Molecular mechanisms of chemoresistance in osteosarcoma (Review).
He Hongtao,Ni Jiangdong,Huang Jun
Due to the emergence of adjuvant and neoadjuvant chemotherapy, the survival rate has been greatly improved in osteosarcoma (OS) patients with localized disease. However, this survival rate has remained unchanged over the past 30 years, and the long-term survival rate for OS patients with metastatic or recurrent disease remains poor. To a certain extent, the reason behind this may be ascribed to the chemoresistance to anti-OS therapy. Chemoresistance in OS appears to be mediated by numerous mechanisms, which include decreased intracellular drug accumulation, drug inactivation, enhanced DNA repair, perturbations in signal transduction pathways, apoptosis- and autophagy-related chemoresistance, microRNA (miRNA) dysregulation and cancer stem cell (CSC)-mediated drug resistance. In addition, methods employed to circumvent these resistance mechanism have been shown to be effective in the treatment of OS. However, almost all the current studies on the mechanisms of chemoresistance in OS are in their infancy. Further studies are required to focus on the following aspects: i) Improving the delivery of efficacy through novel delivery patterns; ii) improving the understanding of the signal transduction pathways that regulate the proliferation and growth of OS cells; iii) elucidating the signaling pathways of autophagy and its association with apoptosis in OS cells; iv) utilizing high-throughput miRNA expression analysis to identify miRNAs associated with chemoresistance in OS; and v) identifying the role that CSCs play in tumor metastasis and in-depth study of the mechanism of chemoresistance in the CSCs of OS.
Hedgehog signalling in the tumourigenesis and metastasis of osteosarcoma, and its potential value in the clinical therapy of osteosarcoma.
Yao Zhihong,Han Lei,Chen Yongbin,He Fei,Sun Bin,Kamar Santosh,Zhang Ya,Yang Yihao,Wang Cao,Yang Zuozhang
Cell death & disease
The Hedgehog (Hh) signalling pathway is involved in cell differentiation, growth and tissue polarity. This pathway is also involved in the progression and invasion of various human cancers. Osteosarcoma, a subtype of bone cancer, is commonly seen in children and adolescents. Typically, pulmonary osteosarcoma metastases are especially difficult to control. In the present paper, we summarise recent studies on the regulation of osteosarcoma progression and metastasis by downregulating Hh signalling. We also summarise the crosstalk between the Hh pathway and other cancer-related pathways in the tumourigenesis of various cancers. We further summarise and highlight the therapeutic value of potential inhibitors of Hh signalling in the clinical therapy of human cancers.
Cyr61 expression in osteosarcoma indicates poor prognosis and promotes intratibial growth and lung metastasis in mice.
Sabile Adam A,Arlt Matthias J E,Muff Roman,Bode Beata,Langsam Bettina,Bertz Josefine,Jentzsch Thorsten,Puskas Gabor J,Born Walter,Fuchs Bruno
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
Osteosarcoma is the most frequent primary malignant bone tumor in children and adolescents with a high propensity for lung metastasis, the major cause of disease-related death. Reliable outcome-predictive markers and targets for osteosarcoma metastasis-suppressing drugs are urgently needed for more effective treatment of metastasizing osteosarcoma, which has a current mean 5-year survival rate of approximately 20%. This study investigated the prognostic value and the biological relevance of the extracellular matrix-associated growth factor Cyr61 of the CCN family of secreted proteins in osteosarcoma and metastasis. The prognostic value of Cyr61 was assessed with Kaplan-Meier analyses based on Cyr61 immunostaining of a tissue microarray of osteosarcoma biopsies collected from 60 patients with local or metastatic disease. Effects of Cyr61 overexpression on intratibial tumor growth and lung metastasis of the low metastatic human SaOS-2 osteosarcoma cell line were examined in severe combined immunodeficiency (SCID) mice. Cyr61-provoked signaling was studied in vitro in nonmanipulated SaOS-2 cells. Cyr61 immunostaining of osteosarcoma tissue cores correlated significantly (p = 0.02) with poor patient survival. Mice intratibially injected with Cyr61-overexpressing SaOS-2 cells showed faster tumor growth and an increase in number and outgrowth of lung metastases and consequently significantly (p = 0.0018) shorter survival than mice injected with control SaOS-2 cells. Cyr61-evoked PI-3K/Akt/GSK3β signaling in SaOS-2 cells resulted in a subcellular redistribution of the cell cycle inhibitor p21(Cip1/WAF1). Cyr61 has considerable potential as a novel marker for poor prognosis in osteosarcoma and is an attractive target for primary tumor- and metastases-suppressing drugs.
Toward a drug development path that targets metastatic progression in osteosarcoma.
Khanna Chand,Fan Timothy M,Gorlick Richard,Helman Lee J,Kleinerman Eugenie S,Adamson Peter C,Houghton Peter J,Tap William D,Welch Danny R,Steeg Patricia S,Merlino Glenn,Sorensen Poul H B,Meltzer Paul,Kirsch David G,Janeway Katherine A,Weigel Brenda,Randall Lor,Withrow Stephen J,Paoloni Melissa,Kaplan Rosandra,Teicher Beverly A,Seibel Nita L,Smith Malcolm,Uren Aykut,Patel Shreyaskumar R,Trent Jeffrey,Savage Sharon A,Mirabello Lisa,Reinke Denise,Barkaukas Donald A,Krailo Mark,Bernstein Mark
Clinical cancer research : an official journal of the American Association for Cancer Research
Despite successful primary tumor treatment, the development of pulmonary metastasis continues to be the most common cause of mortality in patients with osteosarcoma. A conventional drug development path requiring drugs to induce regression of established lesions has not led to improvements for patients with osteosarcoma in more than 30 years. On the basis of our growing understanding of metastasis biology, it is now reasonable and essential that we focus on developing therapeutics that target metastatic progression. To advance this agenda, a meeting of key opinion leaders and experts in the metastasis and osteosarcoma communities was convened in Bethesda, Maryland. The goal of this meeting was to provide a "Perspective" that would establish a preclinical translational path that could support the early evaluation of potential therapeutic agents that uniquely target the metastatic phenotype. Although focused on osteosarcoma, the need for this perspective is shared among many cancer types. The consensus achieved from the meeting included the following: the biology of metastatic progression is associated with metastasis-specific targets/processes that may not influence grossly detectable lesions; targeting of metastasis-specific processes is feasible; rigorous preclinical data are needed to support translation of metastasis-specific agents into human trials where regression of measurable disease is not an expected outcome; preclinical data should include an understanding of mechanism of action, validation of pharmacodynamic markers of effective exposure and response, the use of several murine models of effectiveness, and where feasible the inclusion of the dog with naturally occurring osteosarcoma to define the activity of new drugs in the micrometastatic disease setting.
BMP-2 inhibits lung metastasis of osteosarcoma: an early investigation using an orthotopic model.
Xiong Qisheng,Wang Xuesong,Wang Lizhen,Huang Yan,Tian Xiaodong,Fan Yubo,Lin Chia-Ying
OncoTargets and therapy
Background:Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, are known to regulate cell proliferation, differentiation, apoptosis, chemotaxis, and angiogenesis. BMPs also participate in the development of most tissues and organs in vertebrates. Recombinant human (rh) BMPs, such as rhBMP-2, rhBMP-4, and rhBMP-7, have been recently approved to augment spinal fusion and recalcitrant long-bone non-unions because of their equivalent or superior efficacy to autogenous bone graft in enhancing bony fusion. Nonetheless, the use of BMPs is contraindicated in surgery for bone tumors because of concerns that this anabolic growth factor may cause tumor proliferation. However, we have repeatedly reported that BMP-2 is effective in inducing osteogenic differentiation of a subpopulation of osteosarcoma (OSA) cells that acquire stem cell attributes and are capable of reconstituting tumor masses, which in turn suppress the malignancy of the bone tumor. Methods:3×10/20 µL human OSA 143B cells were inoculated into 5-6 weeks old BABL/c nude mice to establish orthotopic OSA. X-ray device was used to monitor the developed tumors in animals. Necropsy was performed and the pathology of lung metastasis were tested by Haemotoxylin and Eosin. Moreover, bone formation induced by rhBMP-2 was investigated through micro-computed tomography. In addition, immunohistochemistry staining was used to evaluate the tumorigenicity and growth of OSA cells after rhBMP-2 treatment. Results:In the present study, we established an orthotopic model of OSA by inoculating 143B cells into BABL/c mice, which resulted in a tumor occurrence rate of 100%. Following the treatment with rhBMP-2, lung metastasis, which contributes to poor prognosis, was significantly restricted, indicating an additional aspect of rhBMP-2 to suppress expansion of OSA. Concurrently, our micro-computed tomography and radiographic analyses showed that rhBMP-2 reduced the invasion of tumor cells into adjacent bone tissue, which in turn helped to preserve the integrity of the affected bone tissue. Finally, the growth of Ki-67-positive cells and those cells that express high levels of aldehyde dehydrogenase (ALDH) was found to be inhibited in the developed tumors. Conclusion:On the basis of these results, we conclude that rhBMP-2 can impede the malignancy of OSA by reducing lung metastasis of the tumor. Induction of the tumor cells by rhBMP-2 also helps to preserve the impaired skeleton. These results imply that BMP-2 or BMP-2-mimetic drugs, if properly combined with traditional therapies, may provide a new therapeutic option for the treatment of OSA.
Mycophenolic acid is a drug with the potential to be repurposed for suppressing tumor growth and metastasis in osteosarcoma treatment.
Klangjorhor Jeerawan,Chaiyawat Parunya,Teeyakasem Pimpisa,Sirikaew Nutnicha,Phanphaisarn Areerak,Settakorn Jongkolnee,Lirdprapamongkol Kriengsak,Yama Sarawoot,Svasti Jisnuson,Pruksakorn Dumnoensun
International journal of cancer
Our previous review of proteomics data showed that in osteosarcoma, some overexpressed proteins were targets of FDA-approved immunosuppressive and anti-arrhythmic drugs, including mycophenolate mofetil (MMF), ribavirin, leflunomide, azathioprine and digoxin. Here, these drugs were screened for growth inhibitory effects in human osteosarcoma cell lines, including MNNG/HOS, U2OS, SaOS-2, MG-63 and 143B cells. Only mycophenolic acid (MPA), an active metabolite of MMF, efficiently inhibited osteosarcoma cell growth with IC values of 0.46-7.3 μM; these values are in the therapeutic range for organ transplant patients. At a therapeutic dose (10 μM), MPA significantly inhibited colony formation, caused cell cycle arrest in the S phase, and induced apoptosis. Moreover, the in vitro invasion of osteosarcoma cells was reduced by MPA by inhibiting cell migration capability. The in vivo antitumor effect of MMF was determined in nude mice harboring 143B cell xenografts. Daily oral administration of 200 mg/kg/day MMF for 2 weeks significantly suppressed tumor growth in treated mice, achieving 57.4 ± 11.1% tumor growth inhibition. Compared with the vehicle group, the MMF group treated with 50-200 mg/kg/day for 3 weeks had a significant reduction in the number of lung metastatic nodules in a tail vein-lung metastasis model of 143B cells. MMF doses of 50, 100 and 200 mg/kg/day are approximately equivalent to the non-toxic doses of 0.25, 0.5 and 1 g/day in humans, respectively. These findings indicate that MPA/MMF can effectively control osteosarcoma tumor growth and metastasis. Thus, the potential to repurpose MPA/MMF for use in osteosarcoma chemotherapy is of great interest.
Reduction-responsive polypeptide nanomedicines significantly inhibit progression of orthotopic osteosarcoma.
Yin Fei,Wang Zongyi,Jiang Yafei,Zhang Tao,Wang Zhuoying,Hua Yingqi,Song Zhiming,Liu Jianhua,Xu Weiguo,Xu Jing,Cai Zhengdong,Ding Jianxun
Nanomedicine : nanotechnology, biology, and medicine
Osteosarcoma (OS) is the most common malignant bone tumor with high metastasis and mortality. Neoadjuvant chemotherapy is an effective therapeutic regimen, but the clinical application is limited by the unsatisfactory efficacies and considerable side effects. In this study, the reduction-responsive polypeptide micelles based on methoxy poly(ethylene glycol)-block-poly(S-tert-butylmercapto-L-cysteine) copolymers (mPEG-b-PBMLC, P4M, and mPEG-b-PBMLC, P9M) were developed to control the delivery of doxorubicin (DOX) in OS therapy. Compared to free DOX, P4M/DOX and P9M/DOX exhibited 2.6 and 3.5 times increase in the area under the curve of pharmacokinetics, 1.6 and 2.0 times increase in tumor accumulation, and 1.6 and 1.7 times decrease of the distribution in the heart. Moreover, the selective accumulation of micelles, especially P9M/DOX, in tumors induced stronger antitumor effects on both primary and lung metastatic OSs with less systematic toxicity. These micelles with smart responsiveness to intracellular microenvironments are highly promising for the targeted delivery of clinical chemotherapeutic drugs in cancer therapy.
TET2-dependent IL-6 induction mediated by the tumor microenvironment promotes tumor metastasis in osteosarcoma.
Itoh Hitoshi,Kadomatsu Tsuyoshi,Tanoue Hironori,Yugami Masaki,Miyata Keishi,Endo Motoyoshi,Morinaga Jun,Kobayashi Eisuke,Miyamoto Takeshi,Kurahashi Ryoma,Terada Kazutoyo,Mizuta Hiroshi,Oike Yuichi
The tumor microenvironment promotes epigenetic changes in tumor cells associated with tumor aggressiveness. Here we report that in primary tumor cells, increased interleukin-6 (IL-6) expression brought on by DNA demethylation of its promoter by ten-eleven translocation 2 (TET2) promotes lung metastasis in osteosarcoma (OS). Xenograft experiments show increased IL-6 expression and decreased methylation of its promoter in OS cells after implantation relative to before implantation. In addition, changes in IL-6 methylation and expression seen in OS cells at the primary site were maintained at the metastatic site. TET2 knockdown in OS cells suppressed upregulation of IL-6 and demethylation of its promoter in xenograft tumors and decreased tumor metastasis. We also present evidence showing that tumor cell-derived IL-6 facilitates glycolytic metabolism in tumor cells by activating the MEK/ERK1/2/hypoxia-inducible transcription factor-1α (HIF-1α) pathway and increases lung colonization by OS cells by upregulating expression of intercellular adhesion molecule-1 (ICAM-1), enhancing tumor metastasis. Blocking IL-6 signaling with a humanized monoclonal antibody against the IL-6 receptor reduced lung metastasis and prolonged survival of xenografted mice. These findings suggest that TET2-dependent IL-6 induction enables acquisition of aggressive phenotypes in OS cells via the tumor microenvironment and that blocking IL-6 signaling could be serve as a potential therapy to antagonize metastasis.
TH1579, MTH1 inhibitor, delays tumour growth and inhibits metastases development in osteosarcoma model.
Moukengue Brice,Brown Hannah K,Charrier Céline,Battaglia Séverine,Baud'huin Marc,Quillard Thibaut,Pham Therese M,Pateras Ioannis S,Gorgoulis Vassilis G,Helleday Thomas,Heymann Dominique,Berglund Ulrika Warpman,Ory Benjamin,Lamoureux Francois
BACKGROUND:Osteosarcoma (OS) is the most common primary malignant bone tumour. Unfortunately, no new treatments are approved and over the last 30 years the survival rate remains only 30% at 5 years for poor responders justifying an urgent need of new therapies. The Mutt homolog 1 (MTH1) enzyme prevents incorporation of oxidized nucleotides into DNA and recently developed MTH1 inhibitors may offer therapeutic potential as MTH1 is overexpressed in various cancers. METHODS:The aim of this study was to evaluate the therapeutic benefits of targeting MTH1 with two chemical inhibitors, TH588 and TH1579 on human osteosarcoma cells. Preclinical efficacy of TH1579 was assessed in human osteosarcoma xenograft model on tumour growth and development of pulmonary metastases. FINDINGS:MTH1 is overexpressed in OS patients and tumour cell lines, compared to mesenchymal stem cells. In vitro, chemical inhibition of MTH1 by TH588 and TH1579 decreases OS cells viability, impairs their cell cycle and increases apoptosis in OS cells. TH1579 was confirmed to bind MTH1 by CETSA in OS model. Moreover, 90 mg/kg of TH1579 reduces in vivo tumour growth by 80.5% compared to non-treated group at day 48. This result was associated with the increase in 8-oxo-dG integration into tumour cells DNA and the increase of apoptosis. Additionally, TH1579 also reduces the number of pulmonary metastases. INTERPRETATION:All these results strongly provide a pre-clinical proof-of-principle that TH1579 could be a therapeutic option for patients with osteosarcoma. FUNDING:This study was supported by La Ligue Contre le Cancer, la SFCE and Enfants Cancers Santé.
Imaging the inhibition by anti-β1 integrin antibody of lung seeding of single osteosarcoma cells in live mice.
Kimura Hiroaki,Tome Yasunori,Momiyama Masashi,Hayashi Katsuhiro,Tsuchiya Hiroyuki,Bouvet Michael,Hoffman Robert M
International journal of cancer
Integrins play a role in tumor growth and metastasis. However, the effect of integrin inhibition has not been visualized on single cancer cells in vivo. In this study, we used a powerful subcellular in vivo imaging model to demonstrate how an anti-integrin antibody affects seeding and growth of osteosarcoma cells on the lung. The 143B human osteosarcoma cell line, expressing red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nucleus, was established. Such double-labeled cells enable imaging of apoptosis and mitosis and other nuclear-cytoplasmic dynamics. Using the double-labeled osteosarcoma cells, single cancer-cell seeding in the lung after i.v. injection of osteosarcoma cells was imaged. The anti-β1 integrin monoclonal antibody, AIIB2, greatly inhibited the seeding of cancer cells on the lung (experimental metastasis) while a control antibody had no effect. To image the efficacy of the anti-integrin antibody on spontaneous metastasis, mice with orthotopically-growing 143B-RFP cells in the tibia were also treated with AIIB2 or control anti-rat IgG1 antibody. After 3 weeks treatment, mice were sacrificed and primary tumors and lung metastases were evaluated with fluorescence imaging. AIIB2 significantly inhibited spontaneous lung metastasis but not primary tumor growth, possibly due to inhibition of lung seeding of the cancer cells as imaged in the experimental metastasis study. AIIB2 treatment also increased survival of mice with orthotopically growing 143B-RFP.
BMTP-11 is active in preclinical models of human osteosarcoma and a candidate targeted drug for clinical translation.
Lewis Valerae O,Devarajan Eswaran,Cardó-Vila Marina,Thomas Dafydd G,Kleinerman Eugenie S,Marchiò Serena,Sidman Richard L,Pasqualini Renata,Arap Wadih
Proceedings of the National Academy of Sciences of the United States of America
Osteosarcoma occurs predominantly in children and young adults. High-grade tumors require multidisciplinary treatment consisting of chemotherapy in the neoadjuvant and adjuvant settings, along with surgical intervention. Despite this approach, death from respiratory failure secondary to the development and progression of pulmonary metastases remains a significant problem. Here, we identify the IL-11 receptor α subunit (IL-11Rα) as a cell surface marker of tumor progression that correlates with poor prognosis in patients with osteosarcoma. We also show that both IL-11Rα and its ligand, IL-11, are specifically up-regulated in human metastatic osteosarcoma cell lines; engagement of this autocrine loop leads to tumor cell proliferation, invasion, and anchorage-independent growth in vitro. Consistently, IL-11Rα promotes lung colonization by human metastatic osteosarcoma cells in vivo in an orthotopic mouse model. Finally, we evaluate the IL-11Rα-targeted proapoptotic agent bone metastasis-targeting peptidomimetic (BMTP-11) in preclinical models of primary intratibial osteosarcomas, observing marked inhibition of both tumor growth and lung metastases. This effect was enhanced when BMTP-11 was combined with the chemotherapeutic drug gemcitabine. Our combined data support the development of approaches targeting IL-11Rα, and establish BMTP-11 as a leading drug candidate for clinical translation in patients with high-risk osteosarcoma.
All-Trans Retinoic Acid Prevents Osteosarcoma Metastasis by Inhibiting M2 Polarization of Tumor-Associated Macrophages.
Zhou Qian,Xian Miao,Xiang Senfeng,Xiang Danyan,Shao Xuejing,Wang Jincheng,Cao Ji,Yang Xiaochun,Yang Bo,Ying Meidan,He Qiaojun
Cancer immunology research
M2-polarized tumor-associated macrophages (TAM) play a critical role in cancer invasion and metastasis. Here, we report that M2 macrophages enhanced metastasis of K7M2 WT osteosarcoma cells to the lungs in mice, thus establishing M2 TAMs as a therapeutic target for blocking osteosarcoma metastasis. We found that retinoic acid (ATRA) inhibited osteosarcoma metastasis via inhibiting the M2 polarization of TAMs. ATRA suppressed IL13- or IL4-induced M2-type macrophages, and then inhibited migration of osteosarcoma cells as promoted by M2-type macrophages ATRA reduced the number of pulmonary metastatic nodes of osteosarcoma and decreased expression of M2-type macrophages in metastatic nodes both in intravenous injection and orthotopic transplantation models. ATRA's effect was independent of conventional STAT3/6 or C/EBPβ signaling, which regulate M2-like polarization of macrophages. Quantitative genomic and functional analyses revealed that MMP12, a macrophage-secreted elastase, was elevated in IL13-skewed TAM polarization, whereas ATRA treatment downregulated IL13-induced secretion of MMP12. This downregulation correlates with the antimetastasis effect of ATRA. Our results show the role of TAM polarization in osteosarcoma metastasis, identify a therapeutic opportunity for antimetastasis treatment, and indicate ATRA treatment as an approach for preventing osteosarcoma metastasis via M2-type polarization intervention. .
Targeting the CK1α/CBX4 axis for metastasis in osteosarcoma.
Wang Xin,Qin Ge,Liang Xiaoting,Wang Wen,Wang Zhuo,Liao Dan,Zhong Li,Zhang Ruhua,Zeng Yi-Xin,Wu Yuanzhong,Kang Tiebang
Osteosarcoma, an aggressive malignant cancer, has a high lung metastasis rate and lacks therapeutic target. Here, we reported that chromobox homolog 4 (CBX4) was overexpressed in osteosarcoma cell lines and tissues. CBX4 promoted metastasis by transcriptionally up-regulating Runx2 via the recruitment of GCN5 to the Runx2 promoter. The phosphorylation of CBX4 at T437 by casein kinase 1α (CK1α) facilitated its ubiquitination at both K178 and K280 and subsequent degradation by CHIP, and this phosphorylation of CBX4 could be reduced by TNFα. Consistently, CK1α suppressed cell migration and invasion through inhibition of CBX4. There was a reverse correlation between CK1α and CBX4 in osteosarcoma tissues, and CK1α was a valuable marker to predict clinical outcomes in osteosarcoma patients with metastasis. Pyrvinium pamoate (PP) as a selective activator of CK1α could inhibit osteosarcoma metastasis via the CK1α/CBX4 axis. Our findings indicate that targeting the CK1α/CBX4 axis may benefit osteosarcoma patients with metastasis.
Multiregion Sequencing Reveals the Genetic Heterogeneity and Evolutionary History of Osteosarcoma and Matched Pulmonary Metastases.
Wang Di,Niu Xiaohui,Wang Zhijie,Song Cheng-Li,Huang Zhen,Chen Ke-Neng,Duan Jianchun,Bai Hua,Xu Jiachen,Zhao Jun,Wang Yu,Zhuo Minglei,Xie X Sunney,Kang Xiaozheng,Tian Yanhua,Cai Liangliang,Han Jie-Fei,An Tongtong,Sun Yu,Gao Shugeng,Zhao Jun,Ying Jianming,Wang Luhua,He Jie,Wang Jie
Osteosarcoma is the most common primary bone malignancy, and the lung is the most frequent site of metastasis. The limited understanding of the tumoral heterogeneity and evolutionary process of genomic alterations in pulmonary metastatic osteosarcoma impedes development of novel therapeutic strategies. Here we systematically illustrate the genomic disparities between primary tumors and corresponding pulmonary metastatic tumors by multiregional whole-exome and whole-genome sequencing in 86 tumor regions from 10 patients with osteosarcoma. Metastatic tumors exhibited a significantly higher mutational burden and genomic instability compared with primary tumors, possibly due to accumulation of mutations caused by a greater number of alterations in DNA damage response genes in metastatic tumors. Integrated analysis of the architecture and relationships of subclones revealed a dynamic mutational process and diverse dissemination patterns of osteosarcoma during pulmonary metastasis (6/10 with linear and 4/10 with parallel evolutionary patterns). All patients demonstrated more significant intertumoral rather than intratumoral heterogeneity between primary tumors and metastatic tumors. Mutated genes were enriched in the pathway at both the early and late stages of tumor evolution and in the pathway at the metastatic stage. Conversely, metastatic tumors showed improved immunogenicity, including higher neoantigen load, elevated PD-L1 expression, and tumor-infiltrating lymphocytes than the corresponding primary tumors. Our study is the first to report the dynamic evolutionary process and temporospatial tumor heterogeneity of pulmonary metastatic osteosarcoma, providing new insights for diagnosis and potential therapeutic strategies for pulmonary metastasis. SIGNIFICANCE: High-throughput sequencing of primary and metastatic osteosarcoma provides new insights into the diagnosis of and potential clinical therapeutic strategies for pulmonary metastasis.
Genetic and clonal dissection of osteosarcoma progression and lung metastasis.
Xu Huaiyuan,Zhu Xiaojun,Bao Hua,Wh Shek Tony,Huang Zongwen,Wang Yongqian,Wu Xue,Wu Yong,Chang Zhili,Wu Shuyu,Tang Qinglian,Zhang Huizhong,Han Anjia,Mc Cheung Kenneth,Zou Changye,Yau Raymond,Ho Wai-Yip,Huang Gang,Batalha Sellma,Lu Jinchang,Song Guohui,Kang Yao,Shao Yang W,Lam Ying Lee,Shen Jingnan,Wang Jin
International journal of cancer
Osteosarcoma is a primary malignant bone tumor that has a high potential to metastasize to lungs. Little is known about the mechanisms underlying the dissemination of OS cancer cells to lungs. We performed whole exome sequencing of 13 OS primary tumors, with matched lung metastases and normal tissues. Phylogenetic analyses revealed that lung metastatic tumors often harbor clones that are nonexistent or rare in the matched primary OS tumors. Spatially and temporally separated lung metastases were from parallel seeding events with a polyphyletic pattern. Loss of TP53 or RB1 is among the early events during OS tumorigenesis, while loss of PTEN is involved at the later stages associated with lung metastases. Finally, KEAP1 was identified as a novel biomarker for increased metastatic risk. Patients whose primary tumors harbored KEAP1 amplification have significantly poorer lung-metastasis free survival. This finding was validated in two independent datasets. Further, in vitro experiments exhibited that KEAP1 depletion suppressed the invasion of OS cells. Our findings uncover the patterns of clonal evolution during OS progression and highlight KEAP1 as a novel candidate associated with the risk of lung metastasis in OS patients.
Specific Small-Molecule NIR-II Fluorescence Imaging of Osteosarcoma and Lung Metastasis.
Zhou Hui,Yi Wanrong,Li Anguo,Wang Bo,Ding Qihang,Xue Liru,Zeng Xiaodong,Feng Yanzhi,Li Qianqian,Wang Tian,Li Yang,Cheng Xiaoding,Tang Lin,Deng Zixin,Wu Mingfu,Xiao Yuling,Hong Xuechuan
Advanced healthcare materials
Osteosarcoma is an aggressive tumor of mesenchymal origin that is more likely to spread to the lung than others, with a major impact on patients' prognosis. The optimal imaging method that can reliably detect or exclude pulmonary metastases from osteosarcoma is still scarce. Herein, two homologous types of fluorescent probes CH1055-PEG-PT and CH1055-PEG-Affibody, which show highly promising results for targeting imaging of osteosarcoma and its lung metastasis, respectively, are designed and synthesized. It is found that the NIR-II imaging quality of CH1055-PEG-PT is far superior to that of computed tomography for the early in vivo 143B tumor imaging, and this probe-guided surgery for accurate resection of 143B tumor is further performed. The high-resolution visualization of primary and micrometastatic lung lesions of osteosarcoma by using CH1055-PEG-Affibody is also demonstrated. Therefore, the attractive imaging properties of CH1055-PEG-PT and CH1055-PEG-Affibody, including high levels of uptakes, and high spatial and temporal resolution, open up opportunities for molecular imaging and clinical translation of osteosarcoma and its lung metastasis in the unique second near-infrared window.
Chemical Design of Both a Glutathione-Sensitive Dimeric Drug Guest and a Glucose-Derived Nanocarrier Host to Achieve Enhanced Osteosarcoma Lung Metastatic Anticancer Selectivity.
Su Lu,Li Richen,Khan Sarosh,Clanton Ryan,Zhang Fuwu,Lin Yen-Nan,Song Yue,Wang Hai,Fan Jingwei,Hernandez Soleil,Butters Andrew S,Akabani Gamal,MacLoughlin Ronan,Smolen Justin,Wooley Karen L
Journal of the American Chemical Society
Although nanomedicines have been pursued for nearly 20 years, fundamental chemical strategies that seek to optimize both the drug and drug carrier together in a concerted effort remain uncommon yet may be powerful. In this work, two block polymers and one dimeric prodrug molecule were designed to be coassembled into degradable, functional nanocarriers, where the chemistry of each component was defined to accomplish important tasks. The result is a poly(ethylene glycol) (PEG)-protected redox-responsive dimeric paclitaxel (diPTX)-loaded cationic poly(d-glucose carbonate) micelle (diPTX@CPGC). These nanostructures showed tunable sizes and surface charges and displayed controlled PTX drug release profiles in the presence of reducing agents, such as glutathione (GSH) and dithiothreitol (DTT), thereby resulting in significant selectivity for killing cancer cells over healthy cells. Compared to free PTX and diPTX, diPTX@CPGC exhibited improved tumor penetration and significant inhibition of tumor cell growth toward osteosarcoma (OS) lung metastases with minimal side effects both in vitro and in vivo, indicating the promise of diPTX@CPGC as optimized anticancer therapeutic agents for treatment of OS lung metastases.
Positron Emission Tomography Detects Expression of Disialoganglioside GD2 in Mouse Models of Primary and Metastatic Osteosarcoma.
Butch Elizabeth R,Mead Paul E,Amador Diaz Victor,Tillman Heather,Stewart Elizabeth,Mishra Jitendra K,Kim Jieun,Bahrami Armita,Dearling Jason L J,Packard Alan B,Stoddard Shana V,Vāvere Amy L,Han Yuanyuan,Shulkin Barry L,Snyder Scott E
The cell membrane glycolipid GD2 is expressed by multiple solid tumors, including 88% of osteosarcomas and 98% of neuroblastomas. However, osteosarcomas are highly heterogeneous, with many tumors exhibiting GD2 expression on <50% of the individual cells, while some tumors are essentially GD2-negative. Anti-GD2 immunotherapy is the current standard of care for high-risk neuroblastoma, but its application to recurrent osteosarcomas, for which no effective therapies exist, has been extremely limited. This is, in part, because the standard assays to measure GD2 expression in these heterogeneous tumors are not quantitative and are subject to tissue availability and sampling bias. To address these limitations, we evaluated a novel, sensitive radiotracer [Cu]Cu-Bn-NOTA-hu14.18K322A to detect GD2 expression in osteosarcomas (six patient-derived xenografts and one cell line) using positron emission tomography (PET). Tumor uptake of the radiolabeled, humanized anti-GD2 antibody [Cu]Cu-Bn-NOTA-hu14.18K322A was 7-fold higher in modestly GD2-expressing osteosarcomas (32% GD2-positive cells) than in a GD2-negative tumor (9.8% vs. 1.3% of the injected dose per cc, respectively). This radiotracer also identified lesions as small as 29 mm in a 34% GD2-positive model of metastatic osteosarcoma of the lung. Radiolabeled antibody accumulation in patient-derived xenografts correlated with GD2 expression as measured by flow cytometry (Pearson = 0.88, = 0.01), distinguishing moderately GD2-expressing osteosarcomas (32%-69% GD2-positive cells) from high GD2 expressors (>99%, < 0.05). These results support the utility of GD2 imaging with PET to measure GD2 expression in osteosarcoma and thus maximize the clinical impact of anti-GD2 immunotherapy. SIGNIFICANCE: assessment of all GD2-positive osteosarcoma sites with a novel PET radiotracer could significantly impact anti-GD2 immunotherapy patient selection and enable noninvasive probing of correlations between target expression and therapeutic response.
Ex vivo screen identifies CDK12 as a metastatic vulnerability in osteosarcoma.
Bayles Ian,Krajewska Malgorzata,Pontius W Dean,Saiakhova Alina,Morrow James J,Bartels Cynthia,Lu Jim,Faber Zachary J,Fedorov Yuriy,Hong Ellen S,Karnuta Jaret M,Rubin Brian,Adams Drew J,George Rani E,Scacheri Peter C
The Journal of clinical investigation
Despite progress in intensification of therapy, outcomes for patients with metastatic osteosarcoma (OS) have not improved in thirty years. We developed a system that enabled preclinical screening of compounds against metastatic OS cells in the context of the native lung microenvironment. Using this strategy to screen a library of epigenetically targeted compounds, we identified inhibitors of CDK12 to be most effective, reducing OS cell outgrowth in the lung by more than 90% at submicromolar doses. We found that knockout of CDK12 in an in vivo model of lung metastasis significantly decreased the ability of OS to colonize the lung. CDK12 inhibition led to defects in transcription elongation in a gene length- and expression-dependent manner. These effects were accompanied by defects in RNA processing and altered the expression of genes involved in transcription regulation and the DNA damage response. We further identified OS models that differ in their sensitivity to CDK12 inhibition in the lung and provided evidence that upregulated MYC levels may mediate these differences. Our studies provided a framework for rapid preclinical testing of compounds with antimetastatic activity and highlighted CDK12 as a potential therapeutic target in OS.
Positively selected enhancer elements endow osteosarcoma cells with metastatic competence.
Morrow James J,Bayles Ian,Funnell Alister P W,Miller Tyler E,Saiakhova Alina,Lizardo Michael M,Bartels Cynthia F,Kapteijn Maaike Y,Hung Stevephen,Mendoza Arnulfo,Dhillon Gursimran,Chee Daniel R,Myers Jay T,Allen Frederick,Gambarotti Marco,Righi Alberto,DiFeo Analisa,Rubin Brian P,Huang Alex Y,Meltzer Paul S,Helman Lee J,Picci Piero,Versteeg Henri H,Stamatoyannopoulos John A,Khanna Chand,Scacheri Peter C
Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic human tumors and between near isogenic pairs of highly lung metastatic and nonmetastatic osteosarcoma cell lines. We term these regions metastatic variant enhancer loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster nonrandomly in the genome, indicating that activity of these enhancers and expression of their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as that encoding coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for antimetastatic therapies.
Tegavivint and the β-Catenin/ALDH Axis in Chemotherapy-Resistant and Metastatic Osteosarcoma.
Nomura Motonari,Rainusso Nino,Lee Yi-Chien,Dawson Brian,Coarfa Cristian,Han Ruolan,Larson Jeffrey L,Shuck Ryan,Kurenbekova Lyazat,Yustein Jason T
Journal of the National Cancer Institute
BACKGROUND:The Wnt/β-catenin pathway is closely associated with osteosarcoma (OS) development and metastatic progression. We investigated the antitumor activity of Tegavivint, a novel β-catenin/transducin β-like protein 1 (TBL1) inhibitor, against OS employing in vitro, ex vivo, and in vivo cell line and patient-derived xenograft (PDX) models that recapitulate high risk disease. METHODS:The antitumor efficacy of Tegavivint was evaluated in vitro using established OS and PDX-derived cell lines. Use of an ex vivo three-dimensional pulmonary metastasis assay assessed targeting of β-catenin activity during micro- and macrometastatic development. The in vivo activity of Tegavivint was evaluated using chemoresistant and metastatic OS PDX models. Gene and protein expression were quantified by quantitative Reverse transcription polymerase chain reaction or immunoblot analysis. Bone integrity was determined via microCT. All statistical tests were two-sided. RESULTS:Tegavivint exhibited antiproliferative activity against OS cells in vitro and actively reduced micro- and macrometastatic development ex vivo. Multiple OS PDX tumors (n = 3), including paired patient primary and lung metastatic tumors with inherent chemoresistance, were suppressed by Tegavivint in vivo. We identified that metastatic lung OS cell lines (n = 2) exhibited increased stem cell signatures, including enhanced concomitant aldehyde dehydrogenase (ALDH1) and β-catenin expression and downstream activity, which were suppressed by Tegavivint (ALDH1: control group, mean relative mRNA expression = 1.00, 95% confidence interval [CI] = 0.68 to 1.22 vs Tegavivint group, mean = 0.011, 95% CI = 0.0012 to 0.056, P < .001; β-catenin: control group, mean relative mRNA expression = 1.00, 95% CI = 0.71 to 1.36 vs Tegavivint group, mean = 0.45, 95% CI = 0.36 to 0.52, P < .001). ALDH1high PDX-derived lung OS cells, which demonstrated enhanced metastatic potential compared with ALDHlow cells in vivo, were sensitive to Tegavivint. Toxicity studies revealed decreased bone density in male Tegavivint-treated mice (n = 4 mice per group). CONCLUSIONS:Tegavivint is a promising therapeutic agent for advanced stages of OS via its targeting of the β-catenin/ALDH1 axis.
The cancer-related transcription factor RUNX2 modulates expression and secretion of the matricellular protein osteopontin in osteosarcoma cells to promote adhesion to endothelial pulmonary cells and lung metastasis.
Villanueva Francisco,Araya Hector,Briceño Pedro,Varela Nelson,Stevenson Andres,Jerez Sofia,Tempio Fabian,Chnaiderman Jonas,Perez Carola,Villarroel Milena,Concha Emma,Khani Farzaneh,Thaler Roman,Salazar-Onfray Flavio,Stein Gary S,van Wijnen Andre J,Galindo Mario
Journal of cellular physiology
Osteosarcomas are bone tumors that frequently metastasize to the lung. Aberrant expression of the transcription factor, runt-related transcription factor 2 (RUNX2), is a key pathological feature in osteosarcoma and associated with loss of p53 and miR-34 expression. Elevated RUNX2 may transcriptionally activate genes mediating tumor progression and metastasis, including the RUNX2 target gene osteopontin (OPN/SPP1). This gene encodes a secreted matricellular protein produced by osteoblasts to regulate bone matrix remodeling and tissue calcification. Here we investigated whether and how the RUNX2/OPN axis regulates lung metastasis of osteosarcoma. Importantly, RUNX2 depletion attenuates lung metastasis of osteosarcoma cells in vivo. Using next-generation RNA-sequencing, protein-based assays, as well as the loss- and gain-of-function approaches in selected osteosarcoma cell lines, we show that osteopontin messenger RNA levels closely correlate with RUNX2 expression and that RUNX2 controls the levels of secreted osteopontin. Elevated osteopontin levels promote heterotypic cell-cell adhesion of osteosarcoma cells to human pulmonary microvascular endothelial cells, but not in the presence of neutralizing antibodies. Collectively, these findings indicate that the RUNX2/OPN axis regulates the ability of osteosarcoma cells to attach to pulmonary endothelial cells as a key step in metastasis of osteosarcoma cells to the lung.
Genetic determinants of childhood and adult height associated with osteosarcoma risk.
Zhang Chenan,Morimoto Libby M,de Smith Adam J,Hansen Helen M,Gonzalez-Maya Julio,Endicott Alyson A,Smirnov Ivan V,Metayer Catherine,Wei Qingyi,Eward William C,Wiemels Joseph L,Walsh Kyle M
BACKGROUND:Although increased height has been associated with osteosarcoma risk in previous epidemiologic studies, to the authors' knowledge the relative contribution of stature during different developmental timepoints remains unclear. Furthermore, the question of how genetic determinants of height impact osteosarcoma etiology remains unexplored. Genetic variants associated with stature in previous genome-wide association studies may be biomarkers of osteosarcoma risk. METHODS:The authors tested the associations between osteosarcoma risk and polygenic scores for adult height (416 variants), childhood height (6 variants), and birth length (5 variants) in 864 osteosarcoma cases and 1879 controls of European ancestry. RESULTS:Each standard deviation increase in the polygenic score for adult height, corresponding to a 1.7-cm increase in stature, was found to be associated with a 1.10-fold increase in the risk of osteosarcoma (95% confidence interval [95% CI], 1.01-1.19; P =.027). Each standard deviation increase in the polygenic score for childhood height, corresponding to a 0.5-cm increase in stature, was associated with a 1.10-fold increase in the risk of osteosarcoma (95% CI, 1.01-1.20; P =.023). The polygenic score for birth length was not found to be associated with osteosarcoma risk (P =.11). When adult and childhood height scores were modeled together, they were found to be independently associated with osteosarcoma risk (P =.037 and P = .043, respectively). An expression quantitative trait locus for cartilage intermediate layer protein 2 (CILP2), rs8103992, was significantly associated with osteosarcoma risk after adjustment for multiple comparisons (odds ratio, 1.35; 95% CI, 1.16-1.56 [P = 7.93×10 and P =.034]). CONCLUSIONS:A genetic propensity for taller adult and childhood height attainments contributed independently to osteosarcoma risk in the current study data. These results suggest that the biological pathways affecting normal bone growth may be involved in osteosarcoma etiology.
The new insight on the regulatory role of the vitamin D3 in metabolic pathways characteristic for cancerogenesis and neurodegenerative diseases.
Kubis Adriana Maria,Piwowar Agnieszka
Ageing research reviews
Apart from the classical function of regulating intestinal, bone and kidney calcium and phosphorus absorption as well as bone mineralization, there is growing evidence for the neuroprotective function of vitamin D3 through neuronal calcium regulation, the antioxidative pathway, immunomodulation and detoxification. Vitamin D3 and its derivates influence directly or indirectly almost all metabolic processes such as proliferation, differentiation, apoptosis, inflammatory processes and mutagenesis. Such multifactorial effects of vitamin D3 can be a profitable source of new therapeutic solutions for two radically divergent diseases, cancer and neurodegeneration. Interestingly, an unusual association seems to exist between the occurrence of these two pathological states, called "inverse comorbidity". Patients with cognitive dysfunctions or dementia have considerably lower risk of cancer, whereas survivors of cancer have lower prevalence of central nervous system (CNS) disorders. To our knowledge, there are few publications analyzing the role of vitamin D3 in biological pathways existing in carcinogenic and neuropathological disorders.
Interorganellar calcium signaling in the regulation of cell metabolism: A cancer perspective.
Rimessi Alessandro,Pedriali Gaia,Vezzani Bianca,Tarocco Anna,Marchi Saverio,Wieckowski Mariusz R,Giorgi Carlotta,Pinton Paolo
Seminars in cell & developmental biology
Organelles were originally considered to be individual cellular compartments with a defined organization and function. However, recent studies revealed that organelles deeply communicate within each other via Ca exchange. This communication, mediated by specialized membrane regions in close apposition between two organelles, regulate cellular functions, including metabolism and cell fate decisions. Advances in microscopy techniques, molecular biology and biochemistry have increased our understanding of these interorganelle platforms. Research findings suggest that interorganellar Ca signaling, which is altered in cancer, influences tumorigenesis and tumor progression by controlling cell death programs and metabolism. Here, we summarize the available data on the existence and composition of interorganelle platforms connecting the endoplasmic reticulum with mitochondria, the plasma membrane, or endolysosomes. Finally, we provide a timely overview of the potential function of interorganellar Ca signaling in maintaining cellular homeostasis.
Clinical importance of S100A9 in osteosarcoma development and as a diagnostic marker and therapeutic target.
Liu Yongliang,Luo Gongzeng,He Dongyong
OBJECTIVE:S100A9 is a calcium- and zinc-binding molecule of S100 family. The aim of the study was to evaluate the role of S100A9 in osteosarcoma (OS) and its value as a diagnostic and therapeutic target in OS. METHODS:Quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry and microdissection-based mRNA analysis were used to detect S100A9 mRNA and protein expression in OS and normal bone tissues and its potential as a diagnostic marker in OS. In vitro experiments with RNA interference were performed to evaluate the functional role of S100A9 and its potential as a therapeutic target in OS. RESULTS:S100A9 mRNA levels were significantly higher in OS tissues than that of in normal bone tissues. Receiver operating characteristic curves showed that S100A9 could be a useful diagnostic marker in OS. In vitro data showed that inhibition of S100A9 decreased the proliferation and invasiveness of OS cells, and these findings were supported by microarray data. CONCLUSIONS:Assessment of S100A9 mRNA expression is a promising tool for the diagnosis of OS, and S100A9 may be a promising therapeutic target in OS.
Sarcoma-Targeting Peptide-Decorated Polypeptide Nanogel Intracellularly Delivers Shikonin for Upregulated Osteosarcoma Necroptosis and Diminished Pulmonary Metastasis.
Li Suoyuan,Zhang Tao,Xu Weiguo,Ding Jianxun,Yin Fei,Xu Jing,Sun Wei,Wang Hongsheng,Sun Mengxiong,Cai Zhengdong,Hua Yingqi
PURPOSE:Osteosarcoma is the most common primary bone cancer and is notorious for pulmonary metastasis, representing a major threat to pediatric patients. An effective drug targeting osteosarcoma and its lung metastasis is urgently needed. DESIGN:In this study, a sarcoma-targeting peptide-decorated disulfide-crosslinked polypeptide nanogel (STP-NG) was exploited for enhanced intracellular delivery of shikonin (SHK), an extract of a medicinal herb, to inhibit osteosarcoma progression with minimal systemic toxicity. RESULTS:The targeted, loaded nanogel, STP-NG/SHK, killed osteosarcoma cells by inducing RIP1- and RIP3-dependent necroptosis . Necroptosis is a novel cell death form that could be well adapted as an efficient antitumor strategy, the main obstacle of which is its high toxicity. After intravenous injection, STP-NG/SHK efficiently suppressed tumor growth and reduced pulmonary metastasis, offering greater tumor necrosis and higher RIP1 and RIP3 upregulation compared to free SHK or untargeted NG/SHK . Additionally, the treatment with NG/SHK or STP-NG/SHK showed minimal toxicity to normal organs, suggesting low systemic toxicity compared to free SHK. CONCLUSION:The STP-guided intracellular drug delivery system using the necroptosis mechanism showed profound anti-osteosarcoma activity, especially eliminated lung metastasis . This drug formulation may have great potential for treatment of osteosarcoma.
Present Advances and Future Perspectives of Molecular Targeted Therapy for Osteosarcoma.
Shaikh Atik Badshah,Li Fangfei,Li Min,He Bing,He Xiaojuan,Chen Guofen,Guo Baosheng,Li Defang,Jiang Feng,Dang Lei,Zheng Shaowei,Liang Chao,Liu Jin,Lu Cheng,Liu Biao,Lu Jun,Wang Luyao,Lu Aiping,Zhang Ge
International journal of molecular sciences
Osteosarcoma (OS) is a bone cancer mostly occurring in pediatric population. Current treatment regime of surgery and intensive chemotherapy could cure about 60%-75% patients with primary osteosarcoma, however only 15% to 30% can be cured when pulmonary metastasis or relapse has taken place. Hence, novel precise OS-targeting therapies are being developed with the hope of addressing this issue. This review summarizes the current development of molecular mechanisms and targets for osteosarcoma. Therapies that target these mechanisms with updated information on clinical trials are also reviewed. Meanwhile, we further discuss novel therapeutic targets and OS-targeting drug delivery systems. In conclusion, a full insight in OS pathogenesis and OS-targeting strategies would help us explore novel targeted therapies for metastatic osteosarcoma.
Effect of Protein Hydrolysate-Calcium Chelate on Calcium Absorption Metabolism and Gut Microbiota Composition in Low-Calcium Diet-Fed Rats.
Hua Pengpeng,Xiong Yu,Yu Zhiying,Liu Bin,Zhao Lina
In our current investigation, we evaluated the effect of protein hydrolysate (CPPH) and protein hydrolysate-calcium chelate (CPPH-Ca) on calcium absorption and gut microbiota composition, as well as their in vivo regulatory mechanism in SD rats fed low-calcium diets. Potent major compounds in CPPH were characterized by HPLC-MS/MS, and the calcium-binding mechanism was investigated through ultraviolet and infrared spectroscopy. Using high-throughput next-generation 16S rRNA gene sequencing, we analyzed the composition of gut microbiota in rats. Our study showed that HCPPH-Ca increased the levels of body weight gain, serum Ca, bone activity, bone mineral density (BMD) and bone mineral content (BMC), while decreased serum alkaline phosphatase (ALP) and inhibited the morphological changes of bone. HCPPH-Ca up-regulated the gene expressions of transient receptor potential cation V5 (TRPV5), TRPV6, calcium-binding protein-D9k (CaBP-D9k) and a calcium pump (plasma membrane Ca-ATPase, PMCA1b). It also improved the abundances of and . and were both positively correlated with calcium absorption. Collectively, these findings illustrate the potential of HCPPH-Ca as an effective calcium supplement.