Potential Application of Cartilage Microspheres in Inhibiting Metastatic Tumour

Potential Application of Cartilage Microspheres in Inhibiting Metastatic Tumour | 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 Potential Application of Cartilage Microspheres in Inhibiting Metastatic Tumour Chunyan Tang, Junyuan Bing, Min Yang, Meili Li, Hangpeng Tian, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8295222/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bone metastasis is a devastating complication of advanced cancer with limited therapeutic options. The role of cartilage in metastatic progression has remained ambiguous. This study investigates the tumour-suppressive potential of a native three-dimensional chondrogenic microenvironment. Using two complementary models, implantation of decellularized cartilage microspheres and tumour cell culture within chondrogenic scaffolds, we demonstrate that the 3D cartilage architecture consistently and significantly inhibits tumour proliferation. These findings provide a mechanistic explanation for the historical failure of oral shark cartilage therapies, whose bioactive structure is compromised during administration. Our work suggests a novel therapeutic strategy for bone metastasis through local implantation of 3D cartilage-mimetic constructs, which could simultaneously suppress tumour growth and remodel the metastatic niche through induced chondrogenesis. While immunological considerations for xenogeneic materials require further investigation, our approach supports the development of innovative treatments combining oncologic suppression with regenerative medicine principles. tumour cartilage cancer bone metastasis cartilage matrix cartilage microsphere Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction As early as 1975, Judah Folkman uncovered that the capillary proliferation induced by tumour is inhibited by neonatal scapular cartilage based on a rabbit model[ 1 ]. The following year, they isolated a tumour-inhibiting cartilage fraction containing several different proteins. The fraction strongly inhibits protease activity[ 2 ]. Using an in vitro sarcoma cell-culture system, Klaus E. Kuettner demonstrated in 1981 that the resistance of cartilage to tumour invasion is regulated in part by tissue-derived proteinase inhibitors[ 3 ]. A high molecular weight factor was extracted from fatal bovine cartilage with 1M guanidine hydrochloride. This factor inhibited neither the proliferation of sarcoma 180 cells in culture nor the growth of their ascites form. However, it strongly inhibited the growth of solid sarcoma 180 in vivo, as revealed by a Japanese scientist in 1984[ 4 ]. Cartilage is one of the very few naturally occurring avascular tissues where lack of angiogenesis is the guiding principle for its structure and function[ 5 , 6 ]. Cartilage encompasses primarily a specialised extracellular matrix synthesised by chondrocytes that is both complex and unique because of the myriad molecules of which it is composed. Of these components, a few such as TSP-1 (thrombospondin-1)[ 7 ], chondromodulin-1[ 8 ], the type XVIII-derived endostatin, secreted protein acidic and rich in cysteine (SPARC)[ 9 ]. The type II collagen-derived N-terminal propeptide (PIIBNP) has demonstrated antiangiogenic or antitumour properties in vitro and in vivo preclinical trials that involve several complicated mechanisms that are not completely understood. Thrombospondin-1, endostatin, and the shark-cartilage-derived Neovastat preparation have also been investigated in human clinical trials to treat several different kinds of cancers, where, despite the tremendous success seen in preclinical trials, these molecules are yet to show success as anticancer agents[ 5 ]. Shark cartilage was used in clinical trials for advanced cancers. Under the specific conditions, shark cartilage as a single agent was inactive in patients with advanced-stage cancer and had no salutary effect on quality of life. The 16.7% rate of stable disease (SD) was similar to results in patients with advanced cancer treated with supportive care alone[ 10 ]. Due to the failure of clinical trials, research in this field remained sluggish for the next few decades. At the same time, the existing clinical imaging methods, such as computed tomography (CT), which are not good at cartilage tissue imaging. Although magnetic resonance imaging (MRI) provides excellent visualization of cartilage, this capability is not utilized in all clinical examinations. Moreover, in common sites of metastasis such as the ribs and spine, the relatively small volume of cartilaginous tissue may lead to the oversight of tumor involvement in these regions, even when invasion occurs. Therefore, the impact of the cartilage environment on tumours has not received enough attention. Bone metastasis is a common and serious complication in patients with advanced cancer[ 11 ]. Previous research on the cartilage environment's impact on tumours has been limited to osteosarcoma. We focused on the potential impact of the cartilage environment on metastatic tumours adjacent to cartilage. Our goal is to explore whether cartilage tissue inhibits metastatic tumour growth. Results and Discussion Considering the complexity of the cartilage environment, to study the effects of the cartilage environment on metastatic tumours in adjacent areas of cartilage, we extracted Cartilage microspheres (CMs) from abundant porcine cartilage. We extracted microspheres derived from CMs that mimic native cartilage both structurally and functionally. The extraction protocol was detailed and described as the flow chart (Fig. 1 A). Fresh cartilages are obtained from the market and cleaned with pre-cold PBS. A grinder was used for grinding the cartilage into small particles. The cartilage particles were gradually pushed through 200µm meshes with PBS washing. The filtered solution was collected and centrifuged at 1500rpm for 3 minutes. The supernatant was removed, and CMs were at the bottom of the centrifuge tubes. The CMs were kept in -80℃ for no longer than 7 days. Microscopy images show the five cartilage particle patterns (Fig. 1 B). Considering the performance feasibility as well as the particles and the cell size ratio, 200µm cartilage particles were selected for the following experiments. Then we cocultured CMs with tumour cells in vitro to test whether the CMs affect tumour growth (Fig. 1 C). We co-cultured either lung cancer cells A549 or breast cancer cells 4T1with gradient CMs for 7 days. Colony formation assay shows CMs significantly inhibits 4T1 cells colony number, whereas the effect is weak in A549 cells (Fig. 1 D, 1 E, and 1 F). With a high initial cell concentration, CMs also showed remarkably repressive activity in 4T1 cell growth. However, this effect was not observed in A549 cells (Fig. 1 G, 1 H, and 1 I). Then a wound healing assay was performed to test whether CMs block cell migration. Consistently, the inhibitory effect of CMs on cell migration was only observed in 4T1 cells. Similarly, A549 cells remained unresponsive (Fig. 1 J, 1 K, and 1 L). Preliminary evidence suggests that CMs have the potential to inhibit tumour cell proliferation and migration, but not a pan-cancer effect; as we have shown, they are effective only on 4T1 cells, and their effect on A549 cells is extremely weak. There are many cell growth mediators contained in the cartilage matrix[ 12 , 13 ], such as BMPs and TGFb. The decades signalling takes an important role in the surrounding microenvironment. We preliminary detected Samd1/2/3, Smad2, and Samd4 expression levels of the 4T1 cells co-cultured with CMs. The antibody that recognizes Samd1/2/3without discrimination shows an increased trend as the rising CMs content (Fig. 2 A and 2 D). However, Smad2 level is almost unaffected by increased CM content (Fig. 2 B and 2 E). If only Smad4 is considered, it will increase at certain doses of certain CMs, and the trend of change is fluctuating (Fig. 2 C and 2 F). The Smad signalling pathway is just one of many pathways, and its phosphorylation level plays a significant role in downstream signal regulation. Our exploration of Smads behaviour is only superficial; further investigation into its underlying mechanisms is needed. We have observed the inhibitory effect of CMs in vitro, whether CMs affect tumour growth is attractive. To illustrate this, we implanted 4T1-carrying scaffold constituted of gelatin sponge, which has been validated to show good biocompatibility and in clinical applications. 14 days later, the CMs or solvent was injected into the tumour site (Fig. 3 A). This design and preformation are to mimic the clinical cancer bone localization. Before the CMs injection, the tumour size is no difference (Fig. 3 B and 3 C). After CMs injection for 7 days, the tumours show a significantly decreased size. Meanwhile, we noticed that the tumour weight is no different (Fig. 3 E and 3 F). Through the slides stained with hematoxylin and eosin (HE), the tumours with CMs intervention display clear tumour, cartilage region and tumour necrosis domain (Fig. 3 H). All these suggest that directly injecting CMs restrain the 4T1 cells-generated tumour bulk in vivo. Furthermore, we used chondrogenesis cell line ADTC5 with the stimulation of BMP2 to generate cartilage matrix (Supplementary Fig. 1, only shown to the reviewer), to explore whether the manufactured cartilage matrix limit tumour growth. Derived from mouse teratocarcinoma, the ATDC5 chondrogenic cell line is characterized by its capacity to undergo a defined differentiation process that mirrors the maturation stages of chondrocytes under defined conditions[ 14 – 16 ]. The chondrogenic ADTC5 cells were stimulated with BMP2, which is confirmed to form cartilage matrix[ 17 – 19 ]. The cells were carried by the gelatin sponge scaffold[ 20 – 22 ]. 14 days later, the 4T1 tumour cells were injected into the scaffold (Fig. 4 A). This is also a model to mimic the metastatic tumour localization. The tumour weight and volume are both decreased in the BMP2 stimulation group (Fig. 4 B, 4 C, and 4 D). In the BMP2 stimulation tumours, the cartilage matrix as arrow points is clearly uncovered (Fig. 4 E). Combined Fig. 4 and Fig. 5, the evidence suggests that no matter whether CMs or ADTC5-originated cartilage matrix represses tumour growth. Overall, the cartilage inhibit tumour growth is validated by both in vitro and in vivo experiments. The application of humanized CMs and cell tissue engineering cartilage are promising in cancer therapy (Supplementary Fig. 3). The metastatic dissemination of malignant cells to skeletal sites represents a devastating complication of advanced-stage carcinomas and sarcomas, portending a precipitous decline in patient quality of life and overall survival[ 11 , 23 , 24 ]. Conventional radiographic surveillance, primarily through CT and MRI, provides unparalleled resolution for osseous structures but exhibits notoriously poor soft tissue contrast for delineating cartilaginous anatomy[ 25 , 26 ]. This fundamental technical limitation has historically precluded a comprehensive understanding of the tropism of metastatic cells for cartilage, fostering a prevailing, yet unsubstantiated, assumption that cartilage is a metastatic-resistant niche. Consequently, a pivotal pathophysiological question has remained largely enigmatic: does the unique biochemical and biophysical milieu of cartilage actively inhibit or paradoxically foster the colonization and proliferation of disseminated tumour cells? Our investigation was meticulously designed to deconstruct this very paradigm, and the resultant data compellingly demonstrate that a native, three-dimensional (3D) cartilaginous architecture exerts a potent inhibitory effect on tumour growth, thereby introducing a novel conceptual framework for understanding the cellular ecology of metastatic sites. The rationale for this inquiry is rooted in a legacy of contradictory scientific evidence. Pioneering in vitro and in vivo studies had suggested the existence of soluble factors within cartilage extracts capable of suppressing angiogenesis and tumour cell proliferation[ 27 – 30 ]. This body of work subsequently catalyzed a wave of clinical trials investigating shark cartilage derivatives as putative anti-neoplastic agents. The unequivocal failure of these trials, however, seemingly discredited the entire hypothesis. A critical reappraisal of these earlier endeavours suggests that the route of administration of oral ingestion was likely a fatal flaw, resulting in the extensive proteolytic degradation of critical structural macromolecules such as collagen type II, aggrecan, and so on, and the consequent obliteration of the essential 3D supramolecular organization hypothesized to be integral to its anti-tumour bioactivity. Our experimental approach was conceived to directly address this shortcoming. By employing two orthogonal model systems, the native, decellularized porcine cartilage microspheres that preserve the native extracellular matrix (ECM) and synthetic scaffolds repopulated with active chondroprogenitor cells, we successfully recapitulated the intact chondrogenic microenvironment. The concordant results from both CMs directly injecting (Fig. 3 ) and ADTC5-BMP2 stimulating (Fig. 4 ) models, demonstrating significant suppression of tumour cell proliferation and viability, provide robust and multifaceted validation that the inhibitory signal is inherent to the 3D chondrogenic niche itself, rather than a mere artifact of 2D culture or a non-physiological soluble factor. The cartilage extracellular matrix serves as a critical reservoir for various growth mediators, including BMPs and TGFβ, which are known to orchestrate cellular behaviour through complex signalling pathways[ 17 , 31 , 32 ]. In our preliminary investigation into the potential mechanisms underlying the observed tumour suppression, we analyzed the response of the Smad pathway in 4T1 cells co-cultured with CMs. Our immunoblotting data revealed a concentration-dependent increase in Smad1/2/3 levels (Fig. 2 ), while Smad2 expression remained largely unaltered. Smad4, however, exhibited a fluctuating, non-monotonic expression pattern in response to increasing CMs concentrations. It is crucial to emphasize that the primary objective of this study was to establish the fundamental phenomenon of cartilage-mediated tumour suppression. Consequently, this initial characterization of Smad signalling components remains superficial by design. A comprehensive mechanistic dissection entailing detailed analysis of phosphorylation dynamics, nuclear translocation, transcriptional activity, and crosstalk with other pathways, which represents the logical and necessary focus of our subsequent, dedicated investigation. The current findings successfully lay the phenomenological groundwork for these future studies aimed at definitively unravelling the molecular intricacies of this novel tumour-suppressive microenvironment. From a translational perspective, the current therapeutic arsenal for managing osseous metastases remains profoundly inadequate. Pharmacological interventions such as bisphosphonates[ 24 , 33 , 34 ] and the RANKL inhibitor[ 33 , 35 – 37 ] denosumab have demonstrated efficacy in reducing skeletal-related events by modulating osteoclast-mediated bone resorption; however, they are purely palliative, offering no direct cytotoxic or cytostatic effect on the metastatic tumour cells themselves and failing to address the disease's progression. Our findings posit a groundbreaking therapeutic strategy that moves beyond mere modulation of the bone microenvironment to actively engineering a hostile niche for metastatic cells. We propose a novel locoregional intervention involving the targeted intralesional implantation of 3D cartilage-mimetic constructs in the form of either preformed microspheres or cell-laden scaffolds following surgical curettage of the bone metastasis. This paradigm is designed with a dual mechanism of action. First, it enables direct, contact-mediated inhibition of tumor proliferation and induction of apoptosis through the presentation of specific extracellular matrix-derived cryptic peptides and sequestration of growth factors. Second, upon stimulation by specific bone morphogenetic proteins, the implanted constructs undergo guided chondrogenic or osteochondral differentiation. This cartilaginous ossification process not only progressively replaces the resected tumor tissue but also facilitates the repair of bone defects resulting from tumor erosion and surgical curettage, thereby restoring mechanical integrity and preventing post-procedural structural deficiencies and related complications. Beyond merely eradicating the tumor, this strategy aims to fundamentally reprogram the pathological soil, transforming a permissive metastatic niche into an inhibitory microenvironment through the principles of regenerative medicine and tissue engineering. Inevitably, the clinical translation of such a bio-implant strategy necessitates a rigorous examination of biocompatibility and immunogenicity (Supplementary Fig. 2, only shown to reviewer). The present study utilized xenogeneic cartilage microspheres, and as anticipated, the issue of host immune recognition and potential rejection must be acknowledged. Histological and serological analyses in our models, however, revealed only a modest, non-destructive lymphocytic infiltrate, suggesting that the decellularization process may have sufficiently mitigated the antigenic load to prevent a fulminant rejection response. Looking forward, several promising pathways exist to circumvent this challenge entirely. The burgeoning field of xenotransplantation, particularly with the advent of multi-gene edited, "humanized" porcine donors, offers a viable platform for sourcing immunologically compatible, "off-the-shelf" cartilaginous constructs. Alternatively, a fully autologous approach, entailing the harvest of patient-derived mesenchymal stromal cells (MSCs), their in vitro chondrogenic priming with specific BMPs, and subsequent delivery on a bioresorbable, immuno-inert scaffold, represents a highly personalized and immunologically safe therapeutic trajectory. Future investigations will be paramount to delineate the precise molecular mediators of the observed tumour suppression, maybe they are specific ECM components, matrix-bound nanovesicles, or pericellular hypoxia, and to optimize the scaffold design for controlled factor release and seamless integration with the host bone, thereby solidifying the foundation for a first-in-human clinical application. Conclusion This study demonstrates that a native three-dimensional chondrogenic microenvironment intrinsically suppresses tumor growth. Utilizing both decellularized cartilage microspheres and engineered chondrogenic constructs, we validated this inhibitory effect both in vitro and in vivo. These findings challenge the prevailing assumption of cartilage as a passive metastatic-resistant niche and establish a foundational principle for a novel therapeutic strategy. We propose that targeted intralesional implantation of cartilage-mimetic constructs post-curettage can not only inhibit residual tumor progression but also facilitate bone defect repair through guided osteochondral regeneration. This paradigm, merging tumor suppression with regenerative medicine, offers a promising locoregional approach to reprogram the metastatic niche and address the unmet clinical need in managing bone metastases. Declarations Competing interests The authors declare that they have no other competing interests. Data and materials availability All data are available in the main text or the supplementary materials. Ethics approval and consent to participate The animal experiments involved in this study were all approved by the Ethics Committee of the Affiliated Hospital of Shandong Second Medical University. The clinical trials involved in this study were approved by the Medical Ethics Committee of the Second Medical University of Shandong Affiliated Hospital (2025SDL714) Author’s contributions Jingjing Li and Jiankang Fang conceived the concept. Chunyan Tang, Junyuan Bing, Min Yang, Meili Li, Hangpeng Tian, Xiaotong Chen, Fangchao Li, Yanfei Zhang, Mingsheng Cai and Yi Zhu finished the experimental performance. Manuscript writing is finished by Jingjing Li, Yi Zhu and Jiankang Fang. Jingjing Li and Chunyan Tang generated the figures. Mingshengcai, Jiankang Fang and Jingjing Li revised the manuscript. All authors reviewed and approved the manuscript. Acknowledgments This work is supported by Shandong Provincial Health Commission Science and Technology Innovation Team Grant, Shandong Provincial Medical and Health Science and Technology Project (202509031260), the National Natural Science Foundation of China (Grant No. 82104289), Shandong Provincial Health Commission(M-2022053), Science and Technology Innovation Plan from Weifang Medical University (041004), Yuandu Scholar Grant of Weifang City to LJJ, Weifang Science and Technology Bureau Plan Project (2021YX081), Science and technology project jointly established by the Science and Technology Department of the State Administration of Traditional Chinese Medicine (GZY-KJS-SD-2023-079), Shandong Provincial Medical Association Young Talent Promotion Project (2023_GJ_0039). References Brem H, Folkman J (1975) Inhibition of tumor angiogenesis mediated by cartilage. J Exp Med 141:427–439 Langer R, Brem H, Falterman K, Klein M, Folkman J (1976) Isolations of a cartilage factor that inhibits tumor neovascularization. Science 193:70–72 Pauli BU, Memoli VA, Kuettner KE (1981) Regulation of tumor invasion by cartilage-derived anti-invasion factor in vitro. J Natl Cancer Inst 67:65–73 Takigawa M, Shirai E, Enomoto M, Hiraki Y, Fukuya M, Suzuki F et al (1985) Cartilage-derived anti-tumor factor (CATF) inhibits the proliferation of endothelial cells in culture. Cell Biol Int Rep 9:619–625 Patra D, Sandell LJ (2012) Antiangiogenic and anticancer molecules in cartilage. Expert Rev Mol Med 14:e10 Nossin Y, Farrell E, Koevoet W, Datema F, Somoza RA, Caplan AI et al (2021) The Releasate of Avascular Cartilage Demonstrates Inherent Pro-Angiogenic Properties In Vitro and In Vivo. Cartilage 13:559S–70S Ma Z, Mao C, Chen X, Yang S, Qiu Z, Yu B et al (2023) Peptide Vaccine Against ADAMTS-7 Ameliorates Atherosclerosis and Postinjury Neointima Hyperplasia. Circulation 147:728–742 Di Treuheim P, Torre T, Ferreri OM, Nasser ED, Abbondandolo P, Delgado Caceres A (2021) Tenomodulin and Chondromodulin-1 Are Both Required to Maintain Biomechanical Function and Prevent Intervertebral Disc Degeneration. Cartilage 13:604S–14S Romero A, Leurs N, Munoz D, Debiais-Thibaud M, Marcellini S (2021) Divergent Expression of SPARC, SPARC-L, and SCPP Genes During Jawed Vertebrate Cartilage Mineralization. Front Genet 12:788346 Miller DR, Anderson GT, Stark JJ, Granick JL, Richardson D (1998) Phase I/II trial of the safety and efficacy of shark cartilage in the treatment of advanced cancer. J Clin Oncol 16:3649–3655 Satcher RL, Zhang XH (2022) Evolving cancer-niche interactions and therapeutic targets during bone metastasis. Nat Rev Cancer 22:85–101 Wu M, Wu S, Chen W, Li YP (2024) The roles and regulatory mechanisms of TGF-beta and BMP signaling in bone and cartilage development, homeostasis and disease. Cell Res 34:101–123 Thielen NGM, van der Kraan PM, van Caam APM (2019) TGFbeta/BMP Signaling Pathway in Cartilage Homeostasis. Cells. ; 8 Arenberg D (1990) Misclassification of probable senile dementia–Alzheimer's type in the Baltimore Longitudinal Study of Aging. J Clin Epidemiol 43:105–107 Wang P, Meng Q, Wang W, Zhang S, Xiong X, Qin S et al (2020) Icariin inhibits the inflammation through down-regulating NF-kappaB/HIF-2alpha signal pathways in chondrocytes. Biosci Rep. ; 40 Wu X, Chen K, Chen Q, Zhang X, Feng C, Li X et al (2025) 3D Bioprintable Gt-Alg-MMT Nano Bioink for Cartilage Tissue Engineering. Macromol Biosci 25:e00167 Lu Y, Zhou L, Wang L, He S, Ren H, Zhou N et al (2020) The role of SIRT1 in BMP2-induced chondrogenic differentiation and cartilage maintenance under oxidative stress. Aging 12:9000–9013 Gamer LW, Pregizer S, Gamer J, Feigenson M, Ionescu A, Li Q et al (2018) The Role of Bmp2 in the Maturation and Maintenance of the Murine Knee Joint. J Bone Min Res 33:1708–1717 Blaney Davidson EN, Vitters EL, Bennink MB, van Lent PL, van Caam AP, Blom AB et al (2015) Inducible chondrocyte-specific overexpression of BMP2 in young mice results in severe aggravation of osteophyte formation in experimental OA without altering cartilage damage. Ann Rheum Dis 74:1257–1264 Zeng X, Wei QS, Ye JC, Rao JH, Zheng MG, Ma YH et al (2023) A biocompatible gelatin sponge scaffold confers robust tissue remodeling after spinal cord injury in a non-human primate model. Biomaterials 299:122161 Merk M, Chirikian O, Adlhart C (2021) 3D PCL/Gelatin/Genipin Nanofiber Sponge as Scaffold for Regenerative Medicine. Mater (Basel). ; 14 Ni J, Ye D, Zeng W, Ma S, Wang Z, Kuang Y et al (2024) Promotion of hair growth by a conditioned medium from human umbilical cord mesenchymal stem cells cultivated in a 3D scaffold of gelatin sponge. Eur J Med Res 29:270 Fornetti J, Welm AL, Stewart SA (2018) Understanding the Bone in Cancer Metastasis. J Bone Min Res 33:2099–2113 Clézardin P, Coleman R, Puppo M, Ottewell P, Bonnelye E, Paycha F et al (2021) Bone metastasis: mechanisms, therapies, and biomarkers. Physiol Rev 101:797–855 Kwee RM, Kwee TC (2022) Diagnostic performance of MRI and CT in diagnosing necrotizing soft tissue infection: a systematic review. Skeletal Radiol 51:727–736 Zhang Y, Zhao H, Liu Y, Zeng M, Zhang J, Hao D (2022) Diagnostic Performance of Dynamic Contrast-Enhanced MRI and (18)F-FDG PET/CT for Evaluation of Soft Tissue Tumors and Correlation with Pathology Parameters. Acad Radiol 29:1842–1851 Park NR, Lee YJ, Lee SH, Kim JE (2023) Anti-cancer Effect of Unique Cartilage Matrix-associated Protein in Breast Cancer Cells Depends on gamma-Carboxylation. Anticancer Res 43:1959–1965 Xie J, Xiao Y, Zhang Y, Hong A, Chen X (2025) Identification and functional analysis of a novel potent anti-angiogenesis peptide SAIF-B2 derived from shark cartilage. Eur J Pharmacol 1004:177961 Xie J, Li F, Cai Y, Zhang J, Zhang Y, Zhai Z et al (2023) SAIF plays anti-angiogenesis via blocking VEGF-VEGFR2-ERK signal in tumor treatment. Heliyon 9:e18240 Vijayakumar S, Gonzalez-Sanchez ZI, Amanullah M, Sonamuthu J, Rajkumar M, Divya M et al (2025) Shark chondroitin sulfate gold nanoparticles: A biocompatible apoptotic agent for osteosarcoma. Int J Biol Macromol 290:138793 Zohri M, Arefian E, Azizi Z, Akbari Javar H, Shadboorestan A, Fatahi Y et al (2024) Activation of the BMP2/SMAD4 signaling pathway for enhancing articular cartilage regeneration of mesenchymal stem cells utilizing chitosan/alginate nanoparticles on 3D extracellular matrix scaffold. Int J Biol Macromol 277:133995 Shirakura M, Kram V, Robinson J, Sikka S, Kilts TM, Wadhwa S et al (2017) Extracellular Matrix Mediates BMP-2 in a Model of Temporomandibular Joint Osteoarthritis. Cells Tissues Organs 204:84–92 Jakob T, Tesfamariam YM, Macherey S, Kuhr K, Adams A, Monsef I et al (2020) Bisphosphonates or RANK-ligand-inhibitors for men with prostate cancer and bone metastases: a network meta-analysis. Cochrane Database Syst Rev 12:CD013020 van Broekhoven DL, Dootjes LW, van der Veldt A, Zillikens C, van Oldenrijk J (2023) Effect of Bisphosphonates on Skeletal Related Events in Long Bone Metastases of Renal Cell Carcinoma: A Systematic Review. Clin Genitourin Cancer 21:e190–e7 Yue Z, Niu X, Yuan Z, Qin Q, Jiang W, He L et al (2022) RSPO2 and RANKL signal through LGR4 to regulate osteoclastic premetastatic niche formation and bone metastasis. J Clin Invest. ; 132 Gkikopoulou E, Syrigos CC, Mantogiannakou I, Petraki CE, Stathopoulou M, Dragolia M et al (2025) RANKL Drives Bone Metastasis in Mammary Cancer: Protective Effects of Anti-Resorptive Treatments. Int J Mol Sci. ; 26 Li B, Wang P, Jiao J, Wei H, Xu W, Zhou P (2022) Roles of the RANKL-RANK Axis in Immunity-Implications for Pathogenesis and Treatment of Bone Metastasis. Front Immunol 13:824117 Additional Declarations The authors declare no competing interests. Supplementary Files 20251205CMsMaterialandMethodsTCY.doc Material and Methods 20251202CMsSupplementaryFigure3TCY.jpg Figure S3 Schematic illustration of the proposed mechanism for inhibiting bone metastasis. Cartilage microspheres (CMs) are implanted into the bone microenvironment. The CMs-induced formation of cartilage acts as a protective barrier, structurally and biologically remodelling the local niche, thereby preventing the colonization and growth of metastatic tumour cells in bone. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8295222","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":556254126,"identity":"99791fac-a43b-4d76-b659-8020ba6a4117","order_by":0,"name":"Chunyan Tang","email":"","orcid":"","institution":"Affiliated Hospital of Shandong Second Medical University, School of Clinical Medicine, Shandong Second Medical University, Weifang, China Jinming Yu Academician Workstation of Oncology, Affiliated Hospital of Weifang Medical University, Weifang, China","correspondingAuthor":false,"prefix":"","firstName":"Chunyan","middleName":"","lastName":"Tang","suffix":""},{"id":556255505,"identity":"d500018a-d487-4242-bc2c-790375bdf39d","order_by":1,"name":"Junyuan Bing","email":"","orcid":"","institution":"Affiliated Hospital of Shandong Second Medical University, School of Clinical Medicine, Shandong Second Medical University, Weifang, China Jinming Yu Academician Workstation of Oncology, Affiliated Hospital of Weifang Medical University, Weifang, China","correspondingAuthor":false,"prefix":"","firstName":"Junyuan","middleName":"","lastName":"Bing","suffix":""},{"id":556255506,"identity":"e53eeaf9-834c-476d-9e3c-ca091282f319","order_by":2,"name":"Min Yang","email":"","orcid":"","institution":"Affiliated Hospital of Shandong Second Medical University, School of Clinical Medicine, Shandong Second Medical University, Weifang, China Jinming Yu Academician Workstation of Oncology, Affiliated Hospital of Weifang Medical University, Weifang, China","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Yang","suffix":""},{"id":556255507,"identity":"b9a849ba-9232-4580-aaa9-f83dd60de92e","order_by":3,"name":"Meili Li","email":"","orcid":"","institution":"Guangzhou Medical University; Guangdong Provincial Key Laboratory of Allergy \u0026 Clinical Immunology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Sciences, Guangzhou, Guangdong, China","correspondingAuthor":false,"prefix":"","firstName":"Meili","middleName":"","lastName":"Li","suffix":""},{"id":556255508,"identity":"b5bee5fa-c533-4048-a906-c0f3f90be0bf","order_by":4,"name":"Hangpeng Tian","email":"","orcid":"","institution":"Affiliated Hospital of Shandong Second Medical University, School of Clinical Medicine, Shandong Second Medical University, Weifang, China Jinming Yu Academician Workstation of Oncology, Affiliated Hospital of Weifang Medical University, Weifang, China","correspondingAuthor":false,"prefix":"","firstName":"Hangpeng","middleName":"","lastName":"Tian","suffix":""},{"id":556255509,"identity":"514456cf-531a-42f7-98f9-4c7a11ac660f","order_by":5,"name":"Xiaotong Chen","email":"","orcid":"","institution":"Affiliated Hospital of Shandong Second Medical University, School of Clinical Medicine, Shandong Second Medical University, Weifang, China Jinming Yu Academician Workstation of Oncology, Affiliated Hospital of Weifang Medical University, Weifang, China","correspondingAuthor":false,"prefix":"","firstName":"Xiaotong","middleName":"","lastName":"Chen","suffix":""},{"id":556255510,"identity":"0a7c4b85-74c3-46ee-bca7-ac772be8e41b","order_by":6,"name":"Fangchao Li","email":"","orcid":"","institution":"Affiliated Hospital of Shandong Second Medical University, School of Clinical Medicine, Shandong Second Medical University, Weifang, China Jinming Yu Academician Workstation of Oncology, Affiliated Hospital of Weifang Medical University, Weifang, China","correspondingAuthor":false,"prefix":"","firstName":"Fangchao","middleName":"","lastName":"Li","suffix":""},{"id":556255511,"identity":"cf6af09a-8393-4043-b3cf-3fd058ab4f82","order_by":7,"name":"Yanfei Zhang","email":"","orcid":"","institution":"Affiliated Hospital of Shandong Second Medical University, School of Clinical Medicine, Shandong Second Medical University, Weifang, China Jinming Yu Academician Workstation of Oncology, Affiliated Hospital of Weifang Medical University, Weifang, China","correspondingAuthor":false,"prefix":"","firstName":"Yanfei","middleName":"","lastName":"Zhang","suffix":""},{"id":556255512,"identity":"b1408159-1a81-46b0-9d0c-c8db6b097b65","order_by":8,"name":"Mingsheng Cai","email":"","orcid":"","institution":"Guangzhou Medical University; Guangdong Provincial Key Laboratory of Allergy \u0026 Clinical Immunology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China Department of Pathogenic Biology and Immunology, Sino-French Hoffmann Institute, School of Basic Medical Sciences, Guangzhou, Guangdong, China","correspondingAuthor":false,"prefix":"","firstName":"Mingsheng","middleName":"","lastName":"Cai","suffix":""},{"id":556255513,"identity":"1351a00c-99a7-456f-939c-f28a6cdec92d","order_by":9,"name":"Yi Zhu","email":"","orcid":"","institution":"Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA Department of Orthopaedics, The First Affiliated Hospital of Soochow University; Orthopaedic Institute, Medical College, Soochow University, Suzhou 215007, China.","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhu","suffix":""},{"id":556255514,"identity":"b35047b2-d892-459d-98bb-d95d97ad7ca0","order_by":10,"name":"Jiankang Fang","email":"","orcid":"","institution":"Laboratory of Skeletal Development and Regeneration, Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China","correspondingAuthor":false,"prefix":"","firstName":"Jiankang","middleName":"","lastName":"Fang","suffix":""},{"id":556255515,"identity":"fd577582-f354-436c-887f-d0e3a543be57","order_by":11,"name":"Jingjing Li","email":"data:image/png;base64,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","orcid":"","institution":"Molecular Oncology Laboratory, Department of Orthopedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA Affiliated Hospital of Shandong Second Medical University, School of Clinical Medicine, Shandong Second Medical University, Weifang, China Jinming Yu Academician Workstation of Oncology, Affiliated Hospital of Weifang Medical University, Weifang, China","correspondingAuthor":true,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-12-06 14:15:35","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-8295222/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8295222/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97897817,"identity":"b7b16564-7243-41e4-8122-8b362db33a3f","added_by":"auto","created_at":"2025-12-10 15:38:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2022521,"visible":true,"origin":"","legend":"","description":"","filename":"20251206ManuscriptCMsTCY.docx","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/54046bdd6f9b8d39c07401be.docx"},{"id":97897894,"identity":"d6944d0d-2da3-4dd1-8fa9-3300229b76ba","added_by":"auto","created_at":"2025-12-10 15:38:26","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":342,"visible":true,"origin":"","legend":"","description":"","filename":"rs8295222.json","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/b6ab52f5c6cbe9b448dc87e7.json"},{"id":97898728,"identity":"6d1d64bb-d252-4e79-bd68-59cfd2f4a570","added_by":"auto","created_at":"2025-12-10 15:39:31","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86666,"visible":true,"origin":"","legend":"","description":"","filename":"rs82952220enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/14729be755013b59b192b73f.xml"},{"id":97811604,"identity":"e5203e19-cdb9-4096-9482-ca8aa48b4148","added_by":"auto","created_at":"2025-12-09 16:03:06","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1065047,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/989216755cc2e3583ff58d92.jpeg"},{"id":97897849,"identity":"8ca893d1-db77-40f8-9d2f-071f92ad0294","added_by":"auto","created_at":"2025-12-10 15:38:20","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":536629,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/b3febbfe4a342eb82280908d.png"},{"id":97898803,"identity":"378abbc1-055e-4dbb-af3c-fa89cf8d8d37","added_by":"auto","created_at":"2025-12-10 15:39:47","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":425190,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/fea6b95e6e78495e3e178c94.png"},{"id":97811609,"identity":"89ff0427-6bd9-417d-8226-372806f3094a","added_by":"auto","created_at":"2025-12-09 16:03:06","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":459712,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/1f962cf53e1ba0573d3fb57e.png"},{"id":97898595,"identity":"275eb81c-6e22-497c-8624-a39afc14830d","added_by":"auto","created_at":"2025-12-10 15:39:19","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":263714,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/1d205cef92bc708075817aa6.png"},{"id":97898313,"identity":"4b6e5294-663e-4328-8c85-12cdb8fbed61","added_by":"auto","created_at":"2025-12-10 15:38:59","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":296431,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/7e1556f1c1e492931ec43ed8.png"},{"id":97898342,"identity":"8d80bc2a-691b-4f40-b516-39389f91af5f","added_by":"auto","created_at":"2025-12-10 15:39:02","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90211,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/dabec6a7adff7fca46bfdff0.png"},{"id":97811613,"identity":"9817b214-0d4b-499d-a54e-6489c0a34e41","added_by":"auto","created_at":"2025-12-09 16:03:06","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86001,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/477a056515312802e252aecc.png"},{"id":97811611,"identity":"53e22246-8f8e-4779-beea-24d0bf64559a","added_by":"auto","created_at":"2025-12-09 16:03:06","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96588,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/c7a9a53b5b513257c0ecf6d1.png"},{"id":97811614,"identity":"98f72678-ed8b-44a2-b917-5b97cd35d35d","added_by":"auto","created_at":"2025-12-09 16:03:06","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":47548,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/55da96b54fbdc56f8602ff42.png"},{"id":97897536,"identity":"ef28f915-ca49-4daf-aa50-d6527c4281c8","added_by":"auto","created_at":"2025-12-10 15:37:56","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":85093,"visible":true,"origin":"","legend":"","description":"","filename":"rs82952220structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/66b13d1c17f3535edcb881b1.xml"},{"id":97811615,"identity":"de9aab1e-d166-44f7-aa8b-7417bba05567","added_by":"auto","created_at":"2025-12-09 16:03:06","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94617,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/ce909993e9a551090fff7e21.html"},{"id":97898800,"identity":"1fcb08f7-8715-43f4-8ccf-c57a70fceb22","added_by":"auto","created_at":"2025-12-10 15:39:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":275195,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic Illustration of Cartilage Microsphere Generation and Their Inhibitory Effects on Tumour Cell Proliferation and Migration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Schematic diagram illustrating the isolation process of cartilage microspheres. Fresh porcine costal cartilage was harvested, minced into fine fragments, and subsequently ground in a sterile environment. The resulting cartilage slurry was filtered through a cell strainer to remove large debris, followed by centrifugation to collect the purified cartilage microspheres. B. A representative micrograph showing the isolated chondrogenic microspheres with a uniform diameter of approximately 200 µm, illustrating their spherical morphology and uniform size distribution after filtration. C. Subsequent cell experiment to assess the effect of the prepared cartilage microspheres on tumour cells. D. Representative images of clone formation experiments of A549 cells and 4T1 cells treated with CMs of different volumes. E. Quantitative analysis of the colony number of A549 cells treated with different doses of CMs. The data shows that as the volume of CMs increases, the number of colonies decreases compared to the control group. F. Quantitative analysis of colony numbers of 4T1 cells. A dose dependent inhibition of colony formation was observed, with a significant decrease in colony number compared to the control group as the volume of CMs increased. Significant differences are indicated by asterisks: *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. G. The relative cell density of A549 cells and 4T1 cells after CMs treatment indicates that cell proliferation is inhibited. H. Quantitative analysis of relative cell density of A549 cells treated with CMs showed no significant inhibitory effect on proliferation. I. Quantitative analysis of the relative cell density of 4T1 cells treated with CMs showed a significant inhibitory effect on proliferation. J. The relative cell closure rate of 4T1 cells suggests that the wound healing ability of cells decreases with the increase of CMs volume. K. Quantitative analysis of scratch distance on A549 cells treated with CMs for 24 hours showed impaired migration of A549 cells. L. Quantitative analysis of scratch distance in 4T1 cells treated under the same conditions as H. Significant differences are indicated by asterisks: *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/6e7df90427d2200f58af781a.jpg"},{"id":97811600,"identity":"2a8ccc6c-6c7d-4304-9295-c4fb71632b28","added_by":"auto","created_at":"2025-12-09 16:03:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":424543,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of CMs on the Smad protein signalling pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-C. After treatment with cartilage microspheres of different volumes, Smad were labelled by Hochest staining to quantitatively analyze the nuclear localization of Smad. D. Representative immunofluorescence images of Smad1, 2, and 3 signalling pathways. E. Representative immunofluorescence image of Smad 2 signalling pathway. F. Representative immunofluorescence images of the Smad 4 signalling pathway.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/a3804ed5c2870a386e400882.png"},{"id":97811598,"identity":"fe3c9e6a-f216-4fc0-bf0e-0e9912db1376","added_by":"auto","created_at":"2025-12-09 16:03:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":425988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of tumor growth following intratumorally injection of CMs in a 4T1 mouse model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Experimental timeline: Day 0, gelatin sponge with 4T1 cells was subcutaneously implanted into mice. Day 14, mice received intratumorally injection, with the experimental group receiving CMs and the control group receiving an equal amount of PBS injection. All mice were euthanized on day 21 for analysis. B-D. Tumour status on the 14th day of 4T1 tumour formation: Representative gross photographs of subcutaneous tumours and quantitative analysis of tumour volume and weight. Before treatment, no significant differences were observed between the control group and the CMs group. Ns indicates not significant (p≥0.05). E-G. CMs treatment of tumour status after one week. Obtaining representative gross photos of the tumour and quantitative analysis of the relative growth rate and weight of the tumour. Asterisks denote significant differences (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001), and ns indicates not significant (p≥0.05). H. Representative hematoxylin and eosin (HE) stained sections of tumour tissues from 4T1 tumour-bearing mice. The left panel shows the tumour tissue from the solvent control group, intratumorally injection of solvent. The right panel shows the tumour tissue from the group treated with cartilage microspheres, intratumorally injection of CMs. The distinct cartilage region, characterized by its typical lacunar structure and extracellular matrix, is visible adjacent to the tumour region in the treated group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/90fa7a9fe9f1bcd649e5a75a.png"},{"id":97811601,"identity":"ad5b4e92-73ed-4262-b544-ee6ad33833e3","added_by":"auto","created_at":"2025-12-09 16:03:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":420435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBMP2-induced chondrogenic ATDC5 cells inhibit 4T1 tumour growth in a mouse model in a dose-dependent manner.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Experimental timeline: Gelatin sponges containing ATDC5 cells pre-induced with different volumes of BMP2 were implanted subcutaneously in mice. On the 14th day after implanting chondrocytes, inject 50 µl of 4T1 cell suspension into the cartilage tissue. Mice were euthanized 2 weeks after injection for tumour analysis. B. A general photo of the tumour. With the increase of pre-induced BMP2 concentration, there is a significant decreasing trend in tumour size. C-D. Quantitative analysis of tumour volume at Days 21 and 28 revealed that both low and high dose BMP2 pre-induction groups exhibited significantly smaller tumours compared to the BMP2-free control group. Asterisks denote significant differences (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001). E. HE staining revealed that BMP2 pre-conditioning induced the formation of distinct cartilage regions. The maturation of this cartilaginous matrix was dose-dependent, effectively constraining tumour expansion. This confinement effect stands in stark contrast to the invasive and unbounded tumour growth observed in the control group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/3659907fc96c11cdaffbf084.png"},{"id":97903332,"identity":"b78af851-ca3b-4285-ac1d-9732736f01a1","added_by":"auto","created_at":"2025-12-10 15:55:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2028924,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/e13ea1cc-da13-4279-9542-26d6439015c8.pdf"},{"id":97811596,"identity":"4aa692d3-0c80-4e9d-a43b-f7376b68b679","added_by":"auto","created_at":"2025-12-09 16:03:06","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32256,"visible":true,"origin":"","legend":"\u003cp\u003eMaterial and Methods\u003c/p\u003e","description":"","filename":"20251205CMsMaterialandMethodsTCY.doc","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/97fca1773e5b2c9491d13f74.doc"},{"id":97899120,"identity":"de7f602f-d8b0-4947-8431-e494a31fe231","added_by":"auto","created_at":"2025-12-10 15:41:36","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":555367,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S3 Schematic illustration of the proposed mechanism for inhibiting bone metastasis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCartilage microspheres (CMs) are implanted into the bone microenvironment. The CMs-induced formation of cartilage acts as a protective barrier, structurally and biologically remodelling the local niche, thereby preventing the colonization and growth of metastatic tumour cells in bone.\u003c/p\u003e","description":"","filename":"20251202CMsSupplementaryFigure3TCY.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8295222/v1/a2e2659f46724ad5525a241d.jpg"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003ePotential Application of Cartilage Microspheres in Inhibiting Metastatic Tumour\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs early as 1975, Judah Folkman uncovered that the capillary proliferation induced by tumour is inhibited by neonatal scapular cartilage based on a rabbit model[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The following year, they isolated a tumour-inhibiting cartilage fraction containing several different proteins. The fraction strongly inhibits protease activity[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Using an in vitro sarcoma cell-culture system, Klaus E. Kuettner demonstrated in 1981 that the resistance of cartilage to tumour invasion is regulated in part by tissue-derived proteinase inhibitors[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A high molecular weight factor was extracted from fatal bovine cartilage with 1M guanidine hydrochloride. This factor inhibited neither the proliferation of sarcoma 180 cells in culture nor the growth of their ascites form. However, it strongly inhibited the growth of solid sarcoma 180 in vivo, as revealed by a Japanese scientist in 1984[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Cartilage is one of the very few naturally occurring avascular tissues where lack of angiogenesis is the guiding principle for its structure and function[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Cartilage encompasses primarily a specialised extracellular matrix synthesised by chondrocytes that is both complex and unique because of the myriad molecules of which it is composed. Of these components, a few such as TSP-1 (thrombospondin-1)[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], chondromodulin-1[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the type XVIII-derived endostatin, secreted protein acidic and rich in cysteine (SPARC)[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The type II collagen-derived N-terminal propeptide (PIIBNP) has demonstrated antiangiogenic or antitumour properties in vitro and in vivo preclinical trials that involve several complicated mechanisms that are not completely understood. Thrombospondin-1, endostatin, and the shark-cartilage-derived Neovastat preparation have also been investigated in human clinical trials to treat several different kinds of cancers, where, despite the tremendous success seen in preclinical trials, these molecules are yet to show success as anticancer agents[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Shark cartilage was used in clinical trials for advanced cancers. Under the specific conditions, shark cartilage as a single agent was inactive in patients with advanced-stage cancer and had no salutary effect on quality of life. The 16.7% rate of stable disease (SD) was similar to results in patients with advanced cancer treated with supportive care alone[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Due to the failure of clinical trials, research in this field remained sluggish for the next few decades.\u003c/p\u003e\u003cp\u003eAt the same time, the existing clinical imaging methods, such as computed tomography (CT), which are not good at cartilage tissue imaging. Although magnetic resonance imaging (MRI) provides excellent visualization of cartilage, this capability is not utilized in all clinical examinations. Moreover, in common sites of metastasis such as the ribs and spine, the relatively small volume of cartilaginous tissue may lead to the oversight of tumor involvement in these regions, even when invasion occurs. Therefore, the impact of the cartilage environment on tumours has not received enough attention.\u003c/p\u003e\u003cp\u003eBone metastasis is a common and serious complication in patients with advanced cancer[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Previous research on the cartilage environment's impact on tumours has been limited to osteosarcoma. We focused on the potential impact of the cartilage environment on metastatic tumours adjacent to cartilage. Our goal is to explore whether cartilage tissue inhibits metastatic tumour growth.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eConsidering the complexity of the cartilage environment, to study the effects of the cartilage environment on metastatic tumours in adjacent areas of cartilage, we extracted Cartilage microspheres (CMs) from abundant porcine cartilage. We extracted microspheres derived from CMs that mimic native cartilage both structurally and functionally. The extraction protocol was detailed and described as the flow chart (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Fresh cartilages are obtained from the market and cleaned with pre-cold PBS. A grinder was used for grinding the cartilage into small particles. The cartilage particles were gradually pushed through 200\u0026micro;m meshes with PBS washing. The filtered solution was collected and centrifuged at 1500rpm for 3 minutes. The supernatant was removed, and CMs were at the bottom of the centrifuge tubes. The CMs were kept in -80℃ for no longer than 7 days. Microscopy images show the five cartilage particle patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Considering the performance feasibility as well as the particles and the cell size ratio, 200\u0026micro;m cartilage particles were selected for the following experiments. Then we cocultured CMs with tumour cells in vitro to test whether the CMs affect tumour growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe co-cultured either lung cancer cells A549 or breast cancer cells 4T1with gradient CMs for 7 days. Colony formation assay shows CMs significantly inhibits 4T1 cells colony number, whereas the effect is weak in A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). With a high initial cell concentration, CMs also showed remarkably repressive activity in 4T1 cell growth. However, this effect was not observed in A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Then a wound healing assay was performed to test whether CMs block cell migration. Consistently, the inhibitory effect of CMs on cell migration was only observed in 4T1 cells. Similarly, A549 cells remained unresponsive (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). Preliminary evidence suggests that CMs have the potential to inhibit tumour cell proliferation and migration, but not a pan-cancer effect; as we have shown, they are effective only on 4T1 cells, and their effect on A549 cells is extremely weak.\u003c/p\u003e\u003cp\u003eThere are many cell growth mediators contained in the cartilage matrix[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], such as BMPs and TGFb. The decades signalling takes an important role in the surrounding microenvironment. We preliminary detected Samd1/2/3, Smad2, and Samd4 expression levels of the 4T1 cells co-cultured with CMs. The antibody that recognizes Samd1/2/3without discrimination shows an increased trend as the rising CMs content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, Smad2 level is almost unaffected by increased CM content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). If only Smad4 is considered, it will increase at certain doses of certain CMs, and the trend of change is fluctuating (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The Smad signalling pathway is just one of many pathways, and its phosphorylation level plays a significant role in downstream signal regulation. Our exploration of Smads behaviour is only superficial; further investigation into its underlying mechanisms is needed.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe have observed the inhibitory effect of CMs in vitro, whether CMs affect tumour growth is attractive. To illustrate this, we implanted 4T1-carrying scaffold constituted of gelatin sponge, which has been validated to show good biocompatibility and in clinical applications. 14 days later, the CMs or solvent was injected into the tumour site (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This design and preformation are to mimic the clinical cancer bone localization. Before the CMs injection, the tumour size is no difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). After CMs injection for 7 days, the tumours show a significantly decreased size. Meanwhile, we noticed that the tumour weight is no different (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Through the slides stained with hematoxylin and eosin (HE), the tumours with CMs intervention display clear tumour, cartilage region and tumour necrosis domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). All these suggest that directly injecting CMs restrain the 4T1 cells-generated tumour bulk in vivo.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, we used chondrogenesis cell line ADTC5 with the stimulation of BMP2 to generate cartilage matrix (Supplementary Fig.\u0026nbsp;1, only shown to the reviewer), to explore whether the manufactured cartilage matrix limit tumour growth. Derived from mouse teratocarcinoma, the ATDC5 chondrogenic cell line is characterized by its capacity to undergo a defined differentiation process that mirrors the maturation stages of chondrocytes under defined conditions[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The chondrogenic ADTC5 cells were stimulated with BMP2, which is confirmed to form cartilage matrix[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The cells were carried by the gelatin sponge scaffold[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. 14 days later, the 4T1 tumour cells were injected into the scaffold (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This is also a model to mimic the metastatic tumour localization. The tumour weight and volume are both decreased in the BMP2 stimulation group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In the BMP2 stimulation tumours, the cartilage matrix as arrow points is clearly uncovered (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Combined Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;5, the evidence suggests that no matter whether CMs or ADTC5-originated cartilage matrix represses tumour growth.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOverall, the cartilage inhibit tumour growth is validated by both in vitro and in vivo experiments. The application of humanized CMs and cell tissue engineering cartilage are promising in cancer therapy (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\u003cp\u003eThe metastatic dissemination of malignant cells to skeletal sites represents a devastating complication of advanced-stage carcinomas and sarcomas, portending a precipitous decline in patient quality of life and overall survival[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Conventional radiographic surveillance, primarily through CT and MRI, provides unparalleled resolution for osseous structures but exhibits notoriously poor soft tissue contrast for delineating cartilaginous anatomy[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This fundamental technical limitation has historically precluded a comprehensive understanding of the tropism of metastatic cells for cartilage, fostering a prevailing, yet unsubstantiated, assumption that cartilage is a metastatic-resistant niche. Consequently, a pivotal pathophysiological question has remained largely enigmatic: does the unique biochemical and biophysical milieu of cartilage actively inhibit or paradoxically foster the colonization and proliferation of disseminated tumour cells? Our investigation was meticulously designed to deconstruct this very paradigm, and the resultant data compellingly demonstrate that a native, three-dimensional (3D) cartilaginous architecture exerts a potent inhibitory effect on tumour growth, thereby introducing a novel conceptual framework for understanding the cellular ecology of metastatic sites.\u003c/p\u003e\u003cp\u003eThe rationale for this inquiry is rooted in a legacy of contradictory scientific evidence. Pioneering in vitro and in vivo studies had suggested the existence of soluble factors within cartilage extracts capable of suppressing angiogenesis and tumour cell proliferation[\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This body of work subsequently catalyzed a wave of clinical trials investigating shark cartilage derivatives as putative anti-neoplastic agents. The unequivocal failure of these trials, however, seemingly discredited the entire hypothesis. A critical reappraisal of these earlier endeavours suggests that the route of administration of oral ingestion was likely a fatal flaw, resulting in the extensive proteolytic degradation of critical structural macromolecules such as collagen type II, aggrecan, and so on, and the consequent obliteration of the essential 3D supramolecular organization hypothesized to be integral to its anti-tumour bioactivity. Our experimental approach was conceived to directly address this shortcoming. By employing two orthogonal model systems, the native, decellularized porcine cartilage microspheres that preserve the native extracellular matrix (ECM) and synthetic scaffolds repopulated with active chondroprogenitor cells, we successfully recapitulated the intact chondrogenic microenvironment. The concordant results from both CMs directly injecting (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and ADTC5-BMP2 stimulating (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) models, demonstrating significant suppression of tumour cell proliferation and viability, provide robust and multifaceted validation that the inhibitory signal is inherent to the 3D chondrogenic niche itself, rather than a mere artifact of 2D culture or a non-physiological soluble factor.\u003c/p\u003e\u003cp\u003eThe cartilage extracellular matrix serves as a critical reservoir for various growth mediators, including BMPs and TGFβ, which are known to orchestrate cellular behaviour through complex signalling pathways[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In our preliminary investigation into the potential mechanisms underlying the observed tumour suppression, we analyzed the response of the Smad pathway in 4T1 cells co-cultured with CMs. Our immunoblotting data revealed a concentration-dependent increase in Smad1/2/3 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), while Smad2 expression remained largely unaltered. Smad4, however, exhibited a fluctuating, non-monotonic expression pattern in response to increasing CMs concentrations. It is crucial to emphasize that the primary objective of this study was to establish the fundamental phenomenon of cartilage-mediated tumour suppression. Consequently, this initial characterization of Smad signalling components remains superficial by design. A comprehensive mechanistic dissection entailing detailed analysis of phosphorylation dynamics, nuclear translocation, transcriptional activity, and crosstalk with other pathways, which represents the logical and necessary focus of our subsequent, dedicated investigation. The current findings successfully lay the phenomenological groundwork for these future studies aimed at definitively unravelling the molecular intricacies of this novel tumour-suppressive microenvironment.\u003c/p\u003e\u003cp\u003eFrom a translational perspective, the current therapeutic arsenal for managing osseous metastases remains profoundly inadequate. Pharmacological interventions such as bisphosphonates[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and the RANKL inhibitor[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] denosumab have demonstrated efficacy in reducing skeletal-related events by modulating osteoclast-mediated bone resorption; however, they are purely palliative, offering no direct cytotoxic or cytostatic effect on the metastatic tumour cells themselves and failing to address the disease's progression. Our findings posit a groundbreaking therapeutic strategy that moves beyond mere modulation of the bone microenvironment to actively engineering a hostile niche for metastatic cells. We propose a novel locoregional intervention involving the targeted intralesional implantation of 3D cartilage-mimetic constructs in the form of either preformed microspheres or cell-laden scaffolds following surgical curettage of the bone metastasis. This paradigm is designed with a dual mechanism of action. First, it enables direct, contact-mediated inhibition of tumor proliferation and induction of apoptosis through the presentation of specific extracellular matrix-derived cryptic peptides and sequestration of growth factors. Second, upon stimulation by specific bone morphogenetic proteins, the implanted constructs undergo guided chondrogenic or osteochondral differentiation. This cartilaginous ossification process not only progressively replaces the resected tumor tissue but also facilitates the repair of bone defects resulting from tumor erosion and surgical curettage, thereby restoring mechanical integrity and preventing post-procedural structural deficiencies and related complications. Beyond merely eradicating the tumor, this strategy aims to fundamentally reprogram the pathological soil, transforming a permissive metastatic niche into an inhibitory microenvironment through the principles of regenerative medicine and tissue engineering.\u003c/p\u003e\u003cp\u003eInevitably, the clinical translation of such a bio-implant strategy necessitates a rigorous examination of biocompatibility and immunogenicity (Supplementary Fig.\u0026nbsp;2, only shown to reviewer). The present study utilized xenogeneic cartilage microspheres, and as anticipated, the issue of host immune recognition and potential rejection must be acknowledged. Histological and serological analyses in our models, however, revealed only a modest, non-destructive lymphocytic infiltrate, suggesting that the decellularization process may have sufficiently mitigated the antigenic load to prevent a fulminant rejection response. Looking forward, several promising pathways exist to circumvent this challenge entirely. The burgeoning field of xenotransplantation, particularly with the advent of multi-gene edited, \"humanized\" porcine donors, offers a viable platform for sourcing immunologically compatible, \"off-the-shelf\" cartilaginous constructs. Alternatively, a fully autologous approach, entailing the harvest of patient-derived mesenchymal stromal cells (MSCs), their in vitro chondrogenic priming with specific BMPs, and subsequent delivery on a bioresorbable, immuno-inert scaffold, represents a highly personalized and immunologically safe therapeutic trajectory. Future investigations will be paramount to delineate the precise molecular mediators of the observed tumour suppression, maybe they are specific ECM components, matrix-bound nanovesicles, or pericellular hypoxia, and to optimize the scaffold design for controlled factor release and seamless integration with the host bone, thereby solidifying the foundation for a first-in-human clinical application.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that a native three-dimensional chondrogenic microenvironment intrinsically suppresses tumor growth. Utilizing both decellularized cartilage microspheres and engineered chondrogenic constructs, we validated this inhibitory effect both in vitro and in vivo. These findings challenge the prevailing assumption of cartilage as a passive metastatic-resistant niche and establish a foundational principle for a novel therapeutic strategy. We propose that targeted intralesional implantation of cartilage-mimetic constructs post-curettage can not only inhibit residual tumor progression but also facilitate bone defect repair through guided osteochondral regeneration. This paradigm, merging tumor suppression with regenerative medicine, offers a promising locoregional approach to reprogram the metastatic niche and address the unmet clinical need in managing bone metastases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no other competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eData and materials availability\u003c/h2\u003e\u003cp\u003eAll data are available in the main text or the supplementary materials.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003eThe animal experiments involved in this study were all approved by the Ethics Committee of the Affiliated Hospital of Shandong Second Medical University. The clinical trials involved in this study were approved by the Medical Ethics Committee of the Second Medical University of Shandong Affiliated Hospital (2025SDL714)\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor\u0026rsquo;s contributions\u003c/h2\u003e\u003cp\u003eJingjing Li and Jiankang Fang conceived the concept. Chunyan Tang, Junyuan Bing, Min Yang, Meili Li, Hangpeng Tian, Xiaotong Chen, Fangchao Li, Yanfei Zhang, Mingsheng Cai and Yi Zhu finished the experimental performance. Manuscript writing is finished by Jingjing Li, Yi Zhu and Jiankang Fang. Jingjing Li and Chunyan Tang generated the figures. Mingshengcai, Jiankang Fang and Jingjing Li revised the manuscript. All authors reviewed and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThis work is supported by Shandong Provincial Health Commission Science and Technology Innovation Team Grant, Shandong Provincial Medical and Health Science and Technology Project (202509031260), the National Natural Science Foundation of China (Grant No. 82104289), Shandong Provincial Health Commission(M-2022053), Science and Technology Innovation Plan from Weifang Medical University (041004), Yuandu Scholar Grant of Weifang City to LJJ, Weifang Science and Technology Bureau Plan Project (2021YX081), Science and technology project jointly established by the Science and Technology Department of the State Administration of Traditional Chinese Medicine (GZY-KJS-SD-2023-079), Shandong Provincial Medical Association Young Talent Promotion Project (2023_GJ_0039).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrem H, Folkman J (1975) Inhibition of tumor angiogenesis mediated by cartilage. J Exp Med 141:427\u0026ndash;439\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLanger R, Brem H, Falterman K, Klein M, Folkman J (1976) Isolations of a cartilage factor that inhibits tumor neovascularization. Science 193:70\u0026ndash;72\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePauli BU, Memoli VA, Kuettner KE (1981) Regulation of tumor invasion by cartilage-derived anti-invasion factor in vitro. J Natl Cancer Inst 67:65\u0026ndash;73\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTakigawa M, Shirai E, Enomoto M, Hiraki Y, Fukuya M, Suzuki F et al (1985) Cartilage-derived anti-tumor factor (CATF) inhibits the proliferation of endothelial cells in culture. Cell Biol Int Rep 9:619\u0026ndash;625\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePatra D, Sandell LJ (2012) Antiangiogenic and anticancer molecules in cartilage. Expert Rev Mol Med 14:e10\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNossin Y, Farrell E, Koevoet W, Datema F, Somoza RA, Caplan AI et al (2021) The Releasate of Avascular Cartilage Demonstrates Inherent Pro-Angiogenic Properties In Vitro and In Vivo. Cartilage 13:559S\u0026ndash;70S\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa Z, Mao C, Chen X, Yang S, Qiu Z, Yu B et al (2023) Peptide Vaccine Against ADAMTS-7 Ameliorates Atherosclerosis and Postinjury Neointima Hyperplasia. Circulation 147:728\u0026ndash;742\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDi Treuheim P, Torre T, Ferreri OM, Nasser ED, Abbondandolo P, Delgado Caceres A (2021) Tenomodulin and Chondromodulin-1 Are Both Required to Maintain Biomechanical Function and Prevent Intervertebral Disc Degeneration. Cartilage 13:604S\u0026ndash;14S\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRomero A, Leurs N, Munoz D, Debiais-Thibaud M, Marcellini S (2021) Divergent Expression of SPARC, SPARC-L, and SCPP Genes During Jawed Vertebrate Cartilage Mineralization. Front Genet 12:788346\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiller DR, Anderson GT, Stark JJ, Granick JL, Richardson D (1998) Phase I/II trial of the safety and efficacy of shark cartilage in the treatment of advanced cancer. J Clin Oncol 16:3649\u0026ndash;3655\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSatcher RL, Zhang XH (2022) Evolving cancer-niche interactions and therapeutic targets during bone metastasis. Nat Rev Cancer 22:85\u0026ndash;101\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu M, Wu S, Chen W, Li YP (2024) The roles and regulatory mechanisms of TGF-beta and BMP signaling in bone and cartilage development, homeostasis and disease. Cell Res 34:101\u0026ndash;123\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThielen NGM, van der Kraan PM, van Caam APM (2019) TGFbeta/BMP Signaling Pathway in Cartilage Homeostasis. Cells. ; 8\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArenberg D (1990) Misclassification of probable senile dementia\u0026ndash;Alzheimer's type in the Baltimore Longitudinal Study of Aging. J Clin Epidemiol 43:105\u0026ndash;107\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang P, Meng Q, Wang W, Zhang S, Xiong X, Qin S et al (2020) Icariin inhibits the inflammation through down-regulating NF-kappaB/HIF-2alpha signal pathways in chondrocytes. Biosci Rep. ; 40\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu X, Chen K, Chen Q, Zhang X, Feng C, Li X et al (2025) 3D Bioprintable Gt-Alg-MMT Nano Bioink for Cartilage Tissue Engineering. Macromol Biosci 25:e00167\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu Y, Zhou L, Wang L, He S, Ren H, Zhou N et al (2020) The role of SIRT1 in BMP2-induced chondrogenic differentiation and cartilage maintenance under oxidative stress. Aging 12:9000\u0026ndash;9013\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGamer LW, Pregizer S, Gamer J, Feigenson M, Ionescu A, Li Q et al (2018) The Role of Bmp2 in the Maturation and Maintenance of the Murine Knee Joint. J Bone Min Res 33:1708\u0026ndash;1717\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBlaney Davidson EN, Vitters EL, Bennink MB, van Lent PL, van Caam AP, Blom AB et al (2015) Inducible chondrocyte-specific overexpression of BMP2 in young mice results in severe aggravation of osteophyte formation in experimental OA without altering cartilage damage. Ann Rheum Dis 74:1257\u0026ndash;1264\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZeng X, Wei QS, Ye JC, Rao JH, Zheng MG, Ma YH et al (2023) A biocompatible gelatin sponge scaffold confers robust tissue remodeling after spinal cord injury in a non-human primate model. Biomaterials 299:122161\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMerk M, Chirikian O, Adlhart C (2021) 3D PCL/Gelatin/Genipin Nanofiber Sponge as Scaffold for Regenerative Medicine. Mater (Basel). ; 14\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNi J, Ye D, Zeng W, Ma S, Wang Z, Kuang Y et al (2024) Promotion of hair growth by a conditioned medium from human umbilical cord mesenchymal stem cells cultivated in a 3D scaffold of gelatin sponge. Eur J Med Res 29:270\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFornetti J, Welm AL, Stewart SA (2018) Understanding the Bone in Cancer Metastasis. J Bone Min Res 33:2099\u0026ndash;2113\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCl\u0026eacute;zardin P, Coleman R, Puppo M, Ottewell P, Bonnelye E, Paycha F et al (2021) Bone metastasis: mechanisms, therapies, and biomarkers. Physiol Rev 101:797\u0026ndash;855\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKwee RM, Kwee TC (2022) Diagnostic performance of MRI and CT in diagnosing necrotizing soft tissue infection: a systematic review. Skeletal Radiol 51:727\u0026ndash;736\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Zhao H, Liu Y, Zeng M, Zhang J, Hao D (2022) Diagnostic Performance of Dynamic Contrast-Enhanced MRI and (18)F-FDG PET/CT for Evaluation of Soft Tissue Tumors and Correlation with Pathology Parameters. Acad Radiol 29:1842\u0026ndash;1851\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark NR, Lee YJ, Lee SH, Kim JE (2023) Anti-cancer Effect of Unique Cartilage Matrix-associated Protein in Breast Cancer Cells Depends on gamma-Carboxylation. Anticancer Res 43:1959\u0026ndash;1965\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXie J, Xiao Y, Zhang Y, Hong A, Chen X (2025) Identification and functional analysis of a novel potent anti-angiogenesis peptide SAIF-B2 derived from shark cartilage. Eur J Pharmacol 1004:177961\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXie J, Li F, Cai Y, Zhang J, Zhang Y, Zhai Z et al (2023) SAIF plays anti-angiogenesis via blocking VEGF-VEGFR2-ERK signal in tumor treatment. Heliyon 9:e18240\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVijayakumar S, Gonzalez-Sanchez ZI, Amanullah M, Sonamuthu J, Rajkumar M, Divya M et al (2025) Shark chondroitin sulfate gold nanoparticles: A biocompatible apoptotic agent for osteosarcoma. Int J Biol Macromol 290:138793\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZohri M, Arefian E, Azizi Z, Akbari Javar H, Shadboorestan A, Fatahi Y et al (2024) Activation of the BMP2/SMAD4 signaling pathway for enhancing articular cartilage regeneration of mesenchymal stem cells utilizing chitosan/alginate nanoparticles on 3D extracellular matrix scaffold. Int J Biol Macromol 277:133995\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShirakura M, Kram V, Robinson J, Sikka S, Kilts TM, Wadhwa S et al (2017) Extracellular Matrix Mediates BMP-2 in a Model of Temporomandibular Joint Osteoarthritis. Cells Tissues Organs 204:84\u0026ndash;92\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJakob T, Tesfamariam YM, Macherey S, Kuhr K, Adams A, Monsef I et al (2020) Bisphosphonates or RANK-ligand-inhibitors for men with prostate cancer and bone metastases: a network meta-analysis. Cochrane Database Syst Rev 12:CD013020\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Broekhoven DL, Dootjes LW, van der Veldt A, Zillikens C, van Oldenrijk J (2023) Effect of Bisphosphonates on Skeletal Related Events in Long Bone Metastases of Renal Cell Carcinoma: A Systematic Review. Clin Genitourin Cancer 21:e190\u0026ndash;e7\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYue Z, Niu X, Yuan Z, Qin Q, Jiang W, He L et al (2022) RSPO2 and RANKL signal through LGR4 to regulate osteoclastic premetastatic niche formation and bone metastasis. J Clin Invest. ; 132\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGkikopoulou E, Syrigos CC, Mantogiannakou I, Petraki CE, Stathopoulou M, Dragolia M et al (2025) RANKL Drives Bone Metastasis in Mammary Cancer: Protective Effects of Anti-Resorptive Treatments. Int J Mol Sci. ; 26\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi B, Wang P, Jiao J, Wei H, Xu W, Zhou P (2022) Roles of the RANKL-RANK Axis in Immunity-Implications for Pathogenesis and Treatment of Bone Metastasis. Front Immunol 13:824117\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Shandong Second Medical University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"tumour, cartilage, cancer bone metastasis, cartilage matrix, cartilage microsphere","lastPublishedDoi":"10.21203/rs.3.rs-8295222/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8295222/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBone metastasis is a devastating complication of advanced cancer with limited therapeutic options. The role of cartilage in metastatic progression has remained ambiguous. This study investigates the tumour-suppressive potential of a native three-dimensional chondrogenic microenvironment. Using two complementary models, implantation of decellularized cartilage microspheres and tumour cell culture within chondrogenic scaffolds, we demonstrate that the 3D cartilage architecture consistently and significantly inhibits tumour proliferation. These findings provide a mechanistic explanation for the historical failure of oral shark cartilage therapies, whose bioactive structure is compromised during administration. Our work suggests a novel therapeutic strategy for bone metastasis through local implantation of 3D cartilage-mimetic constructs, which could simultaneously suppress tumour growth and remodel the metastatic niche through induced chondrogenesis. While immunological considerations for xenogeneic materials require further investigation, our approach supports the development of innovative treatments combining oncologic suppression with regenerative medicine principles.\u003c/p\u003e","manuscriptTitle":"Potential Application of Cartilage Microspheres in Inhibiting Metastatic Tumour","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-09 16:03:01","doi":"10.21203/rs.3.rs-8295222/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ceb961de-749f-4e19-8621-bbd1ae8eb6ab","owner":[],"postedDate":"December 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-09T16:03:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-09 16:03:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8295222","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8295222","identity":"rs-8295222","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}