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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.

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.

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.

We recently established a unique therapeutic modality for musculoskeletal sarcomas, combining acridine orange (AO) with photodynamic surgery (PDS), photodynamic therapy (PDT) and radiodynamic therapy (RDT); excellent results were obtained in the inhibition of local tumor recurrence after intralesional excision. However, AO is not yet approved for clinical use and intravenous injection. Therefore, methylene blue (MB), which has a very similar chemical structure to AO and is already in clinical use for other diseases, was investigated. In vitro studies using mouse osteosarcoma (LM8) cells revealed that MB-PDT had a strong cytocidal effect and that MB was not radiosensitive, showing no effect in RDT. In vivo studies showed that MB did not specifically accumulate in mouse osteosarcoma tissue and that it did not inhibit tumor growth. MB is not a better photosensitizer than AO in PDS, PDT and RDT for osteosarcoma.

Primary osteosarcoma of the lung is exceedingly rare. A 72-year-old man was admitted to our hospital; a primary osteosarcoma of the lung was diagnosed following computed tomography-guided needle biopsy. He died of rapidly progressing respiratory insufficiency without effective treatment seven months after the onset of symptoms. Macroscopic findings at autopsy showed typical histologic features of osteosarcoma without any bony lesion. Here we present a rare case and review the clinicopathological features of 20 previously reported cases.

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.

Various imaging methods have been employed for the extracorporeal detection of malignant tumors in the human body, such as scintigraphy and PET; however, none is sufficiently accurate and all are also very expensive. To resolve these issues, we attempted to develop a new imaging technique of photodynamic diagnosis (PDD) with acridine orange (AO). AO has the ability to rapidly and specifically accumulate in malignant tumors and emit brilliant green fluorescence after blue light excitation. In this study, we investigated the feasibility of PDD utilizing the fluorovisualization effect of AO, for the extracorporeal detection of mouse osteosarcoma inoculated into the soft tissues. At 2 h after intravenous administration of 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mg/kg AO, the tumor and the surrounding normal tissues were illuminated by blue light. The visual fluorescence contrast and ratio (X) of the difference in fluorescence intensity between the tumor and the surrounding normal tissues were evaluated using a high-resolution digital camera equipped with an absorption filter. In addition, the fluorescence contrast was also detected sequentially at 0.5, 1, 2, 3, 6 and 12 h after intravenous administration of AO at 1.0 mg/kg. The results revealed that the optimal condition for clear detection of the tumor was evaluation 2 h after intravenous injection of AO at 0.1 mg/kg, because it provided the best visual contrast on the digital images, and the fluorescence intensity as well as the value of X were higher as compared to the values under other conditions of dose and timing. Based on the results of an acute toxicity study of AO, the estimated LD50 of this substance following intravenous administration was 27.30 mg/kg. In conclusion, we believe that PDD using AO administered intravenously may be feasible for the detection of human musculoskeletal sarcomas in the soft tissues at extremities, and this technique might be a less invasive, less expensive, quicker and more accurate imaging modality than other previously reported imaging methods for this purpose., (Copyright 2006 S. Karger AG, Basel.)

Synovial sarcoma (SS) is one of common malignant soft-tissue tumors and is encountered most commonly in children and young adults. It frequently involves or invades major neurovascular structures and bones, and its local recurrence rate after simple resection has been reported to be as high as up to 80%. Because major nerves and vessels, as well as an adequate amount of bone, must be preserved to restore excellent limb function in cases of SS, a surgical technique entailing a low risk of local recurrence is needed. Based on the findings of recent experimental studies conducted by us using a mouse osteosarcoma model, we developed a novel therapeutic technique for SS, consisting of reduction surgery followed by photodynamic therapy using acridine orange (AO-PDT), with or without X-ray irradiation at 5 Gy. A preliminary study revealed that low-dose X-rays also excite AO like photons. After an initial study on cell cultures, this novel technique was applied to six cases of SS. A follow-up of the subjects to determine the clinical outcome revealed that none of the cases treated by AO-PDT, including the four cases treated by additional 5 Gy irradiation and the two cases not receiving any radiation, showed any evidence of recurrence or local/systemic complications during the follow-up period of 19-51 months after the surgery. Therefore, we believe that AO-PDT with 5 Gy irradiation may be an excellent novel therapeutic modality with reduction surgery to salvage excellent limb function in SS involving major nerves and vessels or bones.

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.

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.

We examined the effects of inactivation of the RB gene on chemosensitivity of human osteosarcoma cell lines, using the MTT assay and calculating the inhibition index. Although the human osteosarcoma cell lines HOS and MG63 have a wild-type RB gene, SaOS-2 and OSrb (established from retinoblastoma patient) have no active RB gene. We used these 4 cell lines in growth inhibition assays for doxorubicin, cisplatin and methotrexate, and assessed the chemosensitivity. In the growth inhibition assay for methotrexate, cell lines lacking an active RB gene were more resistant than cell lines with an active RB gene.

In the current study, the authors established a novel metastatic model to analyze organ selective metastasis by osteosarcoma, using a murine cell line, Dunn osteosarcoma. The lung, liver, kidney, and spleen were resected from syngeneic donor mice, and a tissue fragment of the respective organs was transplanted subcutaneously into a recipient mouse. Two weeks later, tumor cells were injected intravenously and the formation of metastatic deposits in the ectopic transplants was examined. Ectopic lung transplants had a significantly higher incidence of metastasis than either liver or kidney transplants. When enzymatically dispersed organ cells that were embedded in agar gel were transplanted, intravenous injection of tumor cells also resulted in a higher metastatic rate in lung transplants than in either liver or kidney transplants. Photomicrographs and microangiographs of the transplants showed equivalent revascularization of the different organs. Radiolabeled tumor cells were deposited in equivalent amounts in the ectopic transplants of the different organs after intravenous injection. In addition, in vitro growth of tumor cells was stimulated by medium conditioned with lung tissue in a dose-dependent manner. These results suggest that organ selective metastasis by osteosarcoma is not defined anatomically or hemodynamically, but may be caused by tumor cells responding to possible paracrine factors emanating from the lung.

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.

Purpose: The Japanese Musculoskeletal Oncology Group have developed an original prosthesis called the Kyocera Modular Limb Salvage system (KMLS system). This prosthesis has a semi-rotating hinge joint and is particularly designed for people with an Asian body type. The metallic parts of the prosthesis are made entirely of titanium alloy. The purpose of this study is to evaluate the clinical outcomes of treatment using this system following tumour resection of primary bone sarcoma of the distal femur. Methods: Between 2002 and 2010, 82 patients with primary bone sarcomas of the distal femur were treated. Seventeen patients underwent stem cementation, while 65 patients were treated with cementless prostheses. The mean follow-up period after surgery was 61 months. Results: Complications were observed in 28 of the 82 patients. Forty-one complications occurred in these 28 patients. Thirteen prostheses (16 %) required revision surgery due to complications, including five cases of stem breakage, three deep infections, three cases of aseptic loosening, one case of displacement of the shaft cap and one case of breakage of the tibial tray. The five-year overall prosthetic survival rate was 80.0 %. Four of the 82 patients underwent subsequent amputation due to local recurrence. The five-year limb salvage rate was 94.5 %. The mean function score according to the scoring system of the Musculoskeletal Tumour Society was 21.8 points (72.5 %). Conclusions: Although further follow-up is required to determine the performance, this prosthesis is considered to be satisfactory for reconstruction of the distal femur after resection of bone sarcoma. [ABSTRACT FROM AUTHOR]

Tendon rupture is a common sports injury in adults. However, the mechanical properties of repair tissue are inferior to those of normal tissue. To accelerate tendon healing, an in vivo approach using growth factors has been applied and has shown evidence for the efficacy of biological stimulation of the repair process. Recombinant human osteogenic protein-1 (rhOP-1) has been shown to be effective in stimulating matrix production by various connective tissues. To test the effect of rhOP-1 on the matrix metabolism of tendon cells in vitro, bovine tendon cells were cultured in monolayer with various doses of rhOP-1 for 7 days. The addition of rhOP-1 to cell culture media resulted in significant increases in cell proliferation, DNA content, and the synthesis of proteoglycans (PGs) and collagen, compared to control cultures. The relative percentage of large PGs in the OP-1 culture was higher than that in the control culture. In conclusion, we show for the first time that rhOP-1 stimulates the proliferation of tendon cells and their ability to synthesize and accumulate PGs and collagen in their extracellular matrix. These biological properties may be used in the tissue-engineering of tendon tissues. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:42-48, 2008 [ABSTRACT FROM AUTHOR]