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Background Soft tissue sarcomas are a diverse group of rare malignant tumours, mostly occurring in the lower extremities. Amputations are necessary for achieving local control when the soft tissue sarcomas are too large and/or have neurovascular involvement. Patients who require amputation have a poorer prognosis than those who undergo limb-salvage surgery. Patients and Methods We investigated the tumour characteristics and the clinical outcomes in 55 patients with primary soft tissue sarcomas, who underwent amputation. We excluded patients with amputation performed distal to the wrist or ankle joints and those with recurrent soft tissue sarcomas. Results The mean tumour size was 11.1 cm. Hip disarticulation was performed in 6 patients, 20 underwent above the knee amputation, 8 underwent knee disarticulation and 12 underwent below the knee amputation. Shoulder disarticulation was performed in three patients, five underwent above the elbow amputation, and one underwent below the elbow amputation. The 5-year disease-specific survival rate was 52.8%. The 5-year recurrence-free survival rate and 5-year metastasis-free survival rates were 90.1% and 38.5%, respectively. Larger tumour size, age and the distant metastases at first presentation were predictors of poor prognosis for survival in multivariate analysis. Twenty-eight patients could walk using artificial limbs. The level of amputation (above versus below the knee) showed a significant difference in achieving independent gait. Conclusion Amputation is a useful treatment option for achieving local control in patients with large soft tissue sarcomas. Patients had an opportunity of walking, especially for those who underwent below the knee amputation.

Chondrosarcoma is the second most common primary malignant bone tumor. In this multicenter study, we sought to evaluate the disease-specific survival (DSS) and disease-free survival (DFS), and prognostic factors in patients with dedifferentiated chondrosarcoma (DDCS) or grade 3 chondrosarcoma (G3CS) in Japan. We retrospectively investigated the treatment outcomes and prognostic factors in 62 patients with DDCS and 19 patients with G3CS at 15 institutions participating in the Japanese Musculoskeletal Oncology Group. We also clarified significant clinicopathological factors for oncological outcomes. In surgery for primary lesions aimed at cure, a histologically negative margin (R0) was obtained in 93% (14/15) of patients with G3CS and 100% (49/49) of patients with DDCS. The 5-year DSS was 18.5% in patients with DDCS and 41.7% in patients with G3CS (p = 0.13). Local control was obtained in 80% (12/15) and 79.6% (39/49) of patients with G3CS and DDCS in the primary lesion after surgery with a wide surgical margin, respectively. In multivariate analysis, stage and no treatment/palliative treatment for the primary lesion were independent prognostic factors for DSS of DDCS, and age and no treatment/palliative treatment for DSS of G3CS. The 5-year DFS rate was 22.8% in 26 patients with DDCS who did not receive adjuvant chemotherapy, and 21.4% in 14 patients who received adjuvant chemotherapy. The prognosis of DDCS remains poor, although R0 resection was carried out in most cases. Effective and/or intensive chemotherapeutic regimens or agents should be considered or developed for patients with high-grade chondrosarcoma, particularly for those with DDCS.

Tomoki Nakamura,1 Kunihiro Asanuma,1 Motoshi Takao,2 Takashi Yamanaka,3 Hiroshi Koike,4 Toyofumi F Chen-Yoshikawa,5 Satoshi Tsukushi,6 Hiroaki Kuroda,7 Eiji Kozawa,8 Masaaki Sano,9 Hisaki Aiba,10 Ryoichi Nakanishi,11 Akihito Nagano,12 Kenji Yamada,13 Yoji Shido,14 Katsuhisa Kawanami,15 Yuya Izubuchi,16 Akihiro Sudo,1 Yoshihiro Nishida4,17 1Department of Orthopedic Surgery, Mie University Graduate School of Medicine, Tsu, Mie, Japan; 2Department of Thoracic Surgery, Mie University Graduate School of Medicine, Tsu, Mie, Japan; 3Department of Radiology, Mie University Graduate School of Medicine, Tsu, Mie, Japan; 4Department of Orthopedic Surgery, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; 5Department of Thoracic Surgery, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan; 6Department of Orthopedic Surgery, Aichi Cancer Center Hospital, Nagoya, Aichi, Japan; 7Department of Thoracic Surgery, Aichi Cancer Center Hospital, Nagoya, Aichi, Japan; 8Department of Orthopedic Surgery, Nagoya Memorial Hospital, Nagoya, Aichi, Japan; 9Department of Thoracic Surgery, Nagoya Memorial Hospital, Nagoya, Aichi, Japan; 10Department of Orthopedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Aichi, Japan; 11Department of Oncology, Immunology and Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Aichi, Japan; 12Department of Orthopedic Surgery, Gifu University Graduate School of Medicine, Gifu, Gifu, Japan; 13Department of Musculoskeletal Oncology, Okazaki City Hospital, Okazaki, Aichi, Japan; 14Department of Orthopedic Surgery, Hamamatsu Medical University, Hamamatsu, Sizuoka, Japan; 15Department of Orthopedic Surgery, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan; 16Department of Orthopedics and Rehabilitation Medicine, University of Fukui Faculty of Medical Sciences, Eiheiji, Fukui, Japan; 17Department of Rehabilitation, Nagoya University Hospital, Nagoya, Aichi, JapanCorrespondence: Tomoki NakamuraDepartment of Orthopaedic Surgery, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507, JapanTel +81592315022Fax +81592315211Email tomoki66@med.mie-u.ac.jpPurpose: Here, we investigated the oncological outcomes of lung metastasectomy and/or radiofrequency ablation (RFA) of 92 patients with soft tissue sarcoma (STS) at nine institutions.Methods: The study cohort included 65 men and 27 women with a mean age of 59 years at the time of metastasis. The mean follow-up duration was 51 months. All patients underwent metastasectomy and/or RFA for lung metastasis.Results: The mean maximum size of the initial lung metastasis was 14.6 mm. At the initial evaluation, 41 patients had a single metastasis, whereas 51 patients had multiple metastases. The mean number of metastasectomies and/or RFA was 2 per patient. A total of 70 patients underwent lung metastasectomy, whereas the other 13 underwent lung RFA. The remaining nine patients underwent both RFA and metastasectomy. The 5-year post-metastatic survival rate was 52%. The patients who underwent complete treatment for the initial metastasis had better post-metastatic survival rates than those who underwent incomplete treatment. A univariate analysis of all possible prognostic factors for complete treatment confirmed the predictive value of disease-free interval, metastasis at initial presentation, distribution, tumor size, and number of lung metastases. Of the 92 patients, 74 underwent complete treatment for initial metastasis; in these patients, univariate and multivariate analyses showed that a smaller tumor size and single-lung metastasis were prognostic factors for superior post-metastatic survival. The patients with a smaller (< 11.5 mm) single metastasis had better post-metastasis survival. The 5-year post-metastatic survival rates were 89.9% for patients with a smaller (< 11.5 mm) single metastasis versus 22.7% for patients with larger (> 11.5 mm) and multiple metastases.Discussion: We propose that complete treatment for lung metastasis in patients with STS may improve post-metastatic survival rates. Furthermore, tumor number and size are important variables for clinical decision-making.Keywords: lung metastasis, metastasectomy, radiofrequency ablation, soft tissue sarcoma

Purpose: Osteochondral destruction and high recurrence rate after surgery are major concerns during the treatment course of tenosynovial giant cell tumor. The aims of this study were to elucidate rates of local recurrence and osteochondral destruction, and demographic factors correlated with osteochondral destruction and recurrence rate after surgery. Methods: Eighty surgically treated patients (knee: 49, ankle and foot: 12, hip: 10, others: 9) were included in this study. Factors including age, disease type (diffuse/localized), location, existence of osteochondral destruction were analyzed to be correlated with local recurrence or development/progression of osteochondral destruction. Results: The 5-year local recurrence free survival rate was 71.4 %. Diffuse type (n=59, localized: n=21) (P=0.023) and knee location (P=0.002) were independent risk factors for local recurrence. Diffuse type (P=0. 009) was a significant risk factor, and knee location (P=0. 001) was a negative factor for osteochondral destruction at the initial examination. Progression of osteochondral destruction was observed more often in cases with local recurrence (P=0.040) and findings of osteochondral destruction at the initial examination (P=0.029).Conclusions: Diffuse type is a factor that should be noted for both local recurrence and osteochondral destruction, and local recurrence occurs but osteochondral destruction is less observed for knee location.

The efficacy and safety of eribulin in Japanese patients with advanced soft-tissue sarcomas (STS) have not been evaluated in a large-scale cohort study. Thus, we aimed to investigate the clinical outcome of 82 Japanese patients with STS receiving eribulin across multiple study centers retrospectively. Of 82 STS patients receiving eribulin treatment, 13 were treated for locally unresectable tumor, 46 for metastasis, and 23 for both. The primary endpoint of this study was to evaluate the efficacy of eribulin against STS. The median age was 60 years. Thirty-seven were diagnosed with L-sarcoma (leiomyosarcoma or liposarcoma) and 45 had non-L-sarcoma. The median progression-free survival (PFS) for all patients was 2.7 months, with 3.4 months in those with L-sarcoma and 2.2 months in those with non-L-sarcoma. Patients with L-sarcoma showed a better PFS than those with non-L-sarcoma. Overall, the median survival time was 11.1 months, and 12.3 months and 7.9 months in patients with L-sarcoma and non-L-sarcoma, respectively; however, there was no significant differences between the groups. The prognostic significance of PS = 0 and both existence of local and metastatic STS was evaluated by multivariate analysis. We also evaluated the overall survival (OS) in patients with undifferentiated pleomorphic sarcoma (UPS) and other non-L-sarcomas. Patients with UPS had better OS than those with the other non-L-sarcomas. In conclusion, there was a significant difference in PFS between patients with L-sarcoma and non-L-sarcoma following treatment with eribulin. The anti-tumor potential of eribulin was evident in patients with UPS.

Postoperative complications of thoracic wall resection include respiratory complications, skin necrosis and infection. The aim of the present study was to examine postoperative complications in patients who required combined thoracic wall resection during the surgical removal of a tumor. The present study included 68 patients; there were 50 patients with lung tumors and 18 patients with musculoskeletal tumors. The clinical factors associated with complications were compared between the two groups. Preoperative and postoperative pulmonary function tests were performed to examine the residual pulmonary function in 16 patients. Thoracic cage reconstruction was performed in 46 patients. Postoperative complications occurred in 30 (44.1%) patients, and one patient died from postoperative pneumonitis. Compared with the pulmonary function preoperative test results, the postoperative results revealed a decrease in the mean vital capacity percentage and an increase in the mean forced expiratory volume within 1 sec as a percent of the forced vital capacity. In patients with lung tumors, pneumonectomy can result in an increased rate of complications following thoracic wall resection. Residual pulmonary function is affected by impaired thoracic cage expansion and removal of the lung. However, the results of the present study demonstrated that these complications can be somewhat stabilized by thoracic wall reconstruction.

Several types of soft tissue sarcomas have peripheral infiltrative growth characteristics called tail-like lesions. The efficacy of neoadjuvant therapy for tumors with tail-like lesions has not been elucidated. From 2012 to 2019, we analyzed 36 patients with soft tissue sarcoma with tail-like lesions treated with neoadjuvant therapy, including chemotherapy, radiotherapy, or both. The effect of neoadjuvant therapy on the tail sign was investigated by analyzing the change in tail-like lesions during neoadjuvant therapy and histological responses. The median length of the tail-like lesion reduced from 29.5 mm at initiation to 19.5 mm after neoadjuvant therapy. The extent of shrinkage in tail-like lesions was related to the histopathological responses in the main part of the tumor. Complete disappearance of the tail-like lesion was observed in 12 patients, however, it was not related to achieving a microscopically negative margin. The oncologic outcomes did not significantly differ between cases with and without the complete disappearance of tail-like lesions. This study indicated that the shrinkage of tail-like lesions did not have a significant effect on complete resection or improvements of clinical outcomes. A more comprehensive evaluation is needed to elaborate on the surgical strategy.

Hyaluronan (HA) has been shown to play important roles in the growth, invasion and metastasis of malignant tumors. Our previous study showing that high HA expression in malignant peripheral nerve sheath tumors (MPNST) is predictive of poor patient prognosis, prompted us to speculate that inhibition of HA synthesis in MPNST might suppress the tumorigenicity. The aim of our study was to investigate the antitumor effects of 4-methylumbelliferone (MU), an HA synthesis inhibitor, on human MPNST cells and tissues. The effects of MU on HA accumulation and tumorigenicity in MPNST cells were analyzed in the presence or absence of MU in an in vitro as well as in vivo xenograft model using human MPNST cell lines, sNF96.2 (primary recurrent) and sNF02.2 (metastatic). MU significantly inhibited cell proliferation, migration and invasion in both MPNST cell lines. HA binding protein (HABP) staining, particle exclusion assay and quantification of HA revealed that MU significantly decreased HA accumulation in the cytoplasms and pericellular matrices in both MPNST cell lines. The expression levels of HA synthase2 (HAS2) and HA synthase3 (HAS3) mRNA were downregulated after treatment with MU. MU induced apoptosis of sNF96.2 cells, but not sNF02.2 cells. MU administration significantly inhibited the tumor growth of sNF96.2 cells in the mouse xenograft model. To the best of our knowledge, our study demonstrates for the first time the antitumor effects of MU on human MPNST mediated by inhibition of HA synthesis. Our results suggest that MU may be a promising agent with novel antitumor mechanisms for MPNST.

Desmoid tumors are benign mesenchymal neoplasms with a locally aggressive nature. The mutational status of β‐catenin gene (CTNNB1) is presumed to affect the tumorous activity of the cells. In this study, we isolated three kinds of desmoid cell with different CTNNB1 status, and compared their characteristics. Cells were isolated from three patients with abdominal wall desmoid during surgery, all of which were resistant to meloxicam treatment. The mutational status of the CTNNB1 exon 3 was determined for both parental tumor tissues and isolated cultured cells. β‐catenin expression was determined with immunohistochemistry. Responsiveness to meloxicam was investigated with MTS assay together with COX‐2 immunostaining. mRNA expressions of downstream molecules of Wnt/β‐catenin pathway were determined with real‐time RT‐PCR. Three kinds of cell isolated from desmoid tumors harboring different CTNNB1 mutation status (wild type, T41A, and S45F), all exhibited a spindle shape. These isolated cells could be cultured until the 20th passage with unchanged proliferative activity. Nuclear accumulation of β‐catenin was observed in all cultured cells, particularly in those with S45F. Proliferating activity was significantly suppressed by meloxicam (25 μmol/L, P

Few studies have analyzed Cathepsin K (CatK) expression in human osteoarthritic tissues. We investigated CatK expression and activation in human articular cartilage using clinical specimens. Human osteoarthritic cartilage was obtained during surgery of total hip arthroplasty (n = 10), and control cartilage was from that of femoral head replacement for femoral neck fracture (n = 10). CatB, CatK, CatL, CatS, and Cystatin C (CysC) expressions were evaluated immunohistochemically and by real-time PCR. Intracellular CatK protein was quantified by ELISA. Intracellular CatK activity was also investigated. Osteoarthritis (OA) chondrocytes were strongly stained with CatK, particularly in the superficial layer and more damaged areas. CatB, CatL, CatS, and CysC were weakly stained. CatK mRNA expression was significantly higher in OA group compared to that in control group (p = 0.043), whereas those of CatB, CatL, CatS, and CysC did not differ significantly. Mean CatK concentration (4.83 pmol/g protein) in OA chondrocytes was higher than that (3.91 pmol/g protein) in control chondrocytes (p = 0.001). CatK was enzymatically more activated in OA chondrocytes as compared with control chondrocytes. This study, for the first time, revealed increased CatK expression and activation in human OA cartilage, suggesting possible crucial roles for it in the pathogenesis of osteoarthritic change in articular cartilage.

The association of symptom duration with survival remains controversial in soft-tissue sarcoma (STS).We determined whether the length from initial symptoms to specialist consultation affects prognosis in STSs. We retrospectively reviewed 152 primary STS patients (with 142 non-small round cell sarcomas) who consulted our specialist hospital. The factors that affected the length of the period from the initial symptoms to specialist consultation and the length of the delay at the clinic before specialist hospital referral were investigated. The relation between the length of the period from symptom onset and overall survival was also analyzed.Unplanned excision and superficial tumor were significantly associated with increasing duration from the initial symptoms to specialist hospital referral. Multivariate analysis revealed that tumors over 5 cm (P=0.002 and 0.005) and symptoms within 6 months (P=0.017 and 0.016) were independent poor prognostic factors of overall survival among the pretreatment factors when analyzing all and non-small round cell STSs.This is a first report to show the independent prognostic role of symptom duration in STSs on multivariate analysis. Considering the impact of symptom duration on survival in these heterogenous tumors, careful follow-up and consideration of treatment are necessary for patients with short symptom duration.

Brain metastases (BMs) from soft tissue sarcoma (STS) are rare but lethal. We reviewed 187 consecutive patients with STS treated with definitive surgery in Nagoya University Hospital from 2004 to 2014. There were 10 patients with neurofibromatosis-1 (NF-1). We investigated estimated brain metastasis free survival (BMFS) after surgery and overall survival (OS) after BMs in STS. The factors that affected BMFS were also investigated. Eight of 187 patients (4.3 %) developed BM with a median period of 18.2 (range 8.8–42.6) months after surgery. Seven of 8 BM patients had metastases at other sites. Estimated 5 year BMFS rate after surgery was 95.2 %, and 3 month OS rate after BM was 25.0 %. NF-1 (p

Pigmented villonodular synovitis (PVNS) is a benign, translocation-derived neoplasm. Because of its high local recurrence rate after surgery and occurrence of osteochondral destruction, a novel therapeutic target is required. The present study aimed to evaluate the significance of protein expression possibly associated with the pathogenesis during the clinical course of PVNS. In 40 cases of PVNS, positivity of colony-stimulated factor 1 (CSF1), its receptor (CSF1R), and receptor activator of nuclear factor kappa-B ligand (RANKL) were immunohistochemically determined. The relationship between the positivity and clinical outcomes was investigated. High positivity of CSF1 staining intensity was associated with an increased incidence of osteochondral lesions (bone erosion and osteoarthritis) (p = 0.009), but not with the rate of local recurrence. Positivity of CSF1R and RANKL staining was not associated with any clinical variables. The number of giant cells was not correlated with positivity of any of the three proteins, or with the clinical outcome. Focusing on knee cases, CSF1 positivity was also associated with the incidence of osteochondal change (p = 0.02). CSF1R positivity was high in cases which had local recurrence, but not significantly so (p = 0.129). Determination of CSF1 and CSF1R expression may be useful as a prognosticator of the clinical course and/or outcomes of PVNS.

The catabolism of hyaluronan in articular cartilage remains unclear. The aims of this study were to investigate the effects of hyaluronidase 2 (Hyal2) knockdown in articular cartilage on the development of osteoarthritis (OA) using genetic manipulated mice. Destabilization of the medial meniscus (DMM) model of Col2a promoter specific conditional Hyal2 knockout (Hyal −/− ) mice was established and examined. Age related and DMM induced alterations of articular cartilage of knee joint were evaluated with modified Mankin score and immunohistochemical staining of MMP-13, ADAMTS-5, KIAA11199, and biotinylated- hyaluronan binding protein staining in addition to histomorphometrical analyses. Effects of Hyal2 suppression were also analyzed using explant culture of an IL-1α induced articular cartilage degradation model. The amount and size of hyaluronan in articular cartilage were higher in Hyal2 −/− mice. Hyal2 −/− mice exhibited aggravated cartilage degradation in age-related and DMM induced mice. MMP-13 and ADAMTS-5 positive chondrocytes were significantly higher in Hyal2 −/− mice. Articular cartilage was more degraded in explant cultures obtained from Hyal2 −/− mice. Knockdown of Hyal2 in articular cartilage induced OA development and progression possibly mediated by an imbalance of HA metabolism. This suggests that Hyal2 knockdown exhibits mucopolysaccharidosis-like OA change in articular cartilage similar to Hyal1 knockdown.

The present study investigated the safety and efficacy of neoadjuvant and adjuvant chemotherapy with doxorubicin and ifosfamide for bone sarcoma in adult and older patients. A total of 18 consecutive patients with bone sarcoma (American Joint Committee on Cancer stage II in 14 patients and stage IV in four) treated with neoadjuvant and adjuvant chemotherapy at Nagoya Musculoskeletal Oncology Group hospitals in Japan between 2004 and 2011 were reviewed. The treatment efficacy and side-effects were evaluated. The responses to neoadjuvant chemotherapy were stable disease in 11 patients and progressive disease in three. Among the 12 evaluable patients, there were five with ≥90% tumor necrosis. The estimated overall survival (OS) rate at five years for the patients without metastasis prior to treatment was 56%. Major grade 3 or 4 side-effects included leukopenia in 14 cases, anemia in seven, thrombocytopenia in three, nausea in two and febrile neutropenia in two. One patient discontinued chemotherapy due to a temporarily depressed level of consciousness with arrhythmia (grade 2). The estimated five-year OS rate in this study was acceptable in patients without metastasis prior to treatment. A better coordinated prospective study of this combination regimen for older patients with bone sarcoma will be required to clarify its efficacy and tolerability.

Hyaluronan (HA) regulates malignant tumor growth, invasion, and metastasis. However, few studies have focused on the roles of HA in tumorigenicity in malignant peripheral nerve sheath tumors (MPNST). In this study, we sought to clarify the prognostic value of HA in patients with MPNST. Specimens obtained from 15 patients with neurofibroma and 30 with MPNST were subjected to HA staining and scored as three grades. Protein expressions of HA synthase 1-3 were examined in the 22 MPNST tissue samples available. Statistically higher HA positivity was observed in MPNST as compared with neurofibroma (P = 0.020). The univariate analysis revealed that increased HA expression, age, neurofibromatosis type 1 (NF1) status, large tumor size, and histological grade were significantly associated with reduced overall survival of patients with MPNST; while increased HA expression, NF1 status, tumor size, and histological grade were correlated with disease-free survival. However, HA synthase 1-3 expression related to neither overall survival nor disease-free survival of these patients. In multivariate analysis, large tumor size (P = 0.022) was an independent prognostic factor for overall survival, and HA expression (P = 0.028) and tumor size (P = 0.002) were independent prognostic factors for disease-free survival. Statistically higher levels of HA in the human MPNST cells were observed compared with neurofibroma cells in vitro. Our results demonstrate that HA expression can be a useful marker in differentiating MPNST from neurofibroma, and in identifying patients with a poor prognosis. Hyaluronan-targeting therapy for patients with MPNST may have potential as a therapeutic tool.

Several studies have focused on the relationships between the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and the prognosis of patients with malignant tumors. However, few of these have investigated the expression of EMMPRIN in osteosarcoma. We examined expression levels of EMMPRIN immunohistochemically in 53 cases of high-grade osteosarcoma of the extremities and analyzed the correlation of its expression with patient prognosis. The correlation between matrix metalloproteinases (MMPs) and EMMPRIN expression and the prognostic value of co-expression were also analyzed. Staining positivity for EMMPRIN was negative in 7 cases, low in 17, moderate in 19, and strong in 10. The overall and disease-free survivals (OS and DFS) in patients with higher EMMPRIN expression (strong-moderate) were significantly lower than those in the lower (weak-negative) group (0.037 and 0.024, respectively). In multivariate analysis, age (P = 0.004), location (P = 0.046), and EMMPRIN expression (P = 0.038) were significant prognostic factors for overall survival. EMMPRIN expression (P = 0.024) was also a significant prognostic factor for disease-free survival. Co-expression analyses of EMMPRIN and MMPs revealed that strong co-expression of EMMPRIN and membrane-type 1 (MT1)-MMP had a poor prognostic value (P = 0.056 for DFS, P = 0.006 for OS). EMMPRIN expression and co-expression with MMPs well predict the prognosis of patients with extremity osteosarcoma, making EMMPRIN a possible therapeutic target in these patients.

Objective To clarify the roles of hyaluronan (HA) in joint inflammation and the process of joint destruction, using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, in a mouse model of collagen-induced arthritis (CIA) and in a monolayer culture of fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis. Methods DAB/1J mice were immunized with type II collagen. The effects of 4-MU were evaluated by the physiologic arthritis score, paw swelling, the histologic arthritis score, and expression of matrix metalloproteinase 3 (MMP-3) and MMP-13 in chondrocytes and synovial tissue. In vitro, the effect of 4-MU on messenger RNA and protein expression of MMP-1 and MMP-3 was determined. The effects of 4-MU on HA deposition and on serum/medium concentrations of HA were analyzed using biotinylated HA binding protein staining and an HA binding assay, respectively. Results Treatment with 4-MU in mice with CIA dramatically decreased the severity of arthritis (based on the arthritis score), paw thickness, and histopathologic changes. MMP-3 and MMP-13 expression in chondrocytes and synovial cells was significantly inhibited by 4-MU in vivo. Treatment with 4-MU also inhibited MMP-1 and MMP-3 expression in tumor necrosis factor α–stimulated FLS, in a dose-dependent manner. The 4-MU–induced decreases in the serum HA concentration in mice with CIA and in “medium” and “pericellular” HA concentrations in cultured FLS support the contention that the inhibitory mechanism of 4-MU is mediated by HA suppression. Conclusion Reduced disease activity induced by 4-MU in mice with CIA revealed HA to be a crucial regulator in the course of arthritis. Therefore, 4-MU is a potential therapeutic agent in arthritis, and its inhibitory mechanism is possibly mediated by suppression of HA synthesis.

Several previous reports have described multiple cancers with regard to epithelial tumors, but few reports have focused on multiple primary malignancies including soft tissue sarcomas (STS). The purpose of this study was to analyze the clinical features of patients with high-grade STS with multiple malignancies and possible clinical problems, compared with those with STS as a single malignancy, focusing on elderly patients. This study enrolled 107 patients aged 65 years or over with high-grade STS. Eighty-four patients (79 %) had sarcomas only (S group), and 23 (21 %) had multiple primary malignancies (M group). STS preceded carcinoma in 10 patients, and carcinoma preceded STS in 13. In 7 patients (30 %), the interval between the first and second malignancy was less than a year. Of 7 patients who received treatment for sarcoma and carcinoma at the same time, the presence of other malignancies had an impact on determination of the treatment modality in 5 patients. The overall survival rate at 5 years was higher in M group (79 %) than in S group (69 %), although this difference was not significant (P = 0.095). This study demonstrates that the presence of multiple malignancies was not correlated with a poor prognosis, and was actually associated with a better prognosis in elderly patients with STS. Physicians should be aware of the possible occurrence of a second malignancy, and on occasion the treatment modalities and their logistical aspects need to be well organized and carefully selected for patients with ongoing multiple malignancies.

The aim of this study is to examine the impact of disease free (DF) status on the prognosis in patients with metastatic non-small round cell soft tissue sarcoma (STS). We retrospectively reviewed 51 metastatic STS patients who were treated in Nagoya University Hospital from 2005 to 2015. The relation between various clinical factors and overall survival (OS) was analyzed. The log rank test and Cox’s proportional hazards test were used to evaluate the differences between groups. p-values of

Objective Several studies have shown that cathepsin K (CTK) is overexpressed in osteoarthritic (OA) cartilage and subchondral bone. However, it has not been well established whether CTK expression is harmful or beneficial. We undertook this study to investigate the direct involvement of CTK in OA development using Ctsk-knockout (Ctsk−/−) mice in a joint instability–induced model of OA. Methods We analyzed the natural course of the phenotype of 25-week-old Ctsk−/− mice. OA development was evaluated with a modified Mankin histologic score up to 8 weeks after surgery was performed to destabilize the knee in Ctsk−/− and Ctsk+/+ mice. Histologic analysis was used to evaluate expression of CTK, matrix metalloproteinase 13 (MMP-13), ADAMTS-5, and tartrate-resistant acid phosphatase (TRAP) proteins in chondrocytes, synovial cells, and osteoclasts. Bone architecture was analyzed by histomorphometry. Results Bone mineral content and bone volume were higher in Ctsk−/− mice at 25 weeks, whereas OA did not develop spontaneously in either Ctsk−/− or Ctsk+/+ mice. In a model of destabilization-induced OA, OA progression was significantly delayed in Ctsk−/− mice. CTK was overexpressed in chondrocytes and synovial cells of knee joints developing OA in Ctsk+/+ mice. MMP-13 and ADAMTS-5 were less strongly expressed in chondrocytes of Ctsk−/− mice, and MMP-13 was less strongly expressed in synovial cells. TRAP-positive osteoclasts were overexpressed in Ctsk−/− mice. Conclusion These results indicate that CTK plays crucial direct roles in the early to intermediate stage of OA development. CTK-positive chondrocytes and synovial cells may be a possible target to prevent disease progression in OA.

In chondrogenic differentiation, expression and collaboration of specific molecules, such as aggrecan and type II collagen, in extracellular matrix (ECM) are crucial. However, few studies have clarified the roles of hyaluronan (HA) in proteoglycan aggregation during chondrogenic differentiation. We assessed the roles of HA in sulfated glycosaminoglycans deposition during chondrogenic differentiation by means of 4-methylumbelliferone (4-MU), an HA synthase inhibitor, using ATDC5 cells. ATDC5 cells were treated with 0.5 mM 4-MU for 7 or 21 days after induction of chondrogenic differentiation with insulin. Depositions of sulfated glycosaminoglycans were evaluated with Alcian blue staining. mRNA expression of ECM molecules was determined using real-time RT-PCR. The deposition of aggrecan and versican was investigated with immunohistochemical staining using specific antibodies. Effects of 4-MU on HA concentrations were analyzed by HA binding assay. 4-MU suppressed the positivity of Alcian blue staining, although this delay was reversible. Interestingly, stronger positivity of Alcian blue staining was observed at day 21 in cultures with 4-MU discontinuation than in the control. 4-MU significantly increased the mRNA expression of aggrecan, versican, and type II collagen, which was consistent with increased deposition of aggrecan and versican. The HA concentration in ECM and cell-associated region was significantly suppressed with 4-MU treatment. We conclude that the inhibition of HA synthesis slows sulfated glycosaminoglycans deposition during chondrogenic differentiation despite the increased deposition of other ECM molecules. Transient starvation of HA with 4-MU accelerates chondrogenic ECM formation, suggesting its potential to stimulate chondrogenic differentiation with adequate use.

Few studies have analyzed Cathepsin K (CatK) expression in human osteoarthritic tissues. We investigated CatK expression and activation in human articular cartilage using clinical specimens. Human osteoarthritic cartilage was obtained during surgery of total hip arthroplasty (n = 10), and control cartilage was from that of femoral head replacement for femoral neck fracture (n = 10). CatB, CatK, CatL, CatS, and Cystatin C (CysC) expressions were evaluated immunohistochemically and by real-time PCR. Intracellular CatK protein was quantified by ELISA. Intracellular CatK activity was also investigated. Osteoarthritis (OA) chondrocytes were strongly stained with CatK, particularly in the superficial layer and more damaged areas. CatB, CatL, CatS, and CysC were weakly stained. CatK mRNA expression was significantly higher in OA group compared to that in control group (p = 0.043), whereas those of CatB, CatL, CatS, and CysC did not differ significantly. Mean CatK concentration (4.83 pmol/g protein) in OA chondrocytes was higher than that (3.91 pmol/g protein) in control chondrocytes (p = 0.001). CatK was enzymatically more activated in OA chondrocytes as compared with control chondrocytes. This study, for the first time, revealed increased CatK expression and activation in human OA cartilage, suggesting possible crucial roles for it in the pathogenesis of osteoarthritic change in articular cartilage.

This study was conducted to determine the efficacy and safety of low-dose chemotherapy with methotrexate (MTX) and vinblastine (VBL) for patients with desmoid tumors refractory to meloxicam treatment, focusing in particular on the relationship between the efficacy of this chemotherapy and catenin β-1 (CTNNB1) mutation status. Since March 2003, patients pathologically diagnosed with extraperitoneal desmoid tumors have been prospectively treated with meloxicam, a COX-2 inhibitor, at our institution. Patients with inoperable tumors who were resistant to meloxicam treatment underwent MTX and VBL therapy every other week. The responses of all patients were evaluated, and factors that were correlated with efficacy were analyzed, including CTNNB1 mutation status. Sixty-eight patients were prospectively treated with meloxicam. MTX + VBL therapy was administered in 15 patients. Six patients showed a partial response. Only one patient presented disease progression. A few patients showed grade 3–4 treatment-related toxicity with the administration of MTX and VBL every other week. Intriguingly, CTNNB1 status did not affect the efficacy of this treatment. MTX and VBL treatment every other week is well tolerated and achieved a favorable response in patients resistant to meloxicam treatment, regardless of CTNNB1 mutation status.

Giant cell tumor of bone (GCTB) is a primary benign bone tumor with a locally aggressive character. Definitive descriptions of the site of origin for this type of tumor are not available. The aim of the present study was to evaluate the site of origin of GCTB of long bones with regards to epiphyseal lines by means of radiographic examination. For that purpose, plain X‑ray scans of 71 GCTBs arising in long bones were retrospectively reviewed. The tumor locations were the distal femur in 31 cases, proximal femur in 11 cases, proximal tibia in 13 cases, distal radius in 6 cases, proximal humerus in 5 cases and proximal fibula in 5 cases. The vertical center (VC) of the tumor was determined with X‑ray anteroposterior view, and the correlation between the VC and the epiphyseal line, and between the distance from the epiphyseal line to the VC and tumor area or volume were analyzed using a regression model equation based on scatter plot diagrams. The VC of the tumor was located in the metaphyseal region in 57 cases, in the epiphyseal line in 11 cases and in the epiphyseal region in 3 cases. In cases of GCTB located in the distal femur or proximal tibia, significant correlations between the distance from the VC to the epiphyseal line and tumor area or volume were identified. The site of origin of GCTB was estimated to be located in the metaphyseal region. GCTB often occurs in mature patients, which renders it challenging to estimate the true site of origin of this lesion, since the metaphyseal line has disappeared in mature patients. The results of the present study suggest that GCTB possibly originates in the metaphyseal region.

Several studies have reported that heat stress stimulates the activity of osteoblastic cells in vitro. However, few have addressed the effects of heat stress on osteogenesis in vivo, nor have the optimal temperatures for bone formation been determined. The aim of the present study was to investigate the effects of hyperthermia treatment on osteogenesis in a rat tibial defect model. Forty-four Sprague Dawley rats were divided into two groups with or without hyperthermia treatment. A 3-mm circular defect in the proximal tibia filled with magnetite cationic liposomes embedded in alginate beads was subjected to hyperthermia treatment (43-46 °C). Radiological assessment at 2 weeks after the treatment showed that significantly stimulated osteogenesis was observed in the hyperthermia group as compared to the control group (p = 0.003). Histomorphometrical analysis at 2 weeks revealed a significant increase of newly formed bone in the hyperthermia group, compared with the control group (p < 0.001). Area of newly formed bone in each hyperthermia group was significantly increased as compared with the control group (43 °C; p = 0.005, 44 °C; p = 0.019, 45 °C; p = 0.003, and 46 °C; p = 0.003, respectively). Alkaline phosphatase was overexpressed at the surfaces of newly formed bone adjacent to magnetite cationic liposome implantation. Our results demonstrate for the first time that heat stimulus accelerates osteogenesis in vivo, and may thus be of interest as a novel and promising tool to induce osteogenesis clinically as well.

A recent study reported that heat stress stimulates osteogenesis in an in vivo rat model using alginate gel and magnetite cationic liposomes. However, for clinical use, the efficacy for promoting osteogenesis needs to be investigated using clinically approved materials, and preferably with animals larger than rats. The aim of this study was to evaluate multiple heat stimuli-triggered osteogenesis in rat tibial defect models using already clinically applicable materials (Resovist® and REGENOS®) and determine the efficacy also in the rabbit. Fifty-eight rats and 10 rabbits were divided into two groups, respectively, with or without hyperthermia treatment at 45°C for 15 min. (hyperthermia; 20 rats once a week, 8 rats three times a week, 5 rabbits once a week, control; 30 rats and 5 rabbits). Micro-CT assessment at 4 weeks revealed that a significantly stimulated osteogenesis was observed in the once a week group of both rats and rabbits as compared to the control group (p = 0.018 and 0.036, respectively). In contrast, the three times a week group did not show enhanced osteogenesis. Histological examination and image analysis showed consistent results in which the area of mineralized bone formation in the once a week hyperthermia group was significantly increased compared with that in the control group at four weeks (rat; p = 0.026, rabbit; p = 0.031). Newly formed bone was observed in the grafted materials from the periphery toward the center, and more osteoclasts were found in the once a week group. Heat stress also induced enhanced alkaline phosphatase expression in cultured osteoblastic cells, MC3T3, in vitro (p = 0.03). On the other hand, heat stress had no obvious effects on chondrogenic differentiation using ATDC5 cells. Our study demonstrates that heat-stimuli with clinically applicable novel heating materials can promote significant osteogenesis, and may thus be a promising treatment option for diseases associated with bone defects.

High grade soft tissue sarcomas often show histological invasion to adjacent compartment including bone and vessel. This study aimed to evaluate histological invasion in high grade soft tissue sarcomas, clarify its impact on prognosis and devise treatment strategies. We retrospectively reviewed 133 patients with non-small round cell high grade soft tissue sarcomas surgically treated between 2001 and 2011. Clinical and histological factors examined for prognostic value included age, gender, size, depth, location, adjuvant therapy and invasion to adjacent compartment. Study endpoints included overall survival rate, event free survival rate and local recurrence free survival rate, estimated by the Kaplan-Meier method. Univariate and multivariate analyses were performed using the log-rank test and Cox proportional hazards model. Local recurrence was recognized in 14 cases (11%). The 5-year overall survival rate and 5-year event free survival rate were 82.2% and 63.6% respectively. The metastasis-free survival rate at 5 years and local recurrence-free survival rate at 5 years were 69.8% and 86.8% respectively. Histological invasion to adjacent compartment was noted in 52 cases (39%), and was significantly correlated with histological type (p = 0.01), tumor size (p = 0.009), and depth (p

We hypothesized that patterns of CTNNB1 (β-catenin) mutations would affect the outcome of conservative therapy in patients with desmoid tumors. This study aimed to determine the significance of CTNNB1 (β-catenin) mutations in predicting the treatment outcome in patients with desmoid tumors treated with meloxicam, a cyclooxygenase-2 (COX-2) selective inhibitor. Between 2003 and 2012, consecutive thirty-three patients with extra-peritoneal sporadic desmoid tumors were prospectively treated with meloxicam as the initial systemic medical therapy. The efficacy of meloxicam was evaluated according to Response Evaluation Criteria in Solid Tumors (RECIST). DNA was isolated from frozen tissue or formalin-fixed materials. CTNNB1 mutation analysis was performed by direct sequencing. Positivity of nuclear β-catenin staining by immunohistochemistry was compared with the status of CTNNB1 mutations. The correlation between the efficacy of meloxicam treatment and status of CTNNB1 mutations was analyzed. Of the 33 patients with meloxicam treatment, one showed complete remission (CR), 7 partial remission (PR), 12 stable disease (SD), and 13 progressive disease (PD). The following 3 point mutations were identified in 21 of the 33 cases (64%): T41A (16 cases), S45F (4 cases) and S45P (one case). The nuclear expression of β-catenin correlated significantly with CTNNB1 mutation status (p = 0.035); all four cases with S45F mutation exhibited strong nuclear expression of β-catenin. S45F mutation was significantly associated with a poor response (all cases; PD) (p = 0.017), whereas the other mutations had no impact on efficacy. The CTNNB1 mutation status was of significant prognostic value for meloxicam treatment in patients with sporadic desmoid tumors.

The aims of this study are to investigate clinical and pathological factors associated with the existence of residual tumor in reexcised specimens (RTRS) following unplanned excisions for soft tissue sarcomas and to determine whether RTRS has an impact on the oncologic outcome. A total of 113 patients with soft tissue sarcoma who received unplanned excision at the prereferral hospital were treated with additional reexcision at our institutions from 1986 to 2010. Clinical and pathological variables were analyzed as possible factors correlated with the existence of RTRS. These factors in addition to surgical margin in reexcisions were analyzed as factors possibly correlated with the oncological outcome. RTRS was observed in 58 cases (51 %). Histological subtype tended to associate with the presence of RTRS (P = 0.063), particularly in patients with dermatofibrosarcoma protuberances (DFSP) (83 %) and myxofibrosarcoma (100 %), whereas the other factors did not. Five-year local recurrence-free, metastasis-free, and overall survival (LRFS, MFS, and OS) were 93, 96, and 97 %, respectively. Patients with RTRS tended to have lower LRFS (89 %) than those without (97 %, P = 0.067) and had significantly lower MFS (92 %) than those without (100 %, P = 0.043). Other factors had no impact on prognosis. Patients receiving unplanned excision particularly for myxofibrosarcoma and DFSP require reexcision with an adequate surgical margin. Although patients with unplanned excision have a favorable clinical outcome following additional wide reexcision, those with RTRS require careful follow-up.

Cathepsin K (CatK) is one of the most potent mammalian collagenases. We showed previously the increased expression of CatK in human and animal atherosclerotic lesions. Here, we hypothesized that ablation of CatK mitigates injury-induced neointimal hyperplasia. Male wild-type (CatK +/+ ) and CatK-deficient (CatK −/− ) mice underwent ligation or a combination of ligation and polyethylene cuff-replacement injuries to the right common carotid artery just proximal to its bifurcation, and they were then processed for morphological and biochemical studies at specific time points. On operative day 28, CatK −/− significantly reduced neointimal formation and neovessel formation in both single- and combination-injured arteries compared with the Cat K +/+ mice. At early time points, CatK −/− reduced the lesion macrophage contents and medial smooth muscle cell proliferation, the mRNA levels of monocyte chemoattractant protein-1, toll-like receptor-2, toll-like receptor-4, chemokine ligand-12, and the gelatinolytic activity related to matrix metalloproteinase-2/-9. An aorta-explant assay revealed that smooth muscle cell movement was impaired in the CatK −/− mice compared with the CatK +/+ mice. In addition, the smooth muscle cells and macrophages from CatK −/− mice had less invasive ability through a reconstituted basement membrane barrier. This vasculoprotective effect was mimicked by Cat inhibition with trans -epoxysuccinyl-L-leucylamido-{4-guanidino} butane (E64 d ). These results demonstrate an essential role of CatK in neointimal lesion formation in response to injury, possibly via the reduction of toll-like receptor-2/-4–mediated inflammation and smooth muscle cell proliferation, suggesting a novel therapeutic strategy for the control of endovascular treatment–related restenosis by regulating CatK activity.

This study aimed to determine the prevalence of β-catenin nuclear positivity as a prognostic factor in patients with desmoid tumors (DTs) treated with meloxicam, a cyclooxygenase-2 (COX-2) selective inhibitor. Between 2003 and 2012, consecutive 31 patients with extraabdominal, sporadic DTs were prospectively treated with meloxicam as a systemic medical therapy. Immunohistochemistry was performed on formalin-fixed material to quantify the nuclear expression of β-catenin and Ki-67, and cytoplasmic expression of COX-2. All clinicopathological characteristics including the intensity of immunohistochemical staining were analyzed with respect to their prognostic value for meloxicam treatment. Of the 31 patients with meloxicam treatment, there was 1 with complete remission (CR), 7 with partial remission (PR), 12 with stable disease (SD), and 11 with progressive disease (PD). Higher nuclear expression of β-catenin was significantly associated with a poor response (PD/SD) (p = 0.017). The positivity of COX-2 and Ki-67 and none of the other clinical variables were associated with prognosis. The nuclear expression of β-catenin can predict the efficacy of meloxicam treatment for patients with sporadic DTs.

Background Gastric metastasis from osteosarcoma is very rare and its clinical features are not well recognized. Case presentation A 73-year-old man was diagnosed with osteosarcoma and treated with four cycles of preoperative chemotherapy with ifosfamide and doxorubicin followed by wide resection. Two cycles of postoperative chemotherapy with ifosfamide and doxorubicin and ten cycles of chemotherapy with carboplatin and etoposide were administered. Eleven months after the surgery, he vomited fresh blood. Unusual progression of anemia was observed with the hematemesis. A biopsy was performed by gastrointestinal endoscopy, and the stomach tumor was diagnosed as metastasis of osteosarcoma. Conclusions Even though gastric metastasis from osteosarcoma is very rare, all three previous reports and our case showed the presence of ulcer on the surface of the gastric lesion. We should consider the possibility of gastric metastasis in patients with osteosarcoma in whom progression of anemia or gastric hemorrhage is observed.

Whether to preserve or sacrifice critical structures (bone, major vessels and major nerves) adjacent to soft tissue sarcomas is still controversial. Referring to characteristic imaging and intraoperative findings, we perform planned preservation surgery for these critical structures. The aim of this study was to investigate the clinical outcome of soft tissue sarcomas adjacent to critical structures and to validate this procedure.Of 202 cases of soft tissue sarcomas surgically treated at our department of orthopedic surgery from 2004 to 2010, 57 cases (28 %) whose tumors were adjacent to the critical structures were studied. There were 36 men and 21 women. In 32 cases critical structures were preserved, and in 25 resected together with the tumor. The oncological outcome and histological surgical margin were analyzed.The overall survival and local recurrence-free survival rates at 5 years were 75.9 and 83.2 %. In 26 of the 32 (81 %) preserved cases histologically the surgical margin was negative, and in 17 (53 %) radiotherapy could be avoided. Neither overall survival (p = 0.9669) nor local recurrence-free survival (p = 0.7819) differed significantly between two groups.When soft tissue sarcomas are located adjacent to bone or major vessels, by meticulously detaching the periosteum or neurovascular sheath referring to characteristic imaging and intraoperative findings, a histologically negative surgical margin can be achieved in the majority, allowing avoidance of postoperative radiotherapy. Planned preservation surgery provided no significant adverse effect on survival or local recurrence rates, validating this procedure.

Hyaluronan is known to have pivotal roles in the growth, migration and invasion of malignant tumors. Bone metastases are critical lesions greatly impairing the quality of patients with malignancies. We investigated whether hyaluronan synthesis inhibitor supplements the inhibitory effects of zoledronic acid, which is a conventional therapeutic agent for bone metastasis. We examined the effects of methylumbelliferone, an inhibitor of hyaluronan synthesis and/or ZA on the tumorigenicity of one murine lung carcinoma and two human (A549, SK-MES-1) lung cancer cell lines in vitro. The interaction between methylumbelliferone and zoledronic acid was analyzed using Calcucyn software. With a murine bone metastasis model of lung cancer in vivo, we investigated the inhibitory effects and interaction of the two drugs on the progression of metastatic bone lesions. Methylumbelliferone or zoledronic acid treatment individually suppressed proliferation, migration and invasion of 3 cell lines, and combination treatment showed synergistic effects. Although methylumbelliferone as a single agent did not enhance apoptotic activity, it showed additive effects on apoptotic activity to those of zoledronic acid. Co-localization of CD44 and ezrin, which might be a pathway of hyaluronan signaling, was abrogated by methylumbelliferone treatment. Combination therapy showed additive inhibitory effects on metastatic bone lesions in vivo, which paralleled the inhibition of hyaluronan accumulation by methylumbelliferone, and inhibition of osteoclastogenesis. Although the detailed mechanisms underlying the synergistic or additive inhibitory effects of these two drugs should be further analyzed, inhibition of hyaluronan synthesis by methylumbelliferone is a promising novel therapeutic candidate for bone metastasis of lung cancer in addition to zoledronic acid.

Injection of monoclonal antibody to interleukin-2 (S4B6) into mice depletes regulatory T-cells (Tregs) and exhibits antitumor activities mediated through an autoimmune reaction. In this study, we demonstrate that administration of S4B6 suppresses the murine osteosarcoma cell line, LM8.LM8 osteosarcoma cells were transplanted subcutaneously into C3H mice (n=58). C3H mice were injected intraperitoneally with S4B6 starting at 7 days before LM8 transplantation (pre-S4B6 group), 2 days after transplantation, or 5 days after transplantation. Control group mice were injected with normal rat IgG. Mice were sacrificed and examined 4 weeks later.The number of pulmonary metastatic colonies and the tumor size were significantly reduced in the pre-S4B6 group compared to the control group. In addition, pulmonary metastases were inhibited in mice injected with S4B6 2 days, but not 5 days, after tumor transplantation.S4B6 administration inhibited metastasis even when injected 2 days after LM8 transplantation. Our data suggest that treatment with S4B6 might be suitable in a postoperative clinical setting.

Background: Hyaluronan (HA) plays crucial roles in the tumourigenicity of many types of malignant tumours. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis. Several studies have shown its inhibitory effects on malignant tumours; however, none have focused on its effects on osteosarcoma. Methods: We investigated the effects of MU on HA accumulation and tumourigenicity of highly metastatic murine osteosarcoma cells (LM8) that have HA-rich cell-associated matrix, and human osteosarcoma cell lines (MG-63 and HOS). Results: In vitro, MU inhibited HA retention, thereby reducing the formation of functional cell-associated matrices, and also inhibited cell proliferation, migration, and invasion. Akt phosphorylation was suppressed by MU (1.0 m). In vivo, although MU showed only a mild inhibitory effect on the growth of the primary tumour, it markedly inhibited (75% reduction) the development of lung metastasis. Hyaluronan retention in the periphery of the primary tumour was markedly suppressed by MU. Conclusion: These findings suggested that MU suppressed HA retention and cell-associated matrix formation in osteosarcoma cells, resulting in a reduction of tumourigenicity, including lung metastasis. 4-Methylumbelliferone is a promising therapeutic agent targeting both primary tumours and distant metastasis of osteosarcoma, possibly via suppression of HA retention.

Hyaluronan (HA) oligosaccharides were reported to have suppressive effects on various malignant tumors via disruption of receptor HA interactions. However, no studies have focused on the effects of HA oligosaccharides on bone metastasis of breast cancer. In this study, we clarified the effective size of HA oligosaccharides required to inhibit cell growth in the highly invasive breast cancer cell line, MDA-MB-231 cells. Based on the results of cell growth assay, we subsequently analyzed the effects of HA tetrasaccharides, HA decasaccharides, and high molecular weight HA on the other breast cancer cell behaviors in vitro and breast cancer bone metastasis in vivo. HA decasaccharides significantly inhibited cell growth, motility, and invasion, whereas tetrasaccharides did not. HAS2 mRNA expression was altered after the treatment with both tetrasaccharides and decasaccharides. Phosphorylation of Akt was suppressed after 1 h treatment with HA decasaccharides, and the effect was partially reversed by anti-CD44 monoclonal antibody. In vivo, local application of HA decasaccharides inhibited the expansion of osteolytic lesions in tibia on soft X-rays using mouse bone metastasis model of breast cancer. Histological analysis revealed HA accumulation in bone metastatic lesions was perturbed by decasaccharides. These results suggest that HA oligosaccharides suppressed progression of bone metastasis in breast cancer via interruption of endogenous HA–CD44 interaction, and as such, can be a novel therapeutic candidate to limit bone metastasis of breast cancer. © 2011 Orthopaedic Research Society. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:662–672, 2012

Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and is composed of large extracellular matrix aggregates, has been shown to correlate with tumor progression. No studies have examined the roles of versican in chondrosarcoma nor compared them to those of aggrecan. In clinical specimens of human chondromatous tumors, versican expression was significantly increased in malignant tumors, moreover, as the tumor grade increased. To clarify the roles of versican in chondrosarcoma, versican splicing variant 1, variant 3 or only GFP was stably transfected to Swarm rat chondrosarcoma cells with Trap-In System. Forced expression of versican V1 isoform in Swarm rat chondrosarcoma cells induced a marked increase of cell-associated matrix compared to V3-, GFP- transfected or RCS cells. Versican was immunolocalized in a fashion similar to that of hyaluronan and more diffusively than aggrecan. Anchor-dependent and -independent growth was not affected by versican isoform expression, whereas cell motility and migration were significantly enhanced by V1 isoform transfection. Tumors formed in vivo with V1-transfected cells exhibited more myxomatous area and included more spindle shaped cells. These results support the concept that versican has the capacity to form more extensive cell-associated matrix than aggrecan, and the prominent matrix formation alters the cell behavior of chondrosarcoma more aggressively. These observations suggest that versican expression may serve as a marker of tumor grade determination in chondrosarcoma and possibly help to decide on therapeutic targets in higher grades of chondrosarcoma.

Angiogenesis is involved in the growth and metastasis of most solid tumors. Several reports have demonstrated that angiotensin II stimulates growth and migration of certain cancer cell lines and induces angiogenesis through up-regulation of vascular endothelial growth factor. This study examined whether an angiotensin II type 1 receptor (AT1R) antagonist (CV11974) inhibits osteosarcoma progression and distant metastasis.Osteosarcoma (LM8) was transplanted into subcutaneous dorsal tissue of C3H mice. The mice were administered CV11974 daily by intraperitoneal injections at 0.1 mg/kg, 1 mg/kg, or 10 mg/kg, or saline for 28 days.Subcutaneous tumor size was smaller in the CV11974 treatment groups than in the control group. Lung and liver metastases were significantly reduced in the CV11974 treatment groups when compared with the control group.CV11974 is widely used to treat hypertension clinically and therefore may be a novel antiangiogenic therapy for osteosarcoma through blocking AT1R-mediated signaling.

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Previous studies demonstrated that inhibition of HA suppressed the tumorigenicity of various malignant tumors including breast cancer. 4-methylumbelliferone (MU) has been reported to inhibit HA synthesis in several cell types. However, few studies have focused on the effects of HA inhibition in breast cancer cells by MU, nor the effects on bone metastasis. We hypothesized that MU would suppress the progression of bone metastasis via inhibition of HA synthesis. Here, we investigated the effects of MU on HA expression in MDA-MB-231 breast cancer cell line in addition to their tumorigenicity in vitro and in vivo. HAS2 mRNA expression was downregulated after 6 and 24 hr treatment with MU. Quantitative analysis of HA revealed that MU significantly inhibited the intracellular and cell surface HA. MU significantly inhibited cell growth and induced apoptosis as determined by cell proliferation and TUNEL assays, respectively. Phosphorylation of Akt was suppressed after 12 and 24 hr treatment with MU. MU treatment also inhibited cell motility as well as cell invasiveness. MU also inhibited cell growth and motility in murine fibroblast cell line NIH3T3. In vivo, administration of MU inhibited the expansion of osteolytic lesions on soft X-rays in mouse breast cancer xenograft models. HA accumulation in bone metastatic lesions was perturbed peripherally. These data suggest that MU might be a therapeutic candidate for bone metastasis of breast cancer via suppression of HA synthesis and accumulation.