The Role of Pre-Clinical 3-Dimensional Models of Osteosarcoma.

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

Myoepithelioma of bone: ultrastructural, immunohistochemical and molecular study of three cases.

Primary intraosseous myoepithelial tumors are rare neoplasms with only a handful of cases described in the medical literature. To date, intraosseous variant of benign myoepithelioma, due to its rarity, has not been studied ultrastructurally, and only one case of a malignant intraosseous myoepithelioma has been described. Three cases were retrieved from the files at the Massachusetts General Hospital (MGH). A diagnosis of benign myoepithelioma was made in case 1 and malignant epithelioma in cases 2 and 3. Ultrastructurally, intermediate filaments (without dense bodies) were found in each case with an abundance in case 1 and lesser amounts in cases 2 and 3. Surprisingly, cell junctions were not identified in case 1. However, they were found occasionally as intermediate junctions in case 2 and were easily identified as desmosome like junctions in case 3. The nucleus was irregular in the neoplastic cells of benign myoepithelioma which contrasted with cases 2 and 3 where the nuclei were oval yet had visible nucleoli. Herein, we add three new cases, including two new cases of malignant myoepithelioma. We also provide the first ultrastructural description of benign myoepithelioma of bone.

Modeling Ewing sarcoma tumors in vitro with 3D scaffolds.

The pronounced biological influence of the tumor microenvironment on cancer progression and metastasis has gained increased recognition over the past decade, yet most preclinical antineoplastic drug testing is still reliant on conventional 2D cell culture systems. Although monolayer cultures recapitulate some of the phenotypic traits observed clinically, they are limited in their ability to model the full range of microenvironmental cues, such as ones elicited by 3D cell-cell and cell-extracellular matrix interactions. To address these shortcomings, we established an ex vivo 3D Ewing sarcoma model that closely mimics the morphology, growth kinetics, and protein expression profile of human tumors. We observed that Ewing sarcoma cells cultured in porous 3D electrospun poly(ε-caprolactone) scaffolds not only were more resistant to traditional cytotoxic drugs than were cells in 2D monolayer culture but also exhibited remarkable differences in the expression pattern of the insulin-like growth factor-1 receptor/mammalian target of rapamycin pathway. This 3D model of the bone microenvironment may have broad applicability for mechanistic studies of bone sarcomas and exhibits the potential to augment preclinical evaluation of antineoplastic drug candidates for these malignancies.

Myofibroblastic differentiation of stromal cells in giant cell tumor of bone: an immunohistochemical and ultrastructural study.

The nature of the mononuclear stromal cells (MSCs) in giant cell tumor of bone (GCTB) has not been thoroughly investigated. The purpose of this study was to evaluate the degree and significance of myofibroblastic differentiation in 18 cases of GCTB by immunohistochemistry (IH) and/or electron microscopy (EM). All immunostained cases were found positive for smooth muscle actin (SMA) and/or muscle specific actin (MSA), most in 1-33% of the MSCs. Ultrastructurally, most MSCs were fibroblasts, and a significant number of cells displayed myofibroblastic differentiation. Myofibroblasts are an important component of MSCs in GCTB. The myofibroblastic population may be responsible in part for the production of matrix metalloproteinases (MMPs), which probably play a role in bone destruction, tumor aggression, and recurrence.

Growth plate regulation and osteochondroma formation: insights from tracing proteoglycans in zebrafish models and human cartilage.

Proteoglycans are secreted into the extracellular matrix of virtually all cell types and function in several cellular processes. They consist of a core protein onto which glycosaminoglycans (e.g., heparan or chondroitin sulphates), are attached. Proteoglycans are important modulators of gradient formation and signal transduction. Impaired biosynthesis of heparan sulphate glycosaminoglycans causes osteochondroma, the most common bone tumour to occur during adolescence. Cytochemical staining with positively charged dyes (e.g., polyethyleneimine-PEI) allows, visualisation of proteoglycans and provides a detailed description of how proteoglycans are distributed throughout the cartilage matrix. PEI staining was studied by electron and reflection contrast microscopy in human growth plates, osteochondromas and five different proteoglycan-deficient zebrafish mutants displaying one of the following skeletal phenotypes: dackel (dak/ext2), lacking heparan sulphate and identified as a model for human multiple osteochondromas; hi307 (ß3gat3), deficient for most glycosaminoglycans; pinscher (pic/slc35b2), presenting with defective sulphation of glycosaminoglycans; hi954 (uxs1), lacking most glycosaminoglycans; and knypek (kny/gpc4), missing the protein core of the glypican-4 proteoglycan. The panel of genetically well-characterized proteoglycan-deficient zebrafish mutants serves as a convincing and comprehensive study model to investigate proteoglycan distribution and the relation of this distribution to the model mutation status. They also provide insight into the distributions and gradients that can be expected in the human homologue. Human growth plate, wild-type zebrafish and fish mutants with mild proteoglycan defects (hi307 and kny) displayed proteoglycans distributed in a gradient throughout the matrix. Although the mutants pic and hi954, which had severely impaired proteoglycan biosynthesis, showed no PEI staining, dak mutants demonstrated reduced PEI staining and no gradient formation. Most chondrocytes from human osteochondromas showed normal PEI staining. However, approximately 10% of tumour chondrocytes were similar to those found in the dak mutant (e.g., lack of PEI gradients). The cells in the reduced PEI-stained areas are likely associated with loss-of-function mutations in the EXT genes, and they might contribute to tumour initiation by disrupting the gradients.

Selective effects of hydroxyapatite nanoparticles on osteosarcoma cells and osteoblasts.

The effects of hydroxyapatite nanoparticles (HA-NPs) on two kinds of cells, human MG63 cells and the normal osteoblasts were investigated. According to the MTT assay and fluorescent staining assay, it was proved that HA-NPs could inhibit the growth of MG63 cells but slightly support proliferation of the osteoblasts. Meanwhile, transmission electron microscopy (TEM) was employed to observe the ultrastructural alterations of both cells. The TEM results showed that HA-NPs had entered the two kinds of cells. Typical apoptosis was observed in the MG63 cells, especially in the group of 250 µg/mL with 5 days culture. While no apoptosis could be found in the normal osteoblasts at any concentration group of HA-NPs. Our results suggested that the HA-NPs had selective effects to different kinds of cells: supporting proliferation to the normal bone cells while causing apoptosis to the osteosarcoma cells.

Primary kaposiform hemangioendothelioma of a long bone: two cases in unusual locations with long-term follow up.

Kaposiform hemangioendothelioma (KHE) is a rare vascular neoplasm of low malignant potential that mainly affects infants and adolescents. The tumor almost exclusively occurs in somatic soft tissue or the retroperitoneum. We report herein two cases of primary KHE occurring in a long bone without cutaneous changes with long-term follow up in young patients. The patients were a 9-year-old girl and 5-year-old boy presenting with lytic lesions of the femur and humerus, respectively, without cutaneous lesions. Histologically, the neoplasms were comprised of nodules of spindle- to oval-shaped cells growing in an infiltrative fashion. The neoplastic cells formed poorly canalized or slit-like blood vessels alternating with solid spindle areas. Immunohistochemical studies showed that the tumor cells expressed CD31, CD34 and Fli1, but not HHV8, LNA-1 or GLUT1. D2-40 stained the neoplastic spindle cells and lymphatic channels adjacent to vascular lobules. The girl remains well with 15 years and 6 months follow up after a second complete excision. The boy has no signs of recurrence or metastasis nearly 5 years after local complete excision. To our best knowledge, this is the first report in the English literature of primary long bone occurrences of KHE without cutaneous changes with long-term follow up.

Zoledronic acid inhibits vasculogenic mimicry in murine osteosarcoma cell line in vitro.

BACKGROUND: To study the effects of zoledronic acid (ZA) on the vasculogenic mimicry of osteosarcoma cells in vitro. METHODS: A Three-dimensional culture of LM8 osteosarcoma cells on a type I collagen matrix was used to investigate whether osteosarcoma cells can develop vasculogenic mimicry, and to determine the effects of ZA on this process. In addition, the cellular ultrastructural changes were observed using scanning electron microscopy and laser confocal microscopy. The effects of ZA on the translocation of RhoA protein from the cytosol to the membrane in LM8 cells were measured via immunoblotting. RESULTS: ZA inhibited the development of vasculogenic mimicry by the LM8 osteosarcoma cells, decreased microvilli formation on the cell surface, and disrupted the F-actin cytoskeleton. ZA prevented translocation of RhoA protein from the cytosol to the membrane in LM8 cells. CONCLUSIONS: ZA can impair RhoA membrane localization in LM8 cells, causing obvious changes in the ultrastructure of osteosarcoma cells and induce cell apoptosis, which may be one of the underlying mechanisms by which the agent inhibits the development of vasculogenic mimicry by the LM8 cells.

Bavachin Induces Ferroptosis through the STAT3/P53/SLC7A11 Axis in Osteosarcoma Cells.

Ferroptosis is a new form of regulated cell death, which is mediated by intracellular iron. Although it is reported that bavachin has antitumour effects on several tumour cells and prompts the reactive oxygen species (ROS) generation, it is unclear whether ferroptosis can be induced by bavachin in osteosarcoma (OS) cells. In this study, we found that bavachin inhibits the viability of MG63 and HOS OS cell lines along with an increase in the ferrous iron level, ROS accumulation, malondialdehyde overexpression, and glutathione depletion. Moreover, iron chelators (deferoxamine), antioxidants (Vit E), and ferroptosis inhibitors (ferrostatin-1 and liproxstatin-1) reverse bavachin-induced cell death. Bavachin also altered the mitochondrial morphology of OS cells, leading to smaller mitochondria, higher density of the mitochondrial membrane, and reduced mitochondrial cristae. Further investigation showed that bavachin upregulated the expression of transferrin receptor, divalent metal transporter-1, and P53, along with downregulating the expression of ferritin light chain, ferritin heavy chain, p-STAT3 (705), SLC7A11, and glutathione peroxidase-4 in OS cells. More importantly, STAT3 overexpression, SLC7A11 overexpression, and pretreatment with pifithrin-α (P53 inhibitor) rescued OS cell ferroptosis induced by bavachin. The results show that bavachin induces ferroptosis via the STAT3/P53/SLC7A11 axis in OS cells.

Scaffold-based 3D cellular models mimicking the heterogeneity of osteosarcoma stem cell niche.

The failure of the osteosarcoma conventional therapies leads to the growing need for novel therapeutic strategies. The lack of specificity for the Cancer Stem Cells (CSCs) population has been recently identified as the main limitation in the current therapies. Moreover, the traditional two-dimensional (2D) in vitro models, employed in the drug testing and screening as well as in the study of cell and molecular biology, are affected by a poor in vitro-in vivo translation ability. To overcome these limitations, this work provides two tumour engineering approaches as new tools to address osteosarcoma and improve therapy outcomes. In detail, two different hydroxyapatite-based bone-mimicking scaffolds were used to recapitulate aspects of the in vivo tumour microenvironment, focusing on CSCs niche. The biological performance of human osteosarcoma cell lines (MG63 and SAOS-2) and enriched-CSCs were deeply analysed in these complex cell culture models. The results highlight the fundamental role of the tumour microenvironment proving the mimicry of osteosarcoma stem cell niche by the use of CSCs together with the biomimetic scaffolds, compared to conventional 2D culture systems. These advanced 3D cell culture in vitro tumour models could improve the predictivity of preclinical studies and strongly enhance the clinical translation.

Co-delivery of doxorubicin and paclitaxel by reduction/pH dual responsive nanocarriers for osteosarcoma therapy.

Nanoparticle-based drug delivery system offers a promising platform for combination cancer therapy. However, the inefficient drug release in cells reduces the therapeutic efficacy of cancer nanomedicines. Herein, a PEGylated poly(α-lipoic acid) copolymer (mPEG-PαLA) was prepared and used as a reduction/pH dual responsive nanocarrier to simultaneously deliver paclitaxel (PTX) and doxorubicin (DOX) for osteosarcoma therapy. The amphiphilic mPEG-PαLA could efficiently encapsulate both PTX and DOX during its self-assembly into micelles in aqueous solution to generate PTX and DOX co-loaded nanoparticles (NP-PTX-DOX). The as-prepared NP-PTX-DOX showed enhanced PTX and DOX release in response to reductive and acidic stimuli. Moreover, the dual-drug loaded nanoparticles were efficiently internalized by K7 osteosarcoma cells and released drugs intracellularly, as confirmed by flow cytometry analysis and confocal laser scanning microscopy. Consequently, NP-PTX-DOX exhibited synergistic therapeutic effects and induced enhanced cell apoptosis in K7 cells. Furthermore, NP-PTX-DOX presented improved biodistribution and higher tumor growth inhibition efficacy compared to the control groups in a murine osteosarcoma model. Altogether, the results of this work indicate that the proposed strategy is promising for osteosarcoma therapy using mPEG-PαLA copolymer as a dual-responsive nanocarrier to co-deliver anticancer drugs.

Clear cell chondrosarcoma: an ultrastructural study.

Clear cell chondrosarcoma (CCC) is a rare neoplasm. We report here a case of CCC. A 67-year-old Japanese man presented with right arthralgia for 1 year, and histological examination of the subsequent surgical resection of the right femoral bone showed the finding of CCC. Ultrastructurally, most organelles were observed in the perinuclear area. Clear neoplastic cells contained many glycogen particles in the area of the cytoplasm lacking organelles, although glycogen particles overall seemed to be evenly distributed in the cytoplasm. Some mitochondria, Golgi complex, actin-like filaments, and rough endoplasmic reticulum were also demonstrated in the cytoplasm of clear cells. Well-developed microvilli were also seen on the surface of neoplastic cells. These structures in neoplastic cells corresponded notably to structures of normal chondrocytes. Finally, our ultrastructural findings support further evidence that clear cells in CCC may show chondrocyte differentiation and a lack of an organelles area as well as abundant glycogen particles, may contribute to the clear cell morphology in CCC.

Roles of microRNA-22 in Suppressing Proliferation and Promoting Sensitivity of Osteosarcoma Cells via Metadherin-mediated Autophagy.

OBJECTIVE: To analyze the effect of microRNA-22 on autophagy and proliferation and to investigate the underlying molecular mechanism of osteosarcoma cell chemotherapy sensitivity. METHODS: MG-63 cells were divided into four groups, including a control group, a negative control (NC) group, a cisplatin group, and a cisplatin + miR-22 group. Proliferation of MG-63 cells that had been treated with cisplatin and transfected with miR-22 mimics was determined using MTT assay and colony formation assay. We assessed the degree of autophagy using flow cytometry through cellular staining of the autophagy lysosomal marker monodansylcadaverine (MDC). The effect of microRNA-22 on autophagy was observed along with the expression levels of Beclin1, LC3, metadherin (MTDH) and ATG5 by western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Luciferase reporter assay revealed the targeted binding site between miR-22 and the 3'-untranslated region (3'-UTR) of MTDH mRNA. Western blot and qRT-PCR were used to explore the level of MTDH in the control group, the NC group, the cisplatin group, and the miR-22 group for 6, 12, and 24 h. RESULTS: In the in vitro study, the MTT results indicated that the MG-63 cells with overexpression of miR-22 exhibited a significant decline in the proliferation capacity compared with the control group (0.513 ± 0.001, P < 0.0005). Similar to the MTT results, MG-63 cells that were transfected with miR-22 mimic (101.0 ± 10.58) formed fewer colonies compared with the cisplatin group (129.7 ± 4.163). MDC staining revealed that miR-22-overexpressing osteosarcoma (OS) cells treated with cisplatin showed a significant decrease in the expression of autophagy (7.747 ± 0.117, P < 0.0001). Our data revealed that miR-22 could regulate not only autophagy but also proliferation in the chemosensitivity of osteosarcoma cells. We found that miR-22 sensitized osteosarcoma cells to cisplatin treatment by regulating autophagy-related genes. In addition, Luciferase Reporter Assay revealed that miR-22 negatively regulated autophagy through direct targeting of MTDH. We performed western blot analysis to detect the MTDH expression level. The results revealed that the overexpression of miR-22 obviously decreased the expression of MTDH (1.081 ± 0.023, P < 0.001). CONCLUSION: Inhibition of miR-22 ameliorated the anticancer drug-induced cell proliferation decrease in osteosarcoma cells. MTDH was identified as the miR-22 target in OS cells and MTDH-triggered autophagy played a key function in the miR-22-associated chemotherapy sensitivity.

Osteofibrous dysplasia and adamantinoma in children and adolescents: a clinicopathologic reappraisal.

Osteofibrous dysplasia (OFD) and adamantinoma are rare and most commonly arise in the tibia of young individuals. Although OFD has typical histopathologic features, areas resembling OFD have often been noted at the periphery of otherwise classic adamantinomas, and some have suggested that OFD may be either a precursor to or a regressive phase of adamantinoma. The so-called OFD-like adamantinoma encompasses some features of both OFD and adamantinoma. We studied the clinical, imaging, histopathologic, immunohistochemical, ultrastructural, and molecular features of 16 OFD and 8 adamantinomas (5 OFD-like and 3 classic) in an attempt to further define their morphology, clinical course, and relationship. Patients with OFD were generally younger than those with adamantinoma. Osteoblastic and osteoclastic activity was more prominent in OFD than in OFD-like adamantinoma. In addition to the inconspicuous small clusters of epithelial cells in OFD-like adamantinoma, isolated keratin-positive cells with a unique ultrastructural hybrid fibroblastic-epithelial phenotype were found in the stroma of all OFD and OFD-like adamantinomas. Fluorescence in situ hybridization analysis revealed trisomies 7, 8, and/or 12 in the spindle cell stroma of OFD, OFD-like, and classic adamantinoma, supporting a neoplastic origin of OFD and a common histogenesis for all 3 lesions. Trisomies were not observed in osteoblasts or osteoclasts suggesting that the osseous component is reactive and non-neoplastic. Of the 11 OFD patients with follow-up (median, 4.5 y), all 3 who underwent incisional biopsy had persistent, nonprogressive disease and 2 of 8 who underwent curettage or wide excision had recurrence; none developed adamantinoma. All 6 adamantinoma patients with follow-up (3 classic and 3 OFD-like) were treated with wide excision. One with classic adamantinoma died of pulmonary metastases 9 years after presentation; the other 5 were free of disease with a median follow-up of 12 years. None of the classic adamantinomas evolved into OFD-like adamantinoma or OFD. Although the histopathology, immunohistochemistry, ultrastructure, and cytogenetics indicate that these lesions are closely related, our data and the literature suggest that only classic adamantinoma has malignant potential. OFD, OFD-like adamantinoma, and classic adamantinoma appear to show a progressive complexity of cytogenetic aberrations, perhaps indicative of a multistep neoplastic transformation.

Cellular accumulation and distribution of uranium and lead in osteoblastic cells as a function of their speciation.

Uranium (U) and lead (Pb) are accumulated and fixed for long periods in bone, impairing remodeling processes. Their toxicity to osteoblasts, the cells responsible for bone formation, is poorly documented. It has been previously shown that cytotoxicity and phenotypic effects of both metals on osteoblasts are highly influenced by metal speciation. Differences in sensitivity between cell types have been underlined as well. In this paper, cellular accumulation of U and Pb in cultured and primary osteoblastic cells was assessed by trace element analysis. Distribution of different species at the cell scale was investigated by electron microscopy. Internalization of both metals was shown to be correlated to cytotoxicity and population growth recovery after exposure. For each metal, the amount of metal uptake leading to 50% cell death was shown to be speciation-dependent. Scanning and transmission electron microscopy showed the formation of precipitates with phosphate in lysosomes for both metals, whose role in toxicity or cell defence remains to be clarified. Although a clear link was established between cytotoxicity and accumulation, differences in sensitivity observed in terms of speciation could not be fully explained and other studies seem necessary.

Paeoniflorin induces G2/M cell cycle arrest and caspase-dependent apoptosis through the upregulation of Bcl-2 X-associated protein and downregulation of B-cell lymphoma 2 in human osteosarcoma cells.

Paeoniflorin (PF), extracted from the peony root, has been proved to possess antineoplastic activity in different cancer cell lines. However, it remains unclear whether PF has an antineoplastic effect against osteosarcoma cells. The present study investigated the effects and the specific mechanism of PF on various human osteosarcoma cell lines. Using the multiple methods to detect the activity of PF on HOS and Saos­2 human osteosarcoma cell lines, including an MTS assay, flow cytometry, transmission electron microscopy and western blotting, it was demonstrated that PF induces inhibition of proliferation, G2/M phase cell cycle arrest and apoptosis in the osteosarcoma cell lines in vitro, and activation of cleaved­caspase­3 and cleaved­poly (ADPribose) polymerase in a dose­dependent manner. Furthermore, the pro­apoptotic factors Bcl­2 X­associated protein and BH3 interacting domain death agonist were uregulated, while the anti­apoptotic factors B­cell lymphoma 2 (Bcl­2) and Bcl­2­extra large were downregulated. In conclusion, these results demonstrated that PF has a promising therapeutic potential in for osteosarcoma.

Chondrosarcoma with myxoid change: a study using a quick-freezing and deep-etching method.

A middle-aged Japanese woman visited the Orthopedics Department of Nihon University Nerima Hikarigaoka Hospital complaining of pain in the left hip joint that had started approximately 8 months earlier. Following several examinations, including imaging diagnoses, an incisional biopsy demonstrated a malignant acetabular bone tumor, which was removed and examined by a quick-freezing and deep-etching (QF-DE) method, conventional electron microscopy, and light microscopy. Histologically, the tumor was a chondrosarcoma with marked myxoid changes. An interesting extracellular matrix was observed by the QF-DE method. The myxoid area consisted of a fine meshwork of proteoglycans (PG) without obvious aggrecans, which resembled that of PG usually present in the pericellular matrix of normal cartilage. Thin collagen fibrils with pleated surface structures of regular periodicity were also seen, which were sparsely distributed in wide areas except for the pericellular matrix. These collagen fibrils were of the type that are mainly located in the pericellular side of the territorial matrix in normal cartilage. A myxoid matrix consisting of thin collagen fibrils on the background of pericellular type PG suggested that the myxoid matrix in the chondrosarcoma resembled those of the pericellular and pericellular sides of the territorial matrices in normal cartilage.

Induction of apoptosis in osteogenic sarcoma cells by combination of tumor necrosis factor-related apoptosis inducing ligand and chemotherapeutic agents.

BACKGROUND: Osteosarcoma is one of the most common primary malignant tumors of bone with poor prognosis. TNF-related apoptosis inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. TRAIL induces apoptosis in various tumor cell lines but is not found to be cytotoxic to many normal cell types in vitro. We investigated the cytotoxic activity of TRAIL and chemotherapeutic agents, including methotrexate (MTX), doxorubicin (DOX) and cisplatin (CDDP), on established osteosarcoma cell line--S-732. METHODS: OS-732 cells were incubated with chemotherapeutic agents MTX, DOX and CDDP at various peak plasma concentrations (PPC), 0.1PPC, 1PPC and 10PPC, alone or with 100 ng/ml of TRAIL for 24 hours or 48 hours. MTT was used to evaluate the cytotoxic activity of different agents on OS-732. The apoptosis proportion was assayed by flow cytometry. Cellular morphologic changes were observed by phase contrast microscope, scan electron microscope, and transmission electron microscope. RESULTS: The inhibitory rate was (24.438 +/- 3.414)% with TRAIL of 100 ng/ml for 24 hours. The cells were responsive to DOX and CDDP with a dose-effect relationship (P < 0.05). In OS-732 cells, DOX and CDDP cooperated synergistically with TRAIL when incubated the cells with them for 24 hours (the combined inhibitory rate is (58.360 +/- 2.146)% and (54.101 +/- 2.721)%, respectively). TRAIL alone or drugs alone induced the apoptosis rate was less than 25% (P < 0.05). However, the combination of TRAIL and MTX did not present synergistic effects on OS-732 cells (P > 0.05, compared with TRAIL alone). CONCLUSIONS: Osteosarcoma OS-732 cells were not responsive to TRAIL-induced apoptosis. DOX and CDDP sensitize osteosarcoma OS-732 cells to TRAIL-induced apoptosis. The combination of TRAIL and MTX presented no synergistic effects on killing OS-732 cells.

[Chordomas]. / Les chordomes.

Chordoma is a rare bone tumor, believed to derive from notochordal rests, which generally arises at the two extremities of axial skeleton. We present a literature review on chordomas. Diagnosis has been greatly improved by MRI and immunohistochemistry. Conversely, complementary immunohistochemistry, cytometry and cytogenetic techniques have failed to improve prognosis evaluation. Radical surgery with free surgical margins is the most accurate curative treatment. Progress in radiotherapy should offer new therapeutic perspectives in the future. The recognition of new entities such as giant notochordal rest or hamartoma, and notochordal cells benign tumor, can lead to confusion since there is no consensus regarding their nature and whether or not they correspond to chordoma precursors. Prudence should be the rule in order to avoid overtreatment.

Distribution of macrophages, osteoclasts and the B-lymphocyte lineage in osteolytic metastasis of mouse mammary carcinoma.

The purpose of this study was to examine the localization of macrophages, B-lymphocytes and osteoclasts in tumoral lesions of mammary carcinoma metastasized to bone of non-immunocompromised mice. Mouse mammary carcinoma cells (BALB/c-MC) were injected through the left cardiac ventricle into 5-week-old female wild-type Balb/c mice. The femora and tibiae of mice with metastasized cancer were extracted, and thereafter processed for histochemical analyses. The foci of metastasized tumor cells occupied the metaphyseal area, and the cell death zones could be identified within the tumor mass. Abundant tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were found among the alkaline phosphatase (ALP)-reactive osteoblastic cell layer that covered the bone surface neighboring the metastatic lesion. In contrast, F4/80-positive macrophages/monocytes were localized adjacent to, or invading the metastatic tissue. In addition, some F4/80-positive cells were found in the aforementioned cell death zones. Unlike F4/80-positive cells, CD45R-positive B-lymphocytes did not accumulate at the surfaces of the tumor lesions, nor infiltrate into them, but were found scattered over bone marrow. Interestingly, some CD45R-positive cells were observed close to TRAP-positive osteoclasts in the stromal tissue surrounding the tumor lesion. Our findings suggest that, in the bone metastatic lesions of non-immunocompromised mice, F4/80-positive macrophages/monocytes accumulated on and/or infiltrated into the tumor nests, while CD45R-positive B-lymphocytes were associated with osteoclasts, rather than attacking metastatic tumor cells.