP16\u003c/em\u003e regulates specific senescent states, while \u003cem\u003eP53\u003c/em\u003e governs others based on the cellular response to different stressors [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, endogenous P16 has been shown to inhibit \u003cem\u003eIL-6\u003c/em\u003e expression [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and IL-6-dependent inhibition of G0-G1 progression is mediated through the P21 pathway [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This creates a coherent molecular picture that aligns with our observation regarding the expression levels of \u003cem\u003eP21\u003c/em\u003e and \u003cem\u003eIL-6\u003c/em\u003e. Although IL-8 is recognized as a senescence-associated cytokine [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], our study only detected a trend towards upregulation that did not achieve statistical significance, likely attributable to the small sample size.\u003c/p\u003e\u003cp\u003eThe transcription factors OCT4 and NANOG are critical for the survival and self-renewal of MSCs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. OCT4 establishes a regulatory network that inhibits differentiation-associated genes, thereby preserving pluripotency [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Similarly, NANOG is crucial for maintaining self-renewal and facilitating pluripotent or multipotent differentiation while also suppressing spontaneous senescence [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Consistent with these findings, the current study reveals a significant downregulation of \u003cem\u003eOCT4\u003c/em\u003e and \u003cem\u003eNANOG\u003c/em\u003e expression in MM-MSCs compared to ND-MSCs. Although these genes have been identified as key components of the regulatory network that represses differentiation and maintains MSC pluripotency [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], their reduced expression in MM-MSCs is, to our knowledge, a novel finding. Interestingly, when using publicly available RNA sequencing data from the GEO dataset [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], the findings were concordant with our RT-qPCR results. This concordance further supports the downregulation of critical stemness genes in MM-MSCs. However, a recent study reported no significant differences in the expression of stemness-related genes between HD-MSCs and MM-MSCs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Notably, Lu et al. investigated primarily treated multiple myeloma patients, whereas our study focused on newly diagnosed, untreated MM patients at intermediate to advanced disease stages. This fundamental difference in patient characterization may account for the gene expression discrepancy and suggests that disease stage and treatment status are critical determinants of MSC gene expression profiles in MM. It further emphasizes the potential significance of \u003cem\u003eOCT4\u003c/em\u003e and \u003cem\u003eNANOG\u003c/em\u003e dysregulation in the pathogenesis of newly diagnosed, untreated multiple myeloma.\u003c/p\u003e\u003cp\u003eCrucially, the observed loss of stemness and increased senescence in MM-MSCs are not isolated events but likely represent two faces of the same coin, forming the basis for our \"dual dysregulation\" hypothesis. Our study strongly suggests this link by demonstrating a concurrent downregulation of \u003cem\u003eOCT4\u003c/em\u003e and \u003cem\u003eNANOG\u003c/em\u003e, alongside an upregulation of the cell cycle inhibitor \u003cem\u003eP21.\u003c/em\u003e This finding is well supported by established mechanistic connections; for instance, \u003cem\u003eNANOG\u003c/em\u003e has been shown to counteract senescence [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and delay its onset through specific signaling pathway [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, \u003cem\u003eOCT4\u003c/em\u003e maintains stem cell self-renewal and reverses senescence by inhibiting \u003cem\u003eP21\u003c/em\u003e expression [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, our observation of \u003cem\u003eOCT4\u003c/em\u003e downregulation and \u003cem\u003eP21\u003c/em\u003e upregulation corroborate previous reports of a negative regulatory axis between these two factors. These internally consistent findings reinforce the notion that targeting dysregulated transcription factors could present a novel therapeutic approach to overcoming treatment resistance in MM patients.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides further evidence for the involvement of senescence-related genes in MM-MSCs and confirms that these cells exhibit a more pronounced senescent phenotype compared to ND-MSCs. Furthermore, we present, for the first time, distinct expression patterns of stemness-related genes in MM-MSCs in comparison to ND-MSCs. This dysregulation may contribute to the senescent state of MM-MSCs and potentially underlie the treatment resistance observed in multiple myeloma patients. These findings highlight the potential for novel therapeutic strategies aimed at targeting senescence in MM-MSCs. Specifically, regulating stemness transcription factors may offer a promising avenue for improving treatment outcomes in multiple myeloma. Our study has limitations, primarily a small sample size requiring confirmation in larger cohorts. Additionally, our findings are based on gene expression; future studies should incorporate functional assays to directly assess the consequences of stemness loss and increased senescence. Exploring the signaling pathways driving this phenotype could reveal novel therapeutic targets.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCD markers: Clusters of Differentiation Markers\u003c/p\u003e\n\u003cp\u003eDEGs: Differentially Expressed Genes\u003c/p\u003e\n\u003cp\u003eDM: Diabetes Mellitus\u003c/p\u003e\n\u003cp\u003eGAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase\u003c/p\u003e\n\u003cp\u003eGEO: Gene Expression Omnibus\u003c/p\u003e\n\u003cp\u003eGSEA: Gene Set Enrichment Analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMM: Multiple Myeloma\u003c/p\u003e\n\u003cp\u003eMS: Multiple Sclerosis\u003c/p\u003e\n\u003cp\u003eMSCs: Mesenchymal Stem Cells\u003c/p\u003e\n\u003cp\u003eND : Normal Donors\u003c/p\u003e\n\u003cp\u003ePCs: Plasma Cells\u003c/p\u003e\n\u003cp\u003eRT-qPCR: Reverse Transcription-Quantitative Polymerase Chain Reaction\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u003cspan dir=\"RTL\"\u003e:\u003c/span\u003e\u003c/em\u003e \u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003eM.A.\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e and F.S. designed the study. N.K. was responsible for patient diagnosis, recruitment, and clinical sample acquisition. F.S. and S.K. performed the experiments, data collection, and formal analysis. F.S. wrote the first draft of the manuscript. M.A., and S.K. reviewed and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics Approval\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e:\u003c/em\u003e\u003c/strong\u003e This study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board and\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003ethe Ethics Committee of the Tarbiat Modares University, Tehran, Iran (IR.MODARES.REC.1397.105).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent to\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003earticipate\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u003cspan dir=\"RTL\"\u003e:\u003c/span\u003e\u003c/em\u003e Written Informed consent was obtained from all human donors for the isolation of their stem cells and their subsequent use in this research study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for Publication\u0026nbsp;:\u003c/em\u003e\u003c/strong\u003e Not applicable. (This study does not contain any individual person\u0026rsquo;s data in any form, such as individual details, images, or videos).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eClinical Trial Registration\u0026nbsp;:\u003c/em\u003e\u003c/strong\u003e Not applicable. (This research did not involve a clinical trial).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSEER Program, NCI. Cancer Stat Facts: Myeloma. National Cancer Institute. 2024. Accessed 12 May 2025. https://seer.cancer.gov/statfacts/html/mulmy.html.\u003c/li\u003e\n\u003cli\u003eGiannakoulas N, Ntanasis-Stathopoulos I, Terpos E. 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Stem Cell Res The. 2019;10:28. https://doi.org/10.1186/s13287-018-1120-x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"blood-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"BLOOD RESEARCH","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Multiple Myeloma, Tumor Microenvironment, Bone Marrow Mesenchymal Stem Cells, Stemness, Senescence-Related Genes","lastPublishedDoi":"10.21203/rs.3.rs-7802296/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7802296/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e Multiple myeloma (MM) is a hematologic malignancy with a poor prognosis.\u003cstrong\u003e \u003c/strong\u003eMM-derived MSCs (MM-MSCs) contribute to disease progression by creating a supportive stromal microenvironment for malignant cells. Therefore, elucidating transcriptomic alterations in MSCs may facilitate the development of novel therapeutic strategies for treatment\u003cstrong\u003e \u003c/strong\u003eresistant MM patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Total RNA was extracted from cultured MSCs isolated from bone marrow aspirates of MM patients and normal donors (ND-MSCs). Gene expression of stemness markers (\u003cem\u003eNANOG\u003c/em\u003e, \u003cem\u003eOCT4\u003c/em\u003e) and senescence-related genes (\u003cem\u003eP16\u003c/em\u003e, \u003cem\u003eP21\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eIL-8\u003c/em\u003e) were analyzed using Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), while senescence status was assessed using SA-β-gal staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Compared to ND-MSCs, MM-MSCs demonstrated a higher percentage of SA-β-gal-positive cells. Gene expression analysis revealed upregulation of \u003cem\u003eP21\u003c/em\u003e and \u003cem\u003eIL-6\u003c/em\u003e in MM-MSCs, whereas \u003cem\u003eNANOG\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eand \u003cem\u003eOCT4\u003c/em\u003e were significantly downregulated. Notably, this downregulation was corroborated by analyzing a publicly available RNA-seq dataset, reinforcing our findings\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e While confirming previous reports on increased senescence, our study provides novel evidence for the significant downregulation of stemness-related genes (\u003cem\u003eNANOG\u003c/em\u003e, \u003cem\u003eOCT4\u003c/em\u003e) in MSCs from newly diagnosed, untreated MM patients. This concurrent dysregulation of stemness and increased senescent phenotype may be a key contributor to the pathogenicity of the tumor microenvironment, highlighting a potential axis for therapeutic intervention.\u003c/p\u003e","manuscriptTitle":"Deregulation of Stemness and Senescence Genes in Bone Marrow Mesenchymal Stem Cells of Multiple Myeloma: Implications for Therapeutic Approaches","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 12:17:57","doi":"10.21203/rs.3.rs-7802296/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-02T07:19:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T04:17:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176355579466948786252193122524253268953","date":"2025-10-20T08:43:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-15T12:53:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-15T00:46:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-14T03:04:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"BLOOD RESEARCH","date":"2025-10-07T19:34:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"blood-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"BLOOD RESEARCH","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8ab81915-b464-4a50-ae54-4d9e9d8e090d","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-25T05:24:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-29 12:17:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7802296","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7802296","identity":"rs-7802296","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}