Metastatic promoting role of Mesenchymal Stem Cells in Oral Squamous Cell Carcinoma Revealed by an Improved Bone Marrow Chimeric Model

Metastatic promoting role of Mesenchymal Stem Cells in Oral Squamous Cell Carcinoma Revealed by an Improved Bone Marrow Chimeric Model | 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 Metastatic promoting role of Mesenchymal Stem Cells in Oral Squamous Cell Carcinoma Revealed by an Improved Bone Marrow Chimeric Model Htoo Shwe Eain, Hotaka Kawai, Sho Sanou, Yamin Soe, Tianyan Piao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8323433/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 May, 2026 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract Mesenchymal stem cells (MSCs) are essential stromal regulators that coordinate tissue repair, angiogenesis, and immune balance. Within tumours, MSCs remodel the microenvironment and influence disease progression, yet their systemic contribution remains unclear due to the limited recovery of functional MSCs in conventional bone marrow transplantation models. Here, we developed an improved bone marrow collection (iBMC) method using enzymatic digestion, which markedly increases MSC yield while preserving their native phenotype. GFP bone marrow chimeric mice generated by iBMC and conventional transplantation displayed comparable hematopoietic reconstitution but differed in MSC abundance, enabling direct analysis of MSC-specific effects under physiological conditions. In mouse models of oral squamous cell carcinoma (OSCC), MSCs profoundly affected tumour development. MSC-deficient tumours exhibited necrosis and infiltration of immature myeloid-derived suppressor cells (MDSCs), whereas MSC-rich tumours showed enhanced vascularisation, adaptive immune infiltration, and stromal remodelling. Notably, lung metastasis occurred only in MSC-rich mice, accompanied by bone marrow–derived endothelial activation and TNF-α–driven inflammation. Pharmacological inhibition of LepR signalling using SHU9119 unexpectedly increased metastasis, underscoring the dual, context-dependent functions of MSCs. These findings establish iBMC as a reproducible and physiologically relevant model to study MSC-mediated regulation of tumour immunity, angiogenesis, and metastasis. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Stem cells Mesenchymal stem cells (MSCs) Oral squamous cell carcinoma (OSCC) Bone marrow transplantation Chimeric mouse models Tumour microenvironment (TME). Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Mesenchymal stem cells (MSCs) are multipotent stromal cells that play crucial roles in maintaining tissue homeostasis and promoting regeneration. They migrate to the site of injury and inflammation, where they contribute to repair through stromal support, endothelial differentiation, context-dependent immunomodulation, and the maintenance of balanced inflammation [1–4]. The tumour microenvironment (TME) resembles a chronically inflamed ulcer, often described as "a wound that never heals" [5]. Within this pathological context, MSCs are recruited to tumours, where they adapt to the TME and contribute to stromal remodelling, angiogenesis, and immune modulation [3,6,7]. The multifaceted nature of MSCs suggests that they may be the key stromal component orchestrating the structural and functional dynamics of the TME [7]. However, studying the contribution of MSCs to tumour progression remains challenging due to limitations in experimental models. Conventional approaches rely on the isolation of bone marrow-derived MSCs (BM-MSCs) from femoral tissue, yet the yield of functional MSCs from this source is typically low [8]. Furthermore, in vitro expansion alters their phenotypes and accelerates cellular senescence, leading to inconsistent biological responses [9]. These issues have hindered the establishment of physiologically relevant mouse models that enable the functional tracing of endogenous MSCs within tumours. To overcome these challenges, we optimised an enzyme-treated bone marrow collection protocol that markedly enhances BM-MSC recovery while preserving their native properties [10]. In our previous study, enzymatic treatment during bone marrow collection increased MSC yield compared with conventional non-enzyme-treated methods, resulting in osteoblast differentiation and improved bone marrow formation in fracture models [10]. Based on this approach, we developed two distinct bone marrow-derived chimeric mouse models differing in MSC content but otherwise comparable in hematopoietic composition. These models enable investigation of MSC-specific effects without altering other bone marrow-derived cell populations. Moreover, since MSCs are directly transplanted from donor mice without culture, this can avoid complications such as in vitro ageing. In contrast to studies using localized MSC injections, which fail to mimic the natural recruitment of MSCs to the tumour, our model provides a physiologically similar condition to study endogenous MSC behaviour under systemic homeostasis [11]. In this comprehensive study, we investigated how bone marrow-derived MSCs influence tumour vascularisation, inflammatory activation, and metastasis in oral squamous cell carcinoma (OSCC). Using a GFP bone marrow-chimeric mouse model with varying MSC content, we tracked the dynamics of BM-MSCs and assessed their contributions to immune cell infiltration, vascular development, stromal remodelling, and metastatic dissemination. Furthermore, we examined the inflammatory activation of endothelial cells, focusing on TNF-α–mediated signalling, and evaluated the effects of pharmacological modulation of MSC-associated pathways on metastatic outcomes. Collectively, our findings provide insights into the diverse roles of BM-MSCs in tumour progression and highlight their significance as potential therapeutic targets in OSCC. RESULTS Enzymatic treatment of the femur yields a higher number of mesenchymal stem cells (MSCs) The enzymatically treated bone marrow collection was named iBMC (improved bone marrow collection) and the conventionally collected bone marrow was termed BMC (bone marrow collection). To compare the efficiency of MSC recovery between the two methods, bone marrow cells obtained from iBMC, and BMC were seeded and cultured. The spindle-shaped adherent MSCs were quantified (Fig. 1A). The iBMC method yielded a higher population of spindle-shaped MSCs compared to BMC (Fig. 1B). To establish chimeric models, lethally irradiated C57BL/6J mice were transplanted with the collected bone marrow cells via tail vein injection [10]. The success of bone marrow engraftment was confirmed by GFP immunostaining in the bone marrow tissues of wild-type mice (control), iBMC, and BMC mice. GFP + cells are clearly detected in the bone marrow tissues in iBMC and BMC groups, confirming successful transplantation (Fig.1C). In addition, we found megakaryocytes in iBMC and wild-type mice (Fig.1C, arrows), but they were absent in BMC, suggesting impaired megakaryopoiesis due to dysfunction or lack of MSCs [12]. To analyse the abundance of MSCs in bone marrow, bone marrow tissues were immunostained with MSC markers, LepR and SDF-1 (Fig. 1D, F). BMC group exhibited a markedly lower number of LepR + cells and SDF-1 + cells compared with wild-type and iBMC groups, confirming that enzymatic treatment increases MSC content in the bone marrow (Fig. 1F, G). These results indicate that enzymatic digestion during bone marrow harvest significantly enhances MSC yield, and that transplanted bone marrow cells successfully reconstitute the bone marrow compartment, generating GFP + bone marrow (BM) chimeric mice. MSCs influence the infiltration of bone marrow-derived cells (BMDCs) and immune cells into the tumours. To investigate how MSC abundance affects the TME, GFP BM chimeric mice made from the iBMC, and BMC bone marrow tissue were utilised. Following a two-week recovery period post-transplantation, mouse oral squamous cell line 2 (MOC2) was inoculated into the buccal mucosa of iBMC and BMC mice for 3 weeks (Fig. 2A). GFP immunostaining confirmed infiltration of BMDCs into tumour tissues (Fig. S2A). The iBMC group showed fewer GFP + cells compared with the BMC tumours (Fig. S2B), suggesting differences in BMDC recruitment dynamics. To characterise the immune microenvironment of iBMC and BMC tumours, we compared and studied the immune populations in tumours. We first analysed the T cell population using CD4 and CD8 markers, the B cell population using the CD20 marker, and the monocyte and myeloid-derived suppressor cell (MDSC) using CD11b and Gr-1 markers. Immunostaining revealed that BMC tumours contained significantly fewer CD8 + cytotoxic T cells and CD20 + B cells than wild-type and iBMC tumours (Fig. 2C, D). Although not statistically significant, CD4 + helper T cells were also reduced in BMC tumours (Fig. 2B). In contrast, CD11b single-marker staining showed a significant decrease in both BMC and iBMC tumours (Fig. 2E), while BMC tumours showed an increase in Gr-1 + cells among the three groups (Fig. 2F). However, double immunofluorescence staining for CD11b + Gr-1 + confirmed the MDSC population in BMC tumours was more abundant than that in iBMC tumours (Fig. 2G, H). These results show that MSC deficiency alters the differentiation and infiltration patterns of BMDCs, and iBMC models can more accurately replicate the immune landscape of wild-type tumours while maintaining traceable GFP + BMDCs within the TME. MSCs contribute to tumour vascularisation and maintain the vascular integrity of tumours. MSCs are multipotent and can differentiate into the endothelial cell or pericytes, aiding in angiogenesis and vascular stabilisation [13]. To evaluate tumour vascular characteristics, we compared the necrosis areas and bleeding areas in wild-type, iBMC, and BMC tumours (Fig. 3A, C; red insets). Quantitative analysis revealed that both necrotic and bleeding area-to-total tumour ratios were significantly increased in the BMC tumours (Fig. 3B, D), indicating a higher central necrosis and poorer structural integrity. Histopathological analysis also reveals fewer vessel-like structures within the BMC tumours compared to iBMC tumours (Fig. 3C; yellow insets). These results indicate that a lower number of MSCs compromises tumour vascularisation, leading to necrosis and vascular fragility. Mature vessels, activated vessels, and cancer-associated fibroblasts (CAFs) are recruited into the TME via an MSC-dependent manner. To further investigate the vascular and stromal components formed under different MSC conditions, we used CD34 as a marker for mature vessels, CD105 as a marker for activated vessels, and α-SMA as a marker for CAFs (Fig. 4A, D, G). Double immunofluorescence with GFP confirmed that a subset of these vessels and fibroblasts originated from the bone marrow-derived cells (Fig. 4B, E, H). Quantification revealed significantly higher numbers of CD34 + vessels, CD105 + vessels, and α-SMA + CAFs in iBMC tumours compared to BMC tumours (Fig. 4C, F, I). These results demonstrate that bone marrow-derived MSCs contribute to vascular and stromal formation within the TME, and that an adequate MSC population is essential for establishing functional and organised tumour vasculature. Bone marrow-derived MSCs promote lung metastasis of oral squamous cell carcinoma (OSCC) The most common metastasis sites for OSCC are the cervical lymph nodes and lungs. OSCC spreads to the lungs via hematogenous metastasis, and our previous analyses showed distinct vascular changes between iBMC and BMC tumours. We next examined lung metastasis (Fig. 5A). The number of metastatic islands was significantly lower in BMC groups than in wild-type and iBMC tumours (Fig. 5B), indicating that MSCs enhance metastatic dissemination. BMDCs are known to release inflammatory cytokines that promote endothelial activation, vascular leakage, and metastasis [13–16]. To study this mechanism, we performed double immunofluorescence staining using the bone marrow-derived marker GFP and the inflammatory marker TNF-α on the lung ECs (Fig. 5C, E). iBMC lung ECs showed higher GFP + cells and increased TNF-α expression on ECs compared to that of BMC lung ECs (Fig. 5D, F). Consistent results were observed again in triple fluorescence staining of GFP, TNF-α, and CD105 (endothelial marker), where a higher rate of co-localisation of GFP + and TNF-α + cells was seen on CD105 + vascular structures in the iBMC group compared to the BMC group (Fig. 5G, H). These results suggest that an increase in MSCs promotes lung metastasis, accompanied by the recruitment of BMDCs and the inflammatory activation of lung endothelium. Disturbance of MSC signalling further enhances metastasis via inflammatory activation of lung endothelium. To determine whether disrupting MSC signalling affects metastatic behaviour, we administered SHU9119, a synthetic peptide antagonist of melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R), which mediates leptin-leptin receptor (Lep-LepR) signalling axis. MC3R and MC4R have anti-inflammatory properties, and SHU9119 antagonism promotes the inflammation and disturbs the effects of the leptin-leptin receptor axis by effectively inhibiting MC4R [17,18]. epR + MSCs constitute the major population of bone marrow-derived MSCs [19]. SHU9119 was administered daily to the iBMC mice one week after tumour transplantation to disturb LepR function in MSCs. Lung metastasis was quantified in the SHU9119-treated iBMC group, named iBMC (SHU), and saline-injected control, named iBMC (Saline) (Fig. 6B). The SHU9119-treated group displayed a significantly higher number of lung metastatic colonies (Fig. 6C). To further assess endothelial inflammation by LepR disruption, we repeated the GFP and TNF-α staining in lung tissues (Fig. 6D, 6F). Interestingly, SHU9119 injection further promotes the recruitment of BMDCs and the expression of TNF-α in lung ECs (Fig. 6E, 6G). Similarly, triple staining for GFP, TNF-α, and CD105 revealed accumulation of GFP + TNF-α + cells on CD105 + vascular structures in the lungs following SHU9119 treatment. Together, these results demonstrate that disturbing the LepR + MSC signalling in the bone marrow induces a proinflammatory environment that promotes lung metastasis through the recruitment of BMDCs and the promotion of lung vessel inflammation. Thus, MSC integrity is critical for maintaining vascular homeostasis, and its disturbance accelerates metastatic progression in OSCC. DISCUSSION In our previous work, we established that enzymatic digestion of bone marrow tissue significantly improves MSC recovery, as confirmed by increased LepR + and SDF-1 + stromal cell yield, higher osteoblast recruitment, and enhanced bone formation in the mice transplanted with enzyme-treated bone marrow collection (iBMC) compared with those receiving conventional bone marrow collection (BMC) [10]. Comparative histological analysis showed a lack of megakaryocytes in BMC mice, consistent with a reduced MSC population, as megakaryopoiesis is known to require both HSC- and MSC-derived signals [12]. Together, these results indicate that conventional bone marrow collection impairs MSC retention, leading to models that incompletely reflect the physiological contribution of MSCs to tissue and tumour biology. GFP bone marrow transplantation models are widely used to trace BMDCs in vivo, particularly in studies of tumour biology and tissue regeneration [20–22]. However, our findings highlight a key limitation of traditional methods: 'suboptimal MSC recovery', which compromises model fidelity. iBMC models preserve the physiological diversity of BMDCs while enhancing the representation of MSCs. Importantly, iBMC models resemble wild-type mice without bone marrow transplantation in terms of bone marrow status, immune microenvironment, and tumour histological characteristics, making them more suitable for tracing and studying the BM-MSCs while reflecting normal physiological conditions. On the other hand, our control MSC-deficient BMC mice remain viable, allowing them to serve as appropriate controls for dissecting MSC-specific functions under otherwise equivalent hematopoietic conditions. Using these models, we were able to investigate the tumour microenvironment with a systemic approach by studying the influence of MSCs on immune aspects, vascular characteristics, the source of tumour vessels, and metastatic progression. MOC2 tumours, a poorly differentiated OSCC tumour model transplanted into the immunocompetent C57BL/6 mice, allowed a comprehensive evaluation of immune dynamics [23]. Systemic comparison revealed that MSC abundance shapes the immune microenvironment of OSCC. MSC-deficient BMC tumours exhibited enhanced infiltration of immature myeloid-derived suppressor cells (MDSCs), whereas MSC-rich iBMC tumours displayed increased infiltration of mature adaptive immune cells. This suggests that MSCs regulate BMDC differentiation following their recruitment to tumours, through direct contact and soluble mediators such as SDF-1, TGF-β, and IL-6 [24]. Consistent with previous studies, MSCs orchestrate immune responses in a context-dependent manner, capable of promoting or suppressing immunity depending on the surrounding cytokine milieu [25,26]. Whether this regulation translates into protective immunity or tumour tolerance may depend on the broader stromal and vascular context, underscoring the complex immunoregulatory role of MSCs in OSCC TME. Vascularisation and stromal remodelling are indispensable for tumour growth, survival, and dissemination [27,28]. Although BMC and iBMC tumours exhibited necrotic cores, necrosis was significantly more extensive in BMC tumours. Necrosis in a solid tumour is typically attributed to hypoxia and impaired blood supply, which suggests the role of MSCs in angiogenesis [13]. Consistent with this hypothesis, BMC tumours exhibited a reduced number of CD34 + endothelial cells, CD105 + active endothelial cells, and α-SMA + CAFs compared to iBMC tumours. Double immunofluorescence with GFP revealed that subsets of these stromal cells originated from the bone marrow. These data confirm that BM-MSCs mobilised into the tumours, where they differentiate into stromal components that support angiogenesis and tumour progression. The hemorrhagic regions observed in the BMC tumour further suggest that defective MSC-mediated vascularisation leads to fragile and dysfunctional vessels. Together, these data indicate that MSCs are essential not only for maintaining vascular integrity but also for establishing a stromal milieu that supports tumour expansion [27,29]. Orthotopic implantation of MOC2 tumour also replicated the clinical metastatic pattern of OSCC, with cervical lymph node and lung metastases. Interestingly, metastatic pulmonary spread was observed only in tumours with high MSC content. BMC tumours that lack MSCs rarely produce lung metastases, underscoring the critical contribution of MSCs to hematogenous dissemination. The underlying mechanisms are likely multifactorial. By differentiating into CAFs, they remodel the TME and secrete pro-invasive factors, while MSC-derived endothelial cells enhance angiogenesis and facilitate tumour intravasation [13,15,30]. BM-MSCs themselves can also secrete cytokines and chemokines that prepare pre-metastatic niches, priming sites such as the lungs to be more receptive to colonisation by circulating tumour cells [31–33]. Consistent with these observations, lung endothelial cells of iBMC tumours showed stronger GFP positivity, indicating bone marrow origin, and displayed elevated TNF-α expression on their surface, suggesting inflammatory activation. Triple immunofluorescence further revealed that MSC-derived endothelial cells undergo inflammatory activation, likely increasing vascular permeability and adhesion, thereby facilitating colonisation of circulating tumour cells in the lungs. To explore therapeutic modulation, we targeted MSC signalling using SHU9119, a synthetic peptide antagonist of the melanocortin 3 and 4 receptors (MC3R and MC4R), which disrupts the leptin-leptin receptor axis of MSCs [34–36]. Interestingly, administration of SHU9119 did not suppress lung metastasis as anticipated. Instead, it led to an increase in lung metastases. This paradox may be explained by the proinflammatory consequences of disrupting MSC signalling through MC4R antagonism, as MC4R inhibition skews the immune environment towards a proinflammatory state [37]. These results add a layer of complexity to the therapeutic use of MSCs and caution against manipulating the MSC-associated pathway in therapy. These observations establish iBMC as a superior approach for studying the systemic contribution of MSCs to tumour biology. Taken together, our findings emphasised the multifaceted role of BM-MSCs in the progression of OSCC through their integrated effects on immune regulation, vascular remodelling, and metastatic dissemination. Our iBMC models provide a physiologically relevant platform that overcomes the limitation of conventional bone marrow transplantation by preserving MSC functionality and enabling systemic evaluation of their tumour-promoting or tumour-restraining roles. Therapeutic strategies targeting MSC pathways must therefore be approached with caution, as modulating these biologically complex and multifaceted cells can yield unexpected outcomes depending on context. Future study will focus on identifying the molecular cues that determine whether MSCs adopt tumour-promoting versus tumour-restraining roles, particularly through their crosstalk with inflammatory pathways. This comprehensive study can be critical for developing safe and effective therapeutic approaches that exploit the vulnerabilities of MSC-tumour interactions without compromising their essential roles in normal physiology. METHODS Mice 8-week-old female GFP transgenic mice [C57BL/6-Tg (CAG-EGFP)] and wild-type mice (C57BL/6J) were purchased from Charles River Laboratories. All animals were kept in optimal condition according to the experimental animal guidelines of Okayama University. Mice were used for various in vivo experiments, including cell injection and tumour transplantation, as described in the respective sections. Mice were euthanised following ARRIVE guidelines. Mice were anaesthetised with 5% isoflurane in an enclosed induction chamber and were observed until the complete loss of reflexes, confirming the state of deep anaesthesia. Only after that, cervical dislocation was performed as a secondary physical method to ensure death. Cell lines MOC2 cell line (KER-EWL002-FP) was purchased from Kerafast cell bank. These cells were grown in a 2:1 ratio of Iscove's Modified Dulbecco's Medium (IMDM) (Gibco) and Ham's Nutrient Mixture (F12) (Gibco), 5% FBS (Gibco), 1% anti-anti (Gibco), 5 mg/ mL insulin (Sigma Aldrich), 400 ng/mL hydrocortisone (Sigma Aldrich), and 5 ng/mL EGF (585508 Biolegend) in a humidified incubator at 37℃, 5% CO₂. Bone Marrow Collection (BMC) GFP + bone marrow cells were collected from the femur and tibia of the 8-week-old GFP transgenic mice. The proximal parts of the long bone were cut, and the bone marrow tissues were flushed out with Alpha-Minimum Essential Medium (α-MEM) (Gibco) using 26G needles until the bones turned whitish. The bone marrow tissue suspension was then filtered with a 100µm cell strainer. For transplantation, the bone marrow cells were resuspended in HBSS solution at a concentration of 1 × 10⁷ cells/200 µL. For cell culture, bone marrow cells were resuspended and cultured in α-MEM with 10% FBS (Gibco) and 1% anti-anti (Gibco) in a humidified incubator at 37℃, 5% CO₂. Improved Bone Marrow Collection (iBMC) For the improved method, we modified the BMC method by using an enzymatic α-MEM solution to flush out bone marrow tissues. Enzymatic α-MEM is made by dissolving 0.1% collagenase type 4 and 0.2% dispase in α-MEM. The long bone and the flushed-out bone marrow tissue in enzymatic alpha-MEM were incubated at 37°C for 10 minutes. The bone marrow tissue was then filtered and resuspended in a similar manner to BMC for both transplantation and culture. GFP chimeric mouse model 8-week-old wild-type female recipient mice underwent irradiation of 10Gy of total lethal whole-body irradiation to kill the bone marrow cells. Then, the collected bone marrow cells were transplanted into the irradiated recipient mice via the tail vein. The transplanted mice were kept for at least 14 days to ensure successful bone marrow engraftment and their survival. The success of the engraftment was confirmed again using GFP immunostaining of the bone marrow tissue of the chimeric mice after sacrifice. Orthotopic OSCC mouse models Tumour transplantation was performed 14 days after bone marrow transplantation (iBMC, n=5, BMC, n=3). MOC2 was injected into the right masseter muscle at a concentration of 3×10⁴ cells/50µL HBSS. All mice were euthanised 3 weeks after successful tumour transplantation, and the excised tissues were analysed. SHU9119 injection SHU9119 (Cayman Chemical) was dissolved in saline at a concentration of 50 µg/100 µL and stored at 4 °C. Tumours were implanted into iBMC mice as described above and allowed to settle for one week, after which they were divided into two groups: one group (n=5) that received 50µg/100µL of SHU9119 per iBMC mouse intraperitoneally, and a control group (n=5) that received 100µL of saline per iBMC mouse intraperitoneally. After daily intraperitoneal administration for 2 weeks, all mice were euthanised, and the excised tissues were analysed. Tissue processing for histological examination For the preparation of formalin-fixed paraffin-embedded sections, tumour tissues, organs, and bones of mice were fixed in 4% paraformaldehyde. The bone was demineralised in 10% EDTA at 4 °C for 14 days. Samples were then dehydrated in 70% ethanol, gradually increasing up to 100%. Xylene was used as a clearing agent before being embedded in paraffin. Serial sections of 5µm were prepared. Sections were subjected to hematoxylin-eosin staining before tissue analysis. Immunohistochemistry staining (IHC) IHC was performed using the antibodies described in Supplemental Table S1. Sections were deparaffinised in xylene for 15 min, rehydrated in a graded ethanol solution. The sections were blocked with a 1.2% hydrogen peroxide/methanol solution to inhibit endogenous peroxidase activity. Antigen retrieval was done according to the manufacturer's instructions. After antigen retrieval, the sections are blocked with 10% normal serum for 15 minutes at room temperature and then incubated with primary antibodies overnight at 4 °C. Signals were enhanced by the avidin-biotin complex method (Vector Lab). Colour development was performed with DAB (Histofine DAB substrate), and sections were counterstained with Myer's hematoxylin. Staining results were observed under an optical microscope (BX53, Olympus). Double and triple fluorescent IHC After the antigen retrieval, the sections are blocked using Block Ace (DS Pharma Biomedical) for 20 minutes at RT. The sections were incubated with primary antibodies at 4°C overnight. After washing with TBS, secondary antibodies were diluted 100-fold and reacted for 60 minutes at RT. Secondary antibodies were described in Supplemental Table S2. After reaction, sections were stained with DAPI. Staining results were observed with an All-in-One BZ x700 Fluorescence Microscope (Keyence). Quantification and statistical analysis Quantification was performed in the tumour-implanted right masseter muscle or lung metastases, and the average value was determined for five images taken at uniform magnification for each analysis at 4x, 10x, 20x, or 40x magnification per mouse. Counts and areas were measured using ImageJ (v1.52a). Statistical analysis was then performed using GraphPad Prism 9.1.1. Two-tailed Student's t test with independent samples and equal variance was used for the comparison of the two groups, and One-way ANOVA was used for the comparison of the three groups. Differences were considered significant at two-sided p values of < 0.05. Data are presented as mean ± SEM. Declarations ACKNOWLEDGEMENTS We thank the teachers, professors, and colleagues from Okayama University who provided valuable advice and engaged in scientific discussions throughout the process. AUTHOR CONTRIBUTIONS HK, HSE, SS, KN, and HN designed and conceptualised the study, conceived the experiments and analysed the data. HSE, SS, YS, TP and ZZM carried out experiments. HSE, HK and KT designed the chimeric model experiments. HSE, HK, SS and YS analysed tissue samples. HSE and HK wrote the original draft of the manuscript. HK, KT, KN, SI, and HN acquired the funding and supervised the study. DATA AVAILABILITY STATEMENT T he data analysed during the current study are available from the corresponding author on reasonable request. FUNDING STATEMENT This research is funded by JSPS KAKENHI (grant numbers JP23K09080, JP23K09332, JP23K09397, JP24K02644, JP24K13088, JP24K13130, JP22KK0275, and JP25KF0061). CONFLICT OF INTEREST DISCLOSURE The authors have declared that no conflict of interest exists. ETHICS APPROVAL STATEMENT All procedures performed in studies involving animals were in accordance with the ethical standards of the Okayama University Care and Use of Laboratory Animals guidelines and were approved by the Ethics of Animal Experiments Committee of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (OKU2023534, OKU2024132). We stated that the protocol adheres to the ARRIVE guidelines for reporting animal experiments. References Tang, J. et al. The role of mesenchymal stem cells in cancer and prospects for their use in cancer therapeutics. MedComm (Beijing). 5 (8), e663 (2024). Klimczak, A. & Kozlowska, U. 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Carcinoma-Associated Fibroblast–Like Differentiation of Human Mesenchymal Stem Cells. Cancer Res. 68 (11), 4331–4339 (2008). Wang, H. et al. Characteristics of pre-metastatic niche: the landscape of molecular and cellular pathways. Molecular Biomedicine. 2 (1), 3 (2021). Han, Y. et al. The secretion profile of mesenchymal stem cells and potential applications in treating human diseases. Signal Transduct Target Ther. 7 (1), 92 (2022). Wang, Y. et al. Pre-metastatic niche: formation, characteristics and therapeutic implication. Signal Transduct Target Ther. 9 (1), 236 (2024). Sutton, G. M., Josephine Babin, M., Gu, X., Hruby, V. J. & Butler, A. A. A derivative of the melanocortin receptor antagonist SHU9119 (PG932) increases food intake when administered peripherally. Peptides (N.Y.). 29 (1), 104–111 (2008). Bassi, M. et al. Activation of the brain melanocortin system is required for leptin‐induced modulation of chemorespiratory function. Acta Physiologica. 213 (4), 893–901 (2015). Omoto, A. C. M. et al. Targeting the Brain Leptin-Melanocortin Pathway to Treat Heart Failure. Curr Hypertens Rep. 27 (1), 2 (2025). Catania, A., Gatti, S., Colombo, G. & Lipton, J. M. Targeting Melanocortin Receptors as a Novel Strategy to Control Inflammation. Pharmacol Rev. 56 (1), 1–29 (2004). Additional Declarations No competing interests reported. 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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-8323433","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":581032485,"identity":"5628e803-10a6-4a3a-89bd-6d4f9054a3ce","order_by":0,"name":"Htoo Shwe Eain","email":"","orcid":"","institution":"Okayama University","correspondingAuthor":false,"prefix":"","firstName":"Htoo","middleName":"Shwe","lastName":"Eain","suffix":""},{"id":581032486,"identity":"6c1c6f97-ee38-435e-b232-6b61c8a6ad40","order_by":1,"name":"Hotaka 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20:29:15","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":95842758,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTALFIGURES1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8323433/v1/f9729715ad0569dff4119832.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metastatic promoting role of Mesenchymal Stem Cells in Oral Squamous Cell Carcinoma Revealed by an Improved Bone Marrow Chimeric Model","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMesenchymal stem cells (MSCs) are multipotent stromal cells that play crucial roles in maintaining tissue homeostasis and promoting regeneration. They migrate to the site of injury and inflammation, where they contribute to repair through stromal support, endothelial differentiation, context-dependent immunomodulation, and the maintenance of balanced inflammation [1–4]. The tumour microenvironment (TME) resembles a chronically inflamed ulcer, often described as \"a wound that never heals\" [5]. Within this pathological context, MSCs are recruited to tumours, where they adapt to the TME and contribute to stromal remodelling, angiogenesis, and immune modulation [3,6,7].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe multifaceted nature of MSCs suggests that they may be the key stromal component orchestrating the structural and functional dynamics of the TME [7]. However, studying the contribution of MSCs to tumour progression remains challenging due to limitations in experimental models. Conventional approaches rely on the isolation of bone marrow-derived MSCs (BM-MSCs) from femoral tissue, yet the yield of functional MSCs from this source is typically low [8]. Furthermore, in vitro expansion alters their phenotypes and accelerates cellular senescence, leading to inconsistent biological responses [9]. These issues have hindered the establishment of physiologically relevant mouse models that enable the functional tracing of endogenous MSCs within tumours.\u003c/p\u003e\n\u003cp\u003eTo overcome these challenges, we optimised an enzyme-treated bone marrow collection protocol that markedly enhances BM-MSC recovery while preserving their native properties [10]. In our previous study, enzymatic treatment during bone marrow collection increased MSC yield compared with conventional non-enzyme-treated methods, resulting in osteoblast differentiation and improved bone marrow formation in fracture models [10]. Based on this approach, we developed two distinct bone marrow-derived chimeric mouse models differing in MSC content but otherwise comparable in hematopoietic composition. These models enable investigation of MSC-specific effects without altering other bone marrow-derived cell populations. Moreover, since MSCs are directly transplanted from donor mice without culture, this can avoid complications such as in vitro ageing. In contrast to studies using localized MSC injections, which fail to mimic the natural recruitment of MSCs to the tumour, our model provides a physiologically similar condition to study endogenous MSC behaviour under systemic homeostasis [11].\u003c/p\u003e\n\u003cp\u003eIn this comprehensive study, we investigated how bone marrow-derived MSCs influence tumour vascularisation, inflammatory activation, and metastasis in oral squamous cell carcinoma (OSCC). Using a GFP bone marrow-chimeric mouse model with varying MSC content, we tracked the dynamics of BM-MSCs and assessed their contributions to immune cell infiltration, vascular development, stromal remodelling, and metastatic dissemination. Furthermore, we examined the inflammatory activation of endothelial cells, focusing on TNF-α–mediated signalling, and evaluated the effects of pharmacological modulation of MSC-associated pathways on metastatic outcomes. Collectively, our findings provide insights into the diverse roles of BM-MSCs in tumour progression and highlight their significance as potential therapeutic targets in OSCC.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eEnzymatic treatment of the femur yields a higher number of mesenchymal stem cells (MSCs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe enzymatically treated bone marrow collection was named iBMC (improved bone marrow collection) and the conventionally collected bone marrow was termed BMC (bone marrow collection).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo compare the efficiency of MSC recovery between the two methods, bone marrow cells obtained from iBMC, and BMC were seeded and cultured. The spindle-shaped adherent MSCs were quantified (Fig. 1A). The iBMC method yielded a higher population of spindle-shaped MSCs compared to BMC (Fig. 1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo establish chimeric models, lethally irradiated C57BL/6J mice were transplanted with the collected bone marrow cells via tail vein injection [10].\u003c/p\u003e\n\u003cp\u003eThe success of bone marrow engraftment was confirmed by GFP immunostaining in the bone marrow tissues of wild-type mice (control), iBMC, and BMC mice. GFP\u003csup\u003e+\u003c/sup\u003e cells are clearly detected in the bone marrow tissues in iBMC and BMC groups, confirming successful transplantation (Fig.1C). In addition, we found megakaryocytes in iBMC and wild-type mice (Fig.1C, arrows), but they were absent in BMC, suggesting impaired megakaryopoiesis due to dysfunction or lack of MSCs [12].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo analyse the abundance of MSCs in bone marrow, bone marrow tissues were immunostained with MSC markers, LepR and SDF-1 (Fig. 1D, F). BMC group exhibited a markedly lower number of LepR\u003csup\u003e+\u003c/sup\u003e cells and SDF-1\u003csup\u003e+\u003c/sup\u003e cells compared with wild-type and iBMC groups, confirming that enzymatic treatment increases MSC content in the bone marrow (Fig. 1F, G).\u003c/p\u003e\n\u003cp\u003eThese results indicate that enzymatic digestion during bone marrow harvest significantly enhances MSC yield, and that transplanted bone marrow cells successfully reconstitute the bone marrow compartment, generating GFP\u003csup\u003e+\u003c/sup\u003e bone marrow (BM) chimeric mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMSCs influence the infiltration of bone marrow-derived cells (BMDCs) and immune cells into the tumours.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate how MSC abundance affects the TME, GFP BM chimeric mice made from the iBMC, and BMC bone marrow tissue were utilised. Following a two-week recovery period post-transplantation, mouse oral squamous cell line 2 (MOC2) was inoculated into the buccal mucosa of iBMC and BMC mice for 3 weeks (Fig. 2A).\u003c/p\u003e\n\u003cp\u003eGFP immunostaining confirmed infiltration of BMDCs into tumour tissues (Fig. S2A). The iBMC group showed fewer GFP\u003csup\u003e+\u003c/sup\u003e cells compared with the BMC tumours (Fig. S2B), suggesting differences in BMDC recruitment dynamics.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo characterise the immune microenvironment of iBMC and BMC tumours, we compared and studied the immune populations in tumours. We first analysed the T cell population using CD4 and CD8 markers, the B cell population using the CD20 marker, and the monocyte and myeloid-derived suppressor cell (MDSC) using CD11b and Gr-1 markers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunostaining revealed that BMC tumours contained significantly fewer CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells and CD20\u003csup\u003e+\u003c/sup\u003e B cells than wild-type and iBMC tumours (Fig. 2C, D). Although not statistically significant, CD4\u003csup\u003e+\u003c/sup\u003e helper T cells were also reduced in BMC tumours (Fig. 2B). In contrast, CD11b single-marker staining showed a significant decrease in both BMC and iBMC tumours (Fig. 2E), while BMC tumours showed an increase in Gr-1\u003csup\u003e+\u003c/sup\u003e cells among the three groups (Fig. 2F). However, double immunofluorescence staining for CD11b\u003csup\u003e+\u003c/sup\u003eGr-1\u003csup\u003e+\u003c/sup\u003e confirmed the MDSC population in BMC tumours was more abundant than that in iBMC tumours (Fig. 2G, H).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results show that MSC deficiency alters the differentiation and infiltration patterns of BMDCs, and iBMC models can more accurately replicate the immune landscape of wild-type tumours while maintaining traceable GFP\u003csup\u003e+\u003c/sup\u003e BMDCs within the TME.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMSCs contribute to tumour vascularisation and maintain the vascular integrity of tumours.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMSCs are multipotent and can differentiate into the endothelial cell or pericytes, aiding in angiogenesis and vascular stabilisation [13].\u003c/p\u003e\n\u003cp\u003eTo evaluate tumour vascular characteristics, we compared the necrosis areas and bleeding areas in wild-type, iBMC, and BMC tumours (Fig. 3A, C; red insets). Quantitative analysis revealed that both necrotic and bleeding area-to-total tumour ratios were significantly increased in the BMC tumours (Fig. 3B, D), indicating a higher central necrosis and poorer structural integrity. Histopathological analysis also reveals fewer vessel-like structures within the BMC tumours compared to iBMC tumours (Fig. 3C; yellow insets).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results indicate that a lower number of MSCs compromises tumour vascularisation, leading to necrosis and vascular fragility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMature vessels, activated vessels, and cancer-associated fibroblasts (CAFs) are recruited into the TME via an MSC-dependent manner.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the vascular and stromal components formed under different MSC conditions, we used CD34 as a marker for mature vessels, CD105 as a marker for activated vessels, and α-SMA as a marker for CAFs (Fig. 4A, D, G). Double immunofluorescence with GFP confirmed that a subset of these vessels and fibroblasts originated from the bone marrow-derived cells (Fig. 4B, E, H). Quantification revealed significantly higher numbers of CD34\u003csup\u003e+\u003c/sup\u003e vessels, CD105\u003csup\u003e+\u003c/sup\u003e vessels, and α-SMA\u003csup\u003e+\u003c/sup\u003e CAFs in iBMC tumours compared to BMC tumours (Fig. 4C, F, I).\u003c/p\u003e\n\u003cp\u003eThese results demonstrate that bone marrow-derived MSCs contribute to vascular and stromal formation within the TME, and that an adequate MSC population is essential for establishing functional and organised tumour vasculature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBone marrow-derived MSCs promote lung metastasis of oral squamous cell carcinoma (OSCC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe most common metastasis sites for OSCC are the cervical lymph nodes and lungs. OSCC spreads to the lungs via hematogenous metastasis, and our previous analyses showed distinct vascular changes between iBMC and BMC tumours. We next examined lung metastasis (Fig. 5A). The number of metastatic islands was significantly lower in BMC groups than in wild-type and iBMC tumours (Fig. 5B), indicating that MSCs enhance metastatic dissemination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBMDCs are known to release inflammatory cytokines that promote endothelial activation, vascular leakage, and metastasis [13–16]. To study this mechanism, we performed double immunofluorescence staining using the bone marrow-derived marker GFP and the inflammatory marker TNF-α on the lung ECs (Fig. 5C, E). iBMC lung ECs showed higher GFP\u003csup\u003e+\u003c/sup\u003e cells and increased TNF-α expression on ECs compared to that of BMC lung ECs (Fig. 5D, F). Consistent results were observed again in triple fluorescence staining of GFP, TNF-α, and CD105 (endothelial marker), where a higher rate of co-localisation of GFP\u003csup\u003e+\u003c/sup\u003e and TNF-α\u003csup\u003e+\u003c/sup\u003e cells was seen on CD105\u003csup\u003e+\u003c/sup\u003e vascular structures in the iBMC group compared to the BMC group (Fig. 5G, H).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results suggest that an increase in MSCs promotes lung metastasis, accompanied by the recruitment of BMDCs and the inflammatory activation of lung endothelium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisturbance of MSC signalling further enhances metastasis via inflammatory activation of lung endothelium.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether disrupting MSC signalling affects metastatic behaviour, we administered SHU9119, a synthetic peptide antagonist of melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R), which mediates leptin-leptin receptor (Lep-LepR) signalling axis. MC3R and MC4R have anti-inflammatory properties, and SHU9119 antagonism promotes the inflammation and disturbs the effects of the leptin-leptin receptor axis by effectively inhibiting MC4R [17,18]. epR\u003csup\u003e+\u003c/sup\u003e MSCs constitute the major population of bone marrow-derived MSCs [19].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSHU9119 was administered daily to the iBMC mice one week after tumour transplantation to disturb LepR function in MSCs. Lung metastasis was quantified in the SHU9119-treated iBMC group, named iBMC (SHU), and saline-injected control, named iBMC (Saline) (Fig. 6B). The SHU9119-treated group displayed a significantly higher number of lung metastatic colonies (Fig. 6C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further assess endothelial inflammation by LepR disruption, we repeated the GFP and TNF-α staining in lung tissues (Fig. 6D, 6F). Interestingly, SHU9119 injection further promotes the recruitment of BMDCs and the expression of TNF-α in lung ECs (Fig. 6E, 6G). Similarly, triple staining for\u0026nbsp;GFP, TNF-α, and CD105 revealed accumulation of GFP\u003csup\u003e+\u003c/sup\u003eTNF-α\u003csup\u003e+\u003c/sup\u003e cells on CD105\u003csup\u003e+\u003c/sup\u003e vascular structures in the lungs following SHU9119 treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, these results demonstrate that disturbing the LepR\u003csup\u003e+\u003c/sup\u003e MSC signalling in the bone marrow induces a proinflammatory environment that promotes lung metastasis through the recruitment of BMDCs and the promotion of lung vessel inflammation. Thus, MSC integrity is critical for maintaining vascular homeostasis, and its disturbance accelerates metastatic progression in OSCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn our previous work, we established that enzymatic digestion of bone marrow tissue significantly improves MSC recovery, as confirmed by increased LepR\u003csup\u003e+\u003c/sup\u003e and SDF-1\u003csup\u003e+\u003c/sup\u003e stromal cell yield, higher osteoblast recruitment, and enhanced bone formation in the mice transplanted with enzyme-treated bone marrow collection (iBMC) compared with those receiving conventional bone marrow collection (BMC) [10]. Comparative histological analysis showed a lack of megakaryocytes in BMC mice, consistent with a reduced MSC population, as megakaryopoiesis is known to require both HSC- and MSC-derived signals [12]. Together, these results indicate that conventional bone marrow collection impairs MSC retention, leading to models that incompletely reflect the physiological contribution of MSCs to tissue and tumour biology.\u003c/p\u003e\n\u003cp\u003eGFP bone marrow transplantation models are widely used to trace BMDCs in vivo, particularly in studies of tumour biology and tissue regeneration [20–22]. However, our findings highlight a key limitation of traditional methods: 'suboptimal MSC recovery', which compromises model fidelity. iBMC models preserve the physiological diversity of BMDCs while enhancing the representation of MSCs. Importantly, iBMC models resemble wild-type mice without bone marrow transplantation in terms of bone marrow status, immune microenvironment, and tumour histological characteristics, making them more suitable for tracing and studying the BM-MSCs while reflecting normal physiological conditions. On the other hand, our control MSC-deficient BMC mice remain viable, allowing them to serve as appropriate controls for dissecting MSC-specific functions under otherwise equivalent hematopoietic conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUsing these models, we were able to investigate the tumour microenvironment with a systemic approach by studying the influence of MSCs on immune aspects, vascular characteristics, the source of tumour vessels, and metastatic progression. MOC2 tumours, a poorly differentiated OSCC tumour model transplanted into the immunocompetent C57BL/6 mice, allowed a comprehensive evaluation of immune dynamics [23]. Systemic comparison revealed that MSC abundance shapes the immune microenvironment of OSCC. MSC-deficient BMC tumours exhibited enhanced infiltration of immature myeloid-derived suppressor cells (MDSCs), whereas MSC-rich iBMC tumours displayed increased infiltration of mature adaptive immune cells. This suggests that MSCs regulate BMDC differentiation following their recruitment to tumours, through direct contact and soluble mediators such as SDF-1, TGF-β, and IL-6 [24]. Consistent with previous studies, MSCs orchestrate immune responses in a context-dependent manner, capable of promoting or suppressing immunity depending on the surrounding cytokine milieu [25,26]. Whether this regulation translates into protective immunity or tumour tolerance may depend on the broader stromal and vascular context, underscoring the complex immunoregulatory role of MSCs in OSCC TME.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVascularisation and stromal remodelling are indispensable for tumour growth, survival, and dissemination [27,28]. Although BMC and iBMC tumours exhibited necrotic cores, necrosis was significantly more extensive in BMC tumours. Necrosis in a solid tumour is typically attributed to hypoxia and impaired blood supply, which suggests the role of MSCs in angiogenesis [13]. Consistent with this hypothesis, BMC tumours exhibited a reduced number of CD34\u003csup\u003e+\u003c/sup\u003e endothelial cells, CD105\u003csup\u003e+\u003c/sup\u003e active endothelial cells, and\u0026nbsp;α-SMA\u003csup\u003e+\u003c/sup\u003e CAFs compared to iBMC tumours. Double immunofluorescence with GFP revealed that subsets of these stromal cells originated from the bone marrow.\u0026nbsp;These data confirm that BM-MSCs mobilised into the tumours, where they differentiate into stromal components that support angiogenesis and tumour progression. The hemorrhagic regions observed in the BMC tumour further suggest that defective MSC-mediated vascularisation leads to fragile and dysfunctional vessels. Together, these data indicate that MSCs are essential not only for maintaining vascular integrity but also for establishing a stromal milieu that supports tumour expansion [27,29].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOrthotopic implantation of MOC2 tumour also replicated the clinical metastatic pattern of OSCC, with cervical lymph node and lung metastases. Interestingly, metastatic pulmonary spread was observed only in tumours with high MSC content. BMC tumours that lack MSCs rarely produce lung metastases, underscoring the critical contribution of MSCs to hematogenous dissemination. The underlying mechanisms are likely multifactorial. By differentiating into CAFs, they remodel the TME and secrete pro-invasive factors, while MSC-derived endothelial cells enhance angiogenesis and facilitate tumour intravasation [13,15,30]. BM-MSCs themselves can also secrete cytokines and chemokines that prepare pre-metastatic niches, priming sites such as the lungs to be more receptive to colonisation by circulating tumour cells [31–33]. Consistent with these observations,\u0026nbsp;lung endothelial cells of iBMC tumours showed stronger GFP positivity, indicating bone marrow origin, and displayed elevated TNF-α expression on their surface, suggesting inflammatory activation. Triple immunofluorescence further revealed that MSC-derived endothelial cells undergo inflammatory activation, likely increasing vascular permeability and adhesion, thereby facilitating colonisation of circulating tumour cells in the lungs.\u003c/p\u003e\n\u003cp\u003eTo explore therapeutic modulation, we targeted MSC signalling using SHU9119, a synthetic peptide antagonist of the melanocortin 3 and 4 receptors (MC3R and MC4R), which disrupts the leptin-leptin receptor axis of MSCs [34–36]. Interestingly, administration of SHU9119 did not suppress lung metastasis as anticipated. Instead, it led to an increase in lung metastases. This paradox may be explained by the proinflammatory consequences of disrupting MSC signalling through MC4R antagonism, as MC4R inhibition skews the immune environment towards a proinflammatory state [37]. These results add a layer of complexity to the therapeutic use of MSCs and caution against manipulating the MSC-associated pathway in therapy.\u003c/p\u003e\n\u003cp\u003eThese observations establish iBMC as a superior approach for studying the systemic contribution of MSCs to tumour biology.\u003c/p\u003e\n\u003cp\u003eTaken together, our findings emphasised the multifaceted role of BM-MSCs in the progression of OSCC through their integrated effects on immune regulation, vascular remodelling, and metastatic dissemination. Our iBMC models provide a physiologically relevant platform that overcomes the limitation of conventional bone marrow transplantation by preserving MSC functionality and enabling systemic evaluation of their tumour-promoting or tumour-restraining roles. Therapeutic strategies targeting MSC pathways must therefore be approached with caution, as modulating these biologically complex and multifaceted cells can yield unexpected outcomes depending on context. Future study will focus on identifying the molecular cues that determine whether MSCs adopt tumour-promoting versus tumour-restraining roles, particularly through their crosstalk with inflammatory pathways. This comprehensive study can be critical for developing safe and effective therapeutic approaches that exploit the vulnerabilities of MSC-tumour interactions without compromising their essential roles in normal physiology.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003eMice\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e8-week-old female GFP transgenic mice [C57BL/6-Tg (CAG-EGFP)] and wild-type mice (C57BL/6J) were purchased from Charles River Laboratories. All animals were kept in optimal condition according to the experimental animal guidelines of Okayama University.\u0026nbsp;Mice were used for various in vivo experiments, including cell injection and tumour transplantation, as described in the respective sections.\u0026nbsp;Mice were euthanised following ARRIVE guidelines. Mice were anaesthetised with 5% isoflurane in an enclosed induction chamber and were observed until the complete loss of reflexes, confirming the state of deep anaesthesia. Only after that, cervical dislocation was performed as a secondary physical method to ensure death.\u003c/p\u003e\n\u003cp\u003eCell lines\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMOC2 cell line (KER-EWL002-FP) was purchased from Kerafast cell bank. These cells were grown in a 2:1 ratio of Iscove's Modified Dulbecco's Medium (IMDM) (Gibco) and Ham's Nutrient Mixture (F12) (Gibco), 5% FBS (Gibco), 1% anti-anti (Gibco), 5 mg/ mL insulin (Sigma Aldrich), 400 ng/mL hydrocortisone (Sigma Aldrich), and 5 ng/mL EGF (585508 Biolegend) in a humidified incubator at 37℃, 5% CO₂.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBone Marrow Collection (BMC)\u003c/p\u003e\n\u003cp\u003eGFP\u003csup\u003e+\u003c/sup\u003e bone marrow cells were collected from the femur and tibia of the 8-week-old GFP transgenic mice. The proximal parts of the long bone were cut, and the bone marrow tissues were flushed out with Alpha-Minimum Essential Medium (α-MEM) (Gibco) using 26G needles until the bones turned whitish. The bone marrow tissue suspension was then filtered with a 100µm cell strainer. For transplantation, the bone marrow cells were resuspended in HBSS solution at a concentration of 1 × 10⁷ cells/200 µL. For cell culture, bone marrow cells were resuspended and cultured in α-MEM with 10% FBS (Gibco) and 1% anti-anti (Gibco) in a humidified incubator at 37℃, 5% CO₂.\u003c/p\u003e\n\u003cp\u003eImproved Bone Marrow Collection (iBMC)\u003c/p\u003e\n\u003cp\u003eFor the improved method, we modified the BMC method by using an enzymatic α-MEM solution to flush out bone marrow tissues. Enzymatic α-MEM is made by dissolving 0.1% collagenase type 4 and 0.2% dispase in α-MEM. The long bone and the flushed-out bone marrow tissue in enzymatic alpha-MEM were incubated at 37°C for 10 minutes. The bone marrow tissue was then filtered and resuspended in a similar manner to BMC for both transplantation and culture.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGFP chimeric mouse model\u003c/p\u003e\n\u003cp\u003e8-week-old wild-type female recipient mice underwent irradiation of 10Gy of total lethal whole-body irradiation to kill the bone marrow cells. Then, the collected bone marrow cells were transplanted into the irradiated recipient mice via the tail vein. The transplanted mice were kept for at least 14 days to ensure successful bone marrow engraftment and their survival. The success of the engraftment was confirmed again using GFP immunostaining of the bone marrow tissue of the chimeric mice after sacrifice.\u003c/p\u003e\n\u003cp\u003eOrthotopic OSCC mouse models\u003c/p\u003e\n\u003cp\u003eTumour transplantation was performed 14 days after bone marrow transplantation (iBMC, n=5, BMC, n=3). MOC2 was injected into the right masseter muscle at a concentration of 3×10⁴ cells/50µL HBSS. All mice were euthanised 3 weeks after successful tumour transplantation, and the excised tissues were analysed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSHU9119 injection\u003c/p\u003e\n\u003cp\u003eSHU9119 (Cayman Chemical) was dissolved in saline at a concentration of 50 µg/100 µL and stored at 4 °C. Tumours were implanted into iBMC mice as described above and allowed to settle for one week, after which they were divided into two groups: one group (n=5) that received 50µg/100µL of SHU9119 per iBMC mouse intraperitoneally, and a control group (n=5) that received 100µL of saline per iBMC mouse intraperitoneally. After daily intraperitoneal administration for 2 weeks, all mice were euthanised, and the excised tissues were analysed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTissue processing for histological examination \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the preparation of formalin-fixed paraffin-embedded sections, tumour tissues, organs, and bones of mice were fixed in 4% paraformaldehyde. The bone was demineralised in 10% EDTA at 4 °C for 14 days. Samples were then dehydrated in 70% ethanol, gradually increasing up to 100%. Xylene was used as a clearing agent before being embedded in paraffin. Serial sections of 5µm were prepared. Sections were subjected to hematoxylin-eosin staining before tissue analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry staining (IHC)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIHC was performed using the antibodies described in Supplemental Table S1. Sections were deparaffinised in xylene for 15 min, rehydrated in a graded ethanol solution. The sections were blocked with a 1.2% hydrogen peroxide/methanol solution to inhibit endogenous peroxidase activity. Antigen retrieval was done according to the manufacturer's instructions. After antigen retrieval, the sections are blocked with 10% normal serum for 15 minutes at room temperature and then incubated with primary antibodies overnight at 4 °C. Signals were enhanced by the avidin-biotin complex method (Vector Lab). Colour development was performed with DAB (Histofine DAB substrate), and sections were counterstained with Myer's hematoxylin. Staining results were observed under an optical microscope (BX53, Olympus).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDouble and triple fluorescent IHC\u003c/p\u003e\n\u003cp\u003eAfter the antigen retrieval, the sections are blocked using Block Ace (DS Pharma Biomedical) for 20 minutes at RT. The sections were incubated with primary antibodies at 4°C overnight. After washing with TBS, secondary antibodies were diluted 100-fold and reacted for 60 minutes at RT. Secondary antibodies were described in Supplemental Table S2. After reaction, sections were stained with DAPI. Staining results were observed with an All-in-One BZ x700 Fluorescence Microscope (Keyence).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQuantification and statistical analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQuantification was performed in the tumour-implanted right masseter muscle or lung metastases, and the average value was determined for five images taken at uniform magnification for each analysis at 4x, 10x, 20x, or 40x magnification per mouse.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCounts and areas were measured using ImageJ (v1.52a). Statistical analysis was then performed using GraphPad Prism 9.1.1. Two-tailed Student's t test with independent samples and equal variance was used for the comparison of the two groups, and One-way ANOVA was used for the comparison of the three groups. Differences were considered significant at two-sided p values of \u0026lt; 0.05. Data are presented as mean ± SEM.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the teachers, professors, and colleagues from Okayama University who provided valuable advice and engaged in scientific discussions throughout the process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHK, HSE, SS, KN, and HN designed and conceptualised the study, conceived the experiments and analysed the data. HSE, SS, YS, TP and ZZM carried out experiments. HSE, HK and KT designed the chimeric model experiments. HSE, HK, SS and YS analysed tissue samples. HSE and HK wrote the original draft of the manuscript. HK, KT, KN, SI, and HN acquired the funding and supervised the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003ehe data analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is funded by JSPS KAKENHI (grant numbers JP23K09080, JP23K09332, JP23K09397, JP24K02644, JP24K13088, JP24K13130, JP22KK0275, and JP25KF0061).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST DISCLOSURE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no conflict of interest exists.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving animals were in accordance with the ethical standards of the Okayama University Care and Use of Laboratory Animals guidelines and were approved by the Ethics of Animal Experiments Committee of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (OKU2023534, OKU2024132).\u0026nbsp;We stated that the protocol adheres to the ARRIVE guidelines for reporting animal experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTang, J. \u003cem\u003eet al.\u003c/em\u003e The role of mesenchymal stem cells in cancer and prospects for their use in cancer therapeutics. \u003cem\u003eMedComm (Beijing).\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e(8), e663 (2024).\u003c/li\u003e\n \u003cli\u003eKlimczak, A. \u0026amp; Kozlowska, U. 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Targeting Melanocortin Receptors as a Novel Strategy to Control Inflammation. \u003cem\u003ePharmacol Rev.\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e(1), 1–29 (2004).\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":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal stem cells (MSCs), Oral squamous cell carcinoma (OSCC), Bone marrow transplantation, Chimeric mouse models, Tumour microenvironment (TME). ","lastPublishedDoi":"10.21203/rs.3.rs-8323433/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8323433/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Mesenchymal stem cells (MSCs) are essential stromal regulators that coordinate tissue repair, angiogenesis, and immune balance. Within tumours, MSCs remodel the microenvironment and influence disease progression, yet their systemic contribution remains unclear due to the limited recovery of functional MSCs in conventional bone marrow transplantation models.\nHere, we developed an improved bone marrow collection (iBMC) method using enzymatic digestion, which markedly increases MSC yield while preserving their native phenotype. GFP bone marrow chimeric mice generated by iBMC and conventional transplantation displayed comparable hematopoietic reconstitution but differed in MSC abundance, enabling direct analysis of MSC-specific effects under physiological conditions.\nIn mouse models of oral squamous cell carcinoma (OSCC), MSCs profoundly affected tumour development. MSC-deficient tumours exhibited necrosis and infiltration of immature myeloid-derived suppressor cells (MDSCs), whereas MSC-rich tumours showed enhanced vascularisation, adaptive immune infiltration, and stromal remodelling. Notably, lung metastasis occurred only in MSC-rich mice, accompanied by bone marrow–derived endothelial activation and TNF-α–driven inflammation. Pharmacological inhibition of LepR signalling using SHU9119 unexpectedly increased metastasis, underscoring the dual, context-dependent functions of MSCs.\nThese findings establish iBMC as a reproducible and physiologically relevant model to study MSC-mediated regulation of tumour immunity, angiogenesis, and metastasis.","manuscriptTitle":"Metastatic promoting role of Mesenchymal Stem Cells in Oral Squamous Cell Carcinoma Revealed by an Improved Bone Marrow Chimeric Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 20:29:08","doi":"10.21203/rs.3.rs-8323433/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-24T06:10:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T18:15:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-10T21:36:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168154903008286451738673196868202099612","date":"2026-03-02T11:28:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185064096336801164162199654378649194459","date":"2026-02-23T18:27:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-11T01:47:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336647316882176402164613351120323654630","date":"2026-02-02T11:29:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"226213014498851930463132191132173520520","date":"2026-01-27T09:22:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-27T03:34:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-27T03:26:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-13T10:51:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-08T08:47:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-08T08:34:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"da269864-a3b4-4dce-9b64-b529d4a0f62a","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":61807550,"name":"Biological sciences/Cancer"},{"id":61807551,"name":"Biological sciences/Cell biology"},{"id":61807552,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-05-08T15:19:09+00:00","versionOfRecord":{"articleIdentity":"rs-8323433","link":"https://doi.org/10.1038/s41598-026-50837-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-05-03 15:58:20","publishedOnDateReadable":"May 3rd, 2026"},"versionCreatedAt":"2026-01-28 20:29:08","video":"","vorDoi":"10.1038/s41598-026-50837-z","vorDoiUrl":"https://doi.org/10.1038/s41598-026-50837-z","workflowStages":[]},"version":"v1","identity":"rs-8323433","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8323433","identity":"rs-8323433","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}