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Background: Breast cancer has the potential to metastasize to bone, causing debilitating symptoms. Although many tumor cells have thrombin-generating systems originating from tissue factor (TF), therapy in terms of the coagulation system is not well established. To elucidate the efficacy of the thrombin inhibitor, argatroban, on bone metastasis, we investigated TF activation and vascular endothelial growth factor (VEGF) secretion on treatment with thrombin and argatroban., Methods: MDA-231 breast cancer cells were treated with thrombin in presence or absence of argatroban, and TF activity was measured in the form of activated factor X. Enzyme-linked immunosorbent assay (ELISA) was used to measure VEGF concentrations in the medium. MDA-231 cells were injected into the left heart ventricle of mice, and then argatroban or saline was administered intraperitoneally for 28 days. After 28 days, incidence of bone metastasis was evaluated in the limbs by radiography., Results: TF activity and VEGF secretion were upregulated by thrombin. Argatroban inhibited the enhancement of TF activity and VEGF secretion induced by thrombin. In vivo analysis revealed that the number of metastasized limbs in the argatroban group was significantly lower compared with the saline group (P < 0.05)., Conclusions: Thrombin not only enhances VEGF secretion but also has a positive feedback mechanism to reexpress TF. These results indicate that inhibition of thrombin is of great value in suppression of tumor metastasis. Argatroban is a noteworthy and useful thrombin inhibitor because it has already been used in the clinical setting and has antimetastatic effects in vivo.

Background: Neural-cadherin (N-cadherin) is one of the most important molecules involved in tissue morphogenesis, wound healing, and the maintenance of tissue integrity. Recently, the cleavage of N-cadherin has become a focus of attention in the field of cancer biology. Cadherin and their ectodomain proteolytic shedding play important roles during cancer progression. The aims of this study are to investigate the serum soluble N-cadherin (sN-CAD) levels in patients with malignant bone and soft tissue tumors, and to evaluate the prognostic significance of the sN-CAD levels., Methods: We examined the level of serum sN-CAD using an ELISA in 80 malignant bone and soft tissue tumors (bone sarcoma, n = 23; soft tissue sarcoma, n = 50; metastatic cancer, n = 7) and 87 normal controls. The mean age of the patients was 51 years (range, 10-85 years) and the mean follow-up period was 43 months (range, 1-115 months)., Results: The median serum sN-CAD level was 1,267 ng/ml (range, 135-2,860 ng/ml) in all patients. The mean serum sN-CAD level was 1,269 ng/ml (range, 360-2,860 ng/ml) in sarcoma patients, otherwise 1,246 ng/ml (range, 135-2,140 ng/ml) in cancer patients. The sN-CAD levels in patient were higher than those found in the controls, who had a median serum level of 108 ng/ml (range, 0-540 ng/ml). The patients with tumors larger than 5 cm had higher serum sN-CAD levels than the patients with tumors smaller than 5 cm. The histological grade in the patients with higher serum sN-CAD levels was higher than that in the patients with lower serum sN-CAD levels. A univariate analysis demonstrated that the patients with higher serum sN-CAD levels showed a worse disease-free survival rate, local recurrence-free survival rate, metastasis-free survival rate, and overall survival rate compared to those with lower serum sN-CAD levels. In the multivariate analysis, sN-CAD was an independent factor predicting disease-free survival., Conclusions: sN-CAD is a biomarker for malignant bone and soft tissue tumors, and a potentially valuable pre-therapeutic prognostic factor in patients with bone and soft tissue sarcoma.

Background: The incidence of sarcoma diagnosed in the elderly population may be rising due to the increasing life expectancy. The purpose of our study was to evaluate the efficacy of lung metastasectomy and radiofrequency (RF) ablation in elderly sarcoma patients (65 years or older) with lung metastases, compared with adult sarcoma patients younger than 65 years (20-64 years)., Materials and Methods: We retrospectively evaluated the clinical efficacy of metastasectomy and lung RF ablation in sarcoma patients with lung metastases., Results: Between 2001 and 2010, lung metastases were detected in 66 adult patients with musculoskeletal sarcomas. Twenty-five patients with lung metastasis were elderly. Twelve of the 25 patients were treated with lung metastasectomy and/or RF ablation. In contrast, 41 patients were adult patients younger than 65 years. Twenty of these 41 patients were treated with lung metastasectomy and/or RF ablation. The 1 and 3-year survival rates after initial treatment for lung metastases were 81.8% and 38.4% in 12 elderly patients, respectively. Compared with the control group, the survival rate in the elderly patients was not significantly different., Conclusion: We conclude that elderly sarcoma patients with lung metastases should always be considered for either metastasectomy or RF ablation.

In this study, we analyzed long-term survival, limb function and associated complications after prosthetic limb salvage treatment in patients with bone and soft tissue tumors around the knee joint. A total of 63 patients treated with prosthetic limb salvage surgery around the knee were reviewed. The bone tumors involved the distal femur in 45 patients, the proximal tibia in 14 patients and the soft tissue tumors of the proximal lower leg in 4 patients. The median follow-up period after the first operation was 8.0 years. The medical records of the patients, surgical reports, radiographs and histological specimens were retrospectively reviewed. The 5-year overall survival rate was 63.2% in the patients with distal femur tumors and 86.2% in those with tumors of the proximal lower leg. The 5‑year prosthetic survival rate was 72.8% in the distal femur and 74.6% in the proximal lower leg. The mean functional score according to the scoring system of the Musculoskeletal Tumor Society (MSTS) was 81% in the patients with distal femur tumors and 82% in the patients with proximal lower leg tumors. Post-operative complications occurred in 27 patients. Limb salvage surgery is considered to be an effective treatment option. However, the high complication rate is a major concern for prosthetic replacement. Future improvements of prostheses are very important.

The poliovirus receptor CD155, is essential for poliovirus to infect and induce death in neural cells. Recently, CD155 has been shown to be selectively expressed on certain types of tumor cells originating from the neural crest, including malignant glioma and neuroblastoma. However, the expression pattern of CD155 in soft tissue sarcoma has not been examined. Therefore, we first examined CD155 expression in sarcoma cell lines, and found the expression of both CD155 mRNA and protein in 12 soft and bone tissue sarcoma cell lines. Furthermore, we examined the effect of live attenuated poliovirus (LAPV) on 6 bone and soft tissue sarcoma cell lines in vitro, and found that LAPV induced apoptosis by activating caspases 7 and 3 in all of these cell lines. Furthermore, in BALB/c nu/nu mice xenotransplanted with HT1080 fibrosarcoma cells, administration of live attenuated poliovirus caused growth suppression of the tumors. These results suggest that oncolytic therapy using a LAPV may represent a new option for the treatment of bone and soft tissue sarcomas.

In the evolution of cancer, tumor necrosis factor-alpha (TNF-α) plays a paradoxical role. High doses induce significant anticancer effects, but conversely, physiologic and pathologic levels of TNF-α may be involved in cancer promotion, tumor growth, and metastasis. Infliximab is a chimeric murine monoclonal antibody that binds with high affinity to soluble and membrane TNF-α and inhibits binding of TNF-α to its receptors. In the present study, we investigated the effect of infliximab, a TNF-α antagonist, on breast cancer aggressiveness and bone metastases. Infliximab greatly reduced cell motility and bone metastases in a metastatic breast cancer cell line, MDA-MB-231. The mechanism of bone metastasis inhibition involved decreased expression of CXC chemokine receptor 4 (CXCR4) and increased expression of decorin, which is the prototype of an expanding family of small leucine-rich proteoglycans. These results suggest a novel role for TNF-α inhibition in the reduction or prevention of bone metastases in this breast cancer model. Our study suggests that inhibition of TNF-α using infliximab may become a preventive therapeutic option for breast cancer., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Patients with bone metastases in the extremities sometimes require surgical intervention to prevent deterioration of quality of life due to a pathological fracture. The use of localized radiotherapy combined with surgical reinforcement has been a gold standard for the treatment of bone metastases. However, radiotherapy sometimes induces soft tissue damage, including muscle induration and joint contracture. Moreover, cancer cells are not always radiosensitive. Hyperthermia has been studied since the 1940s using an experimental animal model to treat various types of advanced cancer, and studies have now reached the stage of clinical application, especially in conjunction with radiotherapy or chemotherapy. Nevertheless, bone metastases have several special properties which discourage oncologists from developing hyperthermic therapeutic strategies. First, the bone is located deep in the body, and has low thermal conductivity due to the thickness of cortical bone and the highly vascularized medulla. To address these issues, we developed new hyperthermic strategies which generate heat using magnetic materials under an alternating electromagnetic field, and started clinical application of this treatment modality. The purpose of this review is to summarize the latest studies on hyperthermic treatment in the field of musculoskeletal tumors, and to introduce the treatment strategy employing our novel hyperthermia approach.

Osteosarcoma (OS) is the most common primary malignant tumor of the bone and often forms pulmonary metastases, which are the most important prognostic factor. For further elucidation of the mechanism underlying the progression and metastasis of human OS, a culture system mimicking the microenvironment of the tumor in vivo is needed. We report a novel three-dimensional (3D) alginate spheroid culture system of murine osteosarcoma. Two different metastatic clones, the parental Dunn and its derivative line LM8, which has a higher metastatic potential to the lungs, were encapsulated in alginate beads to develop the 3D culture system. The beads containing murine OS cells were also transplanted into mice to determine their metastatic potential in vivo. In this culture system, murine OS cells encapsulated in alginate beads were able to grow in a 3D structure with cells detaching from the alginate environment. The number of detaching cells was higher in the LM8 cell line than the Dunn cell line. In the in vivo alginate bead transplantation model, the rate of pulmonary metastasis was higher with LM8 cells compared with that of Dunn cells. The cell characteristics and kinetics in this culture system closely reflect the original malignant potential of the cells in vivo.

Background: The authors retrospectively evaluated the impact of lung radiofrequency (RF) ablation on survival in patients with lung metastases from musculoskeletal sarcomas., Methods: Lung RF ablation was done under the real-time computed tomography (CT) fluoroscopy. Safety, local tumor progression, and survival were evaluated in 2 institutions., Results: Lung RF ablation was performed in 20 consecutive patients. The mean maximum tumor diameter was 14 +/- 9 mm (range, 5-40 mm) and the mean tumor number 7 +/- 6 (range, 1-18) per patient. Pneumothorax requiring chest tube placement developed in 24 of 63 RF sessions (38%). During the mean follow-up period of 18 months (range, 7 months to 54 months), 9 of 20 patients died of lung tumor progression. The 1- and 3-year survival rates from RF ablation were 58% (95% confidence interval [CI], 33.3-82.6%) and 29% (95% CI, 0-59.9%) with a median survival time of 12.9 months in all patients. Ablation of all lung tumors was the only significantly better prognostic factors in both the univariate analysis and the multivariate analyses. The 1- and 3-year survival rates were 88.9% (95% CI, 69.3%-100%) and 59.2% (95% CI, 10.2%-100%) in 11 patients with complete tumor ablation., Conclusions: Lung RF ablation is a safe and useful therapeutic option for selected patients. (R#1) Prognostic factors identified in our study will help to stratify those patients who may benefit from lung RF ablation.

Decorin, the prototype of an expanding family of small leucine-rich proteoglycans, is involved in a number of cellular processes including matrix assembly, fibrillogenesis and the control of cell proliferation. In this study, we investigated the role of decorin in suppressing tumor aggressiveness and bone metastases. We used a metastatic breast cancer cell line, MDA-MB-231, to show that decorin causes marked growth suppression bothin vitro and in vivo. A cytomegaloviral vector containing the decorin transgene caused greatly reduced cell growth, motility and observed metastases. Bone metastases were decreased by >90% upon decorin transfection. These results demonstrate a novel role for decorin in the reduction or prevention of tumor metastases in this breast cancer model and could eventually lead to improved therapies for metastatic breast cancer., (Copyright 2009 S. Karger AG, Basel.)

Bone metastasis is one of the most severe cancer complications. To analyze the mechanism of bone metastasis, we established highly invasive cell lines from the human breast cancer cell line MDA-MB-231 using an in vitro sequential selection system. The cell lines, MDA-231-S10 and MDA-231-S5, were more invasive and more motile than the parental cell line. Moreover, MDA-231-S10 metastasized to bone more often when inoculated into the arterial circulation of nude mice. MDA-231-S10-bearing nude mice had a significantly poorer prognosis, and their bony metastatic tumors grew more rapidly than those of the mice bearing the parental cell line (MDA-231-P). Given that a high expression of matrix metalloproteinase (MMP) is reported to be associated with cancer invasiveness, we examined MMP expression. Our results showed that the expression of MMP-3, -5, -7, -9, -13 and -14 was decreased on Multiplex real-time quantitative RT-PCR analysis in the two new cell lines. The zymographic analysis showed no MMP-2 activity and a decreased MMP-9 activity in MDA-231-S10. However, the expression of MMP-1 in MDA-231-S10 was increased. We therefore concluded that MMP-1 plays a crucial role in breast cancer bone metastasis. Furthermore, our MDA-231-derived cell lines are useful analytical models of MMP-1- associated breast cancer bone metastasis.

Purpose: The usefulness of limb salvage surgery for distal lower leg sarcoma remains controversial. We analyzed the long-term prognosis, limb function, and complications after limb salvage treatment of patients with distal lower leg sarcoma., Methods: Ten patients treated with limb salvage surgery for primary distal lower leg sarcoma were retrospectively reviewed. The median follow-up period after the first operation was 9.0 years. We performed three types of reconstructive techniques for the skeletal defect after a wide resection, including (1) arthrodesis with a combination of autograft and intraoperative autoclaved tumor bone graft in two patients, or allograft in one patient, (2) ankle joint preserving surgery using intraoperative extracorporeal irradiated tumor bone graft in five patients, and (3) prosthesis in two patients., Results: The overall survival rate was 80%. The 5-year disease-free survival was 80%. The rate of limb preservation at the final follow-up was 90%. The mean functional score according to the scoring system of the Musculoskeletal Tumor Society was 88% at the final follow-up. Postoperative complications occurred in seven patients. Skin trouble occurred in three patients, infectious non-union in one patient, fracture in three patients, and loosening of prosthesis in one patient., Conclusions: Despite the high rate of complications, patients treated with limb salvage surgery for the distal lower leg sarcoma revealed excellent final functional results without impairing the oncologic results. Limb salvage surgery is therefore considered to be an effective treatment option for distal lower leg sarcoma when adequate informed consent can be obtained from the patient.

Lung metastasis is the most crucial event affecting the therapeutic outcome of osteosarcoma. The prevention of lung metastasis is therefore important in improving the prognosis of patients with osteosarcoma. Decorin is a major extracellular matrix protein which has become the focus of various cancer studies. The biological role of decorin in osteosarcoma has yet to be clarified. The aim of this study was to examine the potential of decorin as a novel biological target for the treatment of osteosarcoma. In this study, the LM8 murine osteosarcoma cell line (LM8) with high metastatic potential to the lung was used. The two cell lines established were LM8-DCN which stably expressed human decorin (hDCN) and LM8-mock, established as a control. The LM8-DCN cell line was subcutaneously injected into the backs of mice. Significantly fewer pulmonary metastases were observed in mice with LM8-DCN compared to mice inoculated with LM8 and LM8-mock (P<0.001). In addition, the mice in the LM8-DCN inoculated group survived significantly longer than those in the LM8 and LM8-mock inoculated group, based on the Kaplan-Meier survival analysis and log-rank tests (P<0.005). The effect of decorin on the growth rates, motility and invasion ability of LM8 was investigated in vitro. There was no difference in the morphology and growth rates, but the motility and invasion of LM8 were inhibited by decorin. These results suggest that decorin has the therapeutic potential to prevent lung metastasis in osteosarcoma.

Background: To maintain excellent limb function after tumor resection in patients with high-grade malignant sarcomas, we developed and established a new surgical adjuvant therapy using acridine orange (AO) after intra-lesional or marginal resection while sparing normal tissues of major nerves, vessels or bones adjacent to the tumor., Method: Our AO therapy procedure was combined with photodynamic surgery (PDS), photodynamic therapy (PDT) and radiodynamic therapy (RDT). In this study, 26 patients with primary high-grade soft tissue sarcomas were treated with AO therapy., Result: Results showed a low local recurrence rate (7.7%) and good local recurrence-free rate (88%) after AO therapy. Limb function of all patients was maintained at 100% of ISOLS criteria., Conclusion: Based on these results, we concluded that AO therapy is useful for local control after margin-positive tumor resection and for preserving excellent limb function in patients with high-grade soft tissue sarcomas., ((c) 2008 Wiley-Liss, Inc.)

Methylthioadenosine phosphorylase (MTAP) is an important enzyme in the salvage pathway of adenosine and methionine synthesis. MTAP is ubiquitously present in all normal cells and tissues, but deficient in a variety of malignant tumors. The enzyme deficiency is caused by either MTAP gene deletion or promoter hypermethylation. We investigated MTAP expression, MTAP gene deletion and promoter abnormality in 40 primary tumor samples from Japanese osteosarcoma patients and determined the frequency of the enzyme deficiency. We also tested whether or not the enzyme deficiency can be exploited for tumor-specific chemotherapy using osteosarcoma cell lines. For MTAP expression, immunohistochemistry (IHC) and Western blotting were used. Real-time quantitative PCR assay was used for the analysis of MTAP gene deletion in fifteen osteosarcoma samples. MTAP promoter abnormality was analyzed by methylation-specific PCR. Then, the relationship between MTAP expression and sensitivity to the inhibitors of de novo AMP synthesis was confirmed in an MTAP-negative and -positive osteosarcoma cell line. The MTAP protein was negative in 11 of 40 samples (27.5%) by IHC and in 4 of 6 osteosarcoma cell lines (66.7%) by Western blot analysis. Among 40 samples, 15 were subjected to quantitative real-time PCR and promoter methylation analysis. Of 6 samples that were negative by IHC, the MTAP gene was deleted in 3 and the MTAP promoter was methylated in 2. These results indicated that MTAP deficiency was caused by MTAP gene deletion or promoter methylation in all MTAP-negative samples except one that was negative with IHC although no deletion or promoter methylation was detected. In in vitro experiments using transfectoma along with the MTAP-negative parental cell line, the MTAP-negative parental cell line was more chemosensitive to the inhibitors of de novo AMP synthesis than MTAP-positive transfectoma. MTAP deficiency frequently found in osteosarcoma can be exploited for selective chemotherapy in MTAP-negative osteosarcoma patients with the inhibitors of de novo purine synthesis.

A photodynamic therapy technique with acridine orange (AO-PDT) was experimentally developed and applied clinically to musculoskeletal sarcoma patients to reduce the surgical margin and obtain good limb function. Furthermore, various modalities to enhance and strengthen the cytocidal effect of AO-PDT were investigated. A recent report revealed that the use of stronger unfiltered xenon light in AO-PDT enhanced the cytocidal efficacy of this treatment modality. Therefore, in this study, we investigated whether the use of a flash wave light (FWL) from a xenon lamp, as compared to that of the conventional continuous wave light (CWL), might enhance the cytocidal effect of AO-PDT, using the mouse osteosarcoma cell line, LM8. For an equal energy dose (79.6 joules/cm2), AO-PDT using FWL (10 minutes excitation) was found to exert a significantly stronger cytocidal effect than that using CWL (18 seconds excitation). For the same excitation time (10 minutes' excitation), the use of FWL (79.6 jouleslcm2) was associated with a significantly stronger cytocidal effect of AO-PDT than that of CWL (3,820 joules/cm2). These results reveal that the use of FWL entails the need for a lower excitation energy and shorter excitation time than that of CWL for the cytocidal effect of AO-PDT to be observed against the osteosarcoma cells. In addition, FWL also has the advantage of generating low heat and of having the ability to homogenously illuminate a wider area. We therefore concluded that FWL is more useful for AO-PDT than CWL in terms of saving on the excitation time and of obtaining good efficacy of destruction of the residual tumor in the treatment of musculoskeletal sarcomas.

We have developed a novel hyperthermic treatment modality using magnetic materials for metastatic bone tumors. The purpose of this study is to show the results of novel hyperthermia for metastatic bone tumors. This novel hyperthermic treatment modality was used for 15 patients with 16 metastatic bone lesions. In seven lesions, after curettage of the metastatic lesion followed by reinforcement with a metal intramedullary nail or plate, calcium phosphate cement (CPC) containing powdery Fe3O4 was implanted into the cavity. In one lesion, prosthetic reconstruction was then performed after an intralesional tumor excision. For the remaining eight lesions, metal intramedullary nails were inserted into the affected bone. Hyperthermic therapy was started at 1 week postoperatively. To comparatively evaluate the radiographic results of patients who underwent hyperthermia (HT group), we also assessed eight patients who received a palliative operation without either radiotherapy or hyperthermia (Op group), and 22 patients who received operation in combination with postoperative radiotherapy (Op + RT group). In HT group, all patients had an acceptable limb function with pain relief without any complications. On radiographs, 87, 38, and 91% were, respectively, considered to demonstrate an effective treatment outcome in HT group, Op group, and Op + RT group. The patients in HT group showed a statistically better radiographic outcome than the patients in Op group (P = 0.0042). But when compared between HT group and Op + RT group, there were no significant difference (P = 0.412). This first series of clinical hyperthermia using magnetic materials achieved good local control of metastatic bone lesion.

This report presents an extremely rare case of extraskeletal myxoid chondrosarcoma (EMC) arising from the clavicular periosteum. To the best of our knowledge, this may be the first detailed report of its clinicopathological findings. The patient was a 48-year-old man. Plain radiography and CT did not demonstrate any osteolytic lesion or periosteal reaction in the right clavicle. However, MRI showed an isosignal-intensity mass on T1-weighted images and a homogeneous high signal intensity lesion on T2-weighted images. The histological findings of the widely resected tumor were consistent with the diagnosis of extraskeletal myxoid chondrosarcoma. Preoperative diagnosis of extraskeletal myxoid chondrosarcoma at an unusual location, as in this case, is difficult not only with imaging examinations alone, but sometimes even after histological examination of biopsy specimens.

Background: Intra-operative photodynamic therapy has been applied with acridine orange (AO-PDT) to human musculoskeletal sarcomas for the past 4 years, resulting in a low local recurrence rate, within 10%, after intra-marginal tumor resection and excellent limb function. However, it is still unclear why acridine orange (AO) specifically accumulates in tumor cells, especially in malignant tumor cells. The purpose of this study was to clarify the mechanism of AO accumulation in malignant musculoskeletal tumors., Materials and Methods: Sixty-two musculoskeletal tumors, including 35 malignant and 27 benign tumors, were studied. Using freshly resected tumor material, the extracellular pH (pHe) was measured and the fluorescence intensity of AO accumulated in the tumors was measured by an image analyzer after ex vivo exposure to 1.0 microg/ml AO, followed by blue excitation. In the in vitro study, bafilomycin A1 was exposed to LM 8 mouse osteosarcoma cells, in order to inhibit V-ATPase, subsequently causing a decrease in the pHgradient (deltapH) between the intracellular pH (pHi) and extracellular pH (pHe) or between the pHi and the pHe. AO accumulation and the cytocidal effect of AO were evaluated., Results: The results of the in vivo study, using human materials freshly resected at surgery, revealed that the pHe of the malignant musculoskeletal tumors was significantly lower than that of the benign tumors and normal muscles or adipose tissues and also showed that the AO fluorescence intensity of the malignant musculoskeletal tumors was significantly stronger than that of the benign tumors and normal muscles or adipose tissues. The results also revealed that the AO fluorescence intensity negatively correlated with the pHe in tumors and normal tissues. The in vitro study showed that bafilomycin A1 inhibited the accumulation of AO in acidic organelles, such as lysosomes, and that the cytocidal effect of AO-PDT was also remarkably inhibited., Discussion: Based on results of the in vivo and in vitro studies, it is suggested that malignant musculoskeletal tumors have a large deltapH between the pHi and the pHe or between the pHi and the vaculolar pH and also that a large ApH increases AO accumulation in tumors. We, therefore, believe that AO-PDT may be more effective in highly malignant musculoskeletal tumors than low grade ones, because of their acidity., Conclusion: AO accumulation in musculoskeletal tumors was dependent on the ApH between the pHi and the pHe, or between the pHi and the vacuolar pH.

When human blood monocytes were cocultured with stromal cells derived from human giant cell tumor of bone (GCTSC) and a Millipore filter (0.4 microm) was interposed between monocytes and GCTSC, multinucleated giant cell formation of monocytes was induced. The multinucleated giant cells have characters as osteoclast-like cells, indicating that a soluble osteoclast-inducing factor(s) is secreted from GCTSC expressing RANK, RANKL/ODF/OPGL and TACE mRNA. Furthermore, OCIF/OPG inhibited GCTSC-induced osteoclastogenesis, showing that the RANK-RANKL system is involved in GCTSC-induced osteoclastogenesis and that soluble form of ODF/RANKL induces osteoclasts from monocytes. GCTSC expressed the cytokine mRNAs such as M-CSF, GM-CSF, IL-3, IL-4, IL-6, and IFN-gamma mRNAs. None of IL-1ralpha, IL-1alpha, IL-1beta, IL-2, IL-4, IL-10, IL-18, TNF-alpha, G-CSF and IFN-gamma could be detected in all culture media. A significant amount of IL-6 could be detected in the culture media of all GCTSC. IL-8 was found in the culture media of two GCTSC and two osteosarcoma-derived cells. M-CSF was detected in all culture media. GCTSC express CaSR, and stimulation of GCTSC with either extracellular Ca(2+) or neomycin, agonist of CaSR, augmented the expression of RANKL. Some lines of GCTSC expressed alkaline phosphatase, osteocalcin and Cbfa1, suggesting that GCTSC are intimately related to osteoblastic lineage.

Most patients with musculoskeletal sarcoma do not recover satisfactory limb function after limb salvage surgery. To achieve satisfactory improvement of limb function, we developed a unique surgical modality of photodynamic therapy using acridine orange (AO-PDT) and clinically applied it to patients with musculoskeletal sarcomas. Ten patients with primary musculoskeletal sarcomas were enrolled in the study. Of these, 6 had primary malignant soft tissue sarcoma and 4 had primary malignant bone tumor. In the AO-PDT procedure, intralesional or partially marginal tumor excision was initially conducted and microscopic curettage of the remnant tumor, which emitted green fluorescence under blue excitation after local administration of 1microg/ml AO solution, was performed using a fluorescence surgical microscope. Subsequently, blue light illuminated there for 10 minutes. The surgical wound was closed, followed by immediate X-ray irradiation of the resected area with 5 Gy in 5 out of 10 patients to enhance the effect of AO-PDT. The follow-up of the patients ranged from 24 to 48 months. All the patients (AO-PDT alone: 5, AO-PDT with 5-Gy radiation: 5) are alive; only one patient showed local recurrence of the tumor. The recurrence rate was 10%. None of the 5 patients treated by AO-PDT with radiation developed local tumor recurrence. The limb function in all the patients, except for one, recovered to the level before surgery. None of the patients clinically showed any local or systemic complications. AO-PDT may be a promising new limb salvage modality for preservation of excellent limb function in patients with musculoskeletal sarcoma.

We recently encountered a very rare case of periosteal Ewing's sarcoma (PES), which was treated by surgery followed by photodynamic therapy using acridine orange with radiodynamic therapy. No more than 15 cases of PES have been reported previously in literature. In our case, MRI revealed the tumor to be localized on the cortical surface of the proximal humerus. Histopathological examination of biopsy specimens revealed a small round cell sarcoma suggestive of Ewing's sarcoma or PNET, and immunohistochemistry showed positive staining for MIC2. Although fusion genes EWS-FLI1 and EWS-ERG were undetectable, PES was still considered to be the most likely diagnosis. Therefore, we administered preoperative chemotherapy, as a result of which the tumor shrank to 48% of its original volume. With a view to preserve excellent shoulder and upper limb function, we attempted intralesional tumor resection supported by photodynamic therapy using acridine orange with radiodynamic therapy. After surgery followed by postoperative chemotherapy, the patient has, until the time of writing, had no local tumor recurrence and no evidence of metastatic disease, and can move his shoulder fully and throw a ball well. Since it has been reported that PES has a better prognosis and responsiveness to chemotherapy than intramedullary Ewing's sarcoma, we believe that such reduction surgery with photodynamic therapy might be the strategy of choice to obtain satisfactory limb function in cases of PES.

Lung metastasis is the most crucial event affecting the treatment of osteosarcoma and is dependent on tumor angiogenesis. To improve the prognosis for patients with osteosarcoma, prevention of lung metastasis is essential. Low-dose methotrexate is a useful drug for treating a variety of diseases. Low-dose methotrexate reportedly plays a role in antiangiogenesis for the synovial blood vessels in rheumatoid arthritis. However, whether low-dose methotrexate is correlated with tumor angiogenesis and metastasis is unclear. We investigated the inhibitory effect of methotrexate on lung metastasis in a rat osteosarcoma cell line with high metastatic potential, S-SLM. Two weeks after inoculation of S-SLM cells into male Fischer 344 rats, low-dose methotrexate (1.2 mg/kg once or twice a week) or saline was intraperitonealy injected for 4 weeks and the antimetastatic effect was evaluated. Low-dose methotrexate significantly reduced the number of lung metastatic nodules and the wet weight of the lungs. Immunohistochemical staining showed a decrease in microvessel density in the metastatic nodules. We also evaluated the effect of methotrexate on the proliferation of endothelial cells and S-SLM osteosarcoma cells in vitro. Methotrexate significantly inhibited the proliferation of endothelial cells at a lower concentration than that of S-SLM osteosarcoma cells. These data suggest that low-dose methotrexate inhibited lung metastasis of osteosarcoma through its antiangiogenic activity. Our results indicate that low-dose methotrexate is a promising drug for tumor dormancy therapy in patients with osteosarcoma and lung metastasis.

In the present study, we investigated chondrocytic characterization for newly established human chondrosarcoma cell lines. A chondrosarcoma cell line, HCS-TG, was established by the implantation of grade-2 human chondrosarcoma into athymic mice. Cloning of HCS-TG cells from passage 17 was performed. After cell cloning, two clonal-cell lines (HCS-TG C3 and E2) with good proliferative activities were obtained. These cell lines in monolayer culture retained a polygonal morphology. Their doubling times were 68 h and 45 h, respectively. mRNA expressions of type-I, -II, -X, and -XI collagens and aggrecan core protein were detected on reverse-transcription polymerase chain reaction. Protein expression of type-II collagen was confirmed in each cell line using Western blotting. However, there was no expression of type-I collagen. Moreover, gelatin zymography revealed that both cell lines produced extracellular matrices with matrix metalloproteinases 2 and 9. The parental HCS-TG cells had tumorigenicity in athymic mice; however, C3 and E2 were not tumorigenic. New clonal-cell lines HCS-TG C3 and E2 derived from human chondrosarcoma are morphologically chondrocytic in serial monolayer cultures and express chondrocytic phenotypes., (Copyright 2004 Springer-Verlag)

A 23-year-old man had eight cycles of adjuvant chemotherapy including doxorubicin, cisplatin, methotrexate, and ifosfamide and radical surgery for biopsy-proved osteosarcoma of the right fibular shaft. Two years after the initial diagnosis, he noticed a mass in the medial aspect of his right knee. Magnetic resonance imaging scans revealed a soft tissue tumor measuring 2 x 2 cm in the pericapsular region of the right knee. Histologically, this soft tissue tumor was composed of spindle cells with occasional atypical mitoses and without matrix formation. Immunohistochemically, the tumor cells were positive for vimentin, cytokeratin, and epithelial membrane antigen, and negative for alpha smooth muscle actin. A fusion gene, SYT-SSX was detected with reverse transcription-polymerase chain reaction. From the results, the secondary tumor was diagnosed as a synovial sarcoma. The current case of double sarcomas is rare. Both sarcomas were diagnosed accurately using immunohistochemical and molecular procedures. This case suggests a positive association between a second tumor and chemotherapy including intraarterial perfusion of doxorubicin.

It is well known that malignant cells show procoagulant activity, which is associated with their metastatic potential. Thrombin, the key enzyme of the blood coagulation system, is generated around tumor cells, promoting the migration and metastasis of tumor cells. In this study, we evaluated the effect of argatroban, a specific thrombin inhibitor, on the migration and metastasis of B16BL6 melanoma cells. In vitro argatroban dose-dependently inhibited cell migration, the maximum inhibition being observed in the presence of 10 microM argatroban (p < 0.0001). In order to investigate the antimetastatic effect of the thrombin inhibitor, we used an animal model that we have reported previously. C57BL6 mice which had received a bone (femur or tibia) transplanted into the dorsal subcutis were injected with B16 melanoma cells into the left heart ventricle. Intraperitoneal injection of argatroban (9 mg/kg/day for 4 weeks) significantly reduced the number of limbs with metastatic lesions as compared to a placebo (p < 0.05). These results suggest that argatroban was associated with reduced melanoma metastases by inhibiting cell migration. Our results showed that argatroban is effective for treatment of bone metastasis., (2004 S. Karger AG, Basel.)

We recently encountered a case with local recurrence of malignant fibrous histiocytoma (MFH) in the bone after wide resection, caused by minute intravenous tumor emboli which were retrospectively detected in MR imaging. The patient was a 69-year-old woman who initially noticed a mass in her left thigh. The tumor was diagnosed to be MFH, therefore a wide resection was performed; although the tumor was closely attached to the periosteum, it was not difficult to dissect the tumor subperiosteally from the cortex of the femur. The patient received postoperative brachytherapy, but no chemotherapy. Two years later, the tumor recurred with bony destruction of the femur. We reviewed the pre-operative films obtained by various imaging modalities, as well as the histology of the primary tumor, and found minute intravenous tumor emboli in the MR imaging obtained before surgery. Tumor emboli were also observed histologically in the small vessels of the surgically resected tumor. Such intravenous tumor emboli have recently been implicated in the development of regional bone metastasis near the site of the primary lesion in cases of malignant soft tissue tumors. Therefore, we concluded that the tumor recurrence in our case was caused by small tumor emboli invading the perforating veins of the femur. It is therefore emphasized that MR images should be carefully reviewed for the presence of such intravenous tumor emboli before surgery in cases of high-grade malignant sarcomas. As at the time of writing, our patient remains alive and disease-free, with no evidence of any local recurrence or distant metastasis after wide tumor resection for the recurrent tumor.

This report presents an extremely rare case of chondromyxoid fibroma arising at the clavicle. To the best of our knowledge, this may be the first case report demonstrating in detail the clinicopathological findings of chondromyxoid fibroma at the clavicle. The patient was a 34-year-old housewife. Radiography and CT demonstrated an osteolytic lesion with cortical thinning and expansion with partial destruction at the diaphysis of the left clavicle. MRI showed a homogeneous iso-signal intensity mass in T1-weighted imaging and a heterogeneous high-signal intensity in T2-weighted imaging. Histological findings of the widely resected tumor were consistent with chondromyxoid fibroma. The pre-operative diagnosis of chondromyxoid fibroma at an unusual location, as in this case, is difficult from the imaging examinations, or sometimes even from the histological examination of biopsy materials.

We evaluated the effect of gene therapy in the murine osteosarcoma cell line, LM8, which preferentially metastasizes to the lungs. LM8 cells were transduced with the gene for a herpes simplex virus thymidine kinase (HSV-tk) or Escherichia coli beta-galactosidase (lacZ). We investigated the cytotoxicity of LM8 cells bearing an HSV-tk gene after treatment with ganciclovir (GCV). LM8 cells bearing an HSV-tk gene were more sensitive than non-transduced cells. The remarkable inhibition of tumor growth and pulmonary metastases was confirmed in vivo. Our findings indicated that GCV kills tumor cells transduced with HSV-tk in vitro and in vivo.

When monocytes were cocultured with human osteosarcoma-derived cells (HOS cells), multinucleated giant cell formation of monocytes was induced. Intriguingly, even when a filter was interposed between monocytes and HOS cells, polykaryocytes also appeared. The multinucleated giant cells have characters similar to osteoclast-like cells. These findings indicate that soluble factor(s) secreted from HOS cells play an important role in polykaryocyte formation from monocytes. Twelve cloned cells were established from HSOS-1 cells and their capacities of inducing osteoclasts were investigated. Three cloned cells inducing nos. 4 and 9 had an ability of inducing osteoclasts (multinucleated giant cells, TRAP, calcitonin receptor and c-src mRNAs, osteoresorbing activity), and three cells, including nos. 1 and 5, did not show the ability. HOS cells and the cloned cells expressed several cytokine mRNAs. M-CSF was detected in the culture fluids of HOS cells, which also expressed RANK and RANK/ODF/OPGL mRNAs. Intriguingly, HOS cells secreting a soluble osteoclast inducing factors(s) expressed TNF-alpha converting enzyme mRNA. Furthermore, OCIF/OPG inhibited HOS cell-induced osteoclastogenesis and soluble RANKL could be detected in the culture fluids of HOS cells expressing TACE, suggesting that one of soluble osteoclast-inducing factor(s) is soluble RANKL. When blood monocytes were indirectly cocultured with HSOS-1 cells or cloned no. 9 cells in the presence of OCIF for 14 days, HOS cell-mediated osteoclastogenesis was suppressed, indicating that RANK-RANKL system is involved in the HOS cell-mediated osteoclastogenesis.

Our study focuses on the antineoplasmic action of hyperthermia. In this study, use of a heat-generating cement was exploited for the management of pathological and impending fractures in malignancies. The cement contains magnetic powder in ceramic particles and generates well-regulated heat when a magnetic field is applied externally. Eighteen rabbits were inoculated with blocks of VX2 tumor into one of their tibia. One week after the procedure, 9 rabbits were exposed to a magnetic field (HT group) while the remaining 9 rabbits were not (non-HT group). In rabbits killed between 20 and 42 days after the VX2 inoculation, the circumference of the operated leg was 7.9 +/- 0.3 cm and 10.2 +/- 1.0 cm (mean +/- SE) in HT and non-HT groups, respectively ( P < 0.05). Histological findings revealed the regressive change in tumor tissue of the HT group. By radiographs, pathological fractures and cortical bone destruction were seen in 5 and 8 rabbits in the non-HT group, respectively, but in the HT group these effects were absent in all the rabbits except 1 in which a definite diagnosis has not been made. Our findings demonstrate that controlled hyperthermia therapy using a newly developed bone cement suppresses tumor growth and prevents local bone destruction caused by VX2 tumors.