Establishment and characterization of a novel chordoma cell line: CH22.

Chordoma is a rare primary malignant bone tumor and there exist only a few established human chordoma cell lines. The scarcity of robust chordoma cell lines has limited the ability to study this tumor. In this report, we describe the establishment of a novel chordoma cell line and characterize its in vitro and in vivo behaviors. The tumor tissue was isolated from a patient with recurrent chordoma of the sacrum. After 6 months in culture, the chordoma cell line, referred here as CH22, was established. Microscopic analysis of two-dimensional culture confirmed that the CH22 cells exhibited a typical vacuolated cytoplasm similar to the well-established chordoma cell line U-CH1. Electron microscopy showed cohesive cells with numerous surface filopodia, pockets of glycogen and aggregates of intermediate tonofilaments in cytoplasm. Three-dimensional culture revealed that the CH22 cells could grow and form clusters by day 8. The MTT assays demonstrated that, compared with sensitive osteosarcoma cell lines, CH22 cells were relatively resistant to conventional chemotherapeutic drugs. Western blotting and immunofluorescence analysis confirmed that the CH22 cells expressed brachyury, vimentin, and cytokeratin. Finally, histological analysis of CH22 xenograft tumor tissues demonstrated the appearance of physaliphorous cells and positive staining of brachyury, cytokeratin, and S100. By CT and MRI, imaging xenografts showed the typical appearances seen in human chordomas. These findings suggest that the established novel human chordoma cell line CH22 and its tumorigenecity in SCID nude mice may serve as an important model for studying chordoma cell biology and the development of new therapeutic modalities.

Efficacy of sunitinib and radiotherapy in genetically engineered mouse model of soft-tissue sarcoma.

PURPOSE: Sunitinib (SU) is a multitargeted receptor tyrosine kinase inhibitor of the vascular endothelial growth factor and platelet-derived growth factor receptors. The present study examined SU and radiotherapy (RT) in a genetically engineered mouse model of soft tissue sarcoma (STS). METHODS AND MATERIALS: Primary extremity STSs were generated in genetically engineered mice. The mice were randomized to treatment with SU, RT (10 Gy x 2), or both (SU+RT). Changes in the tumor vasculature before and after treatment were assessed in vivo using fluorescence-mediated tomography. The control and treated tumors were harvested and extensively analyzed. RESULTS: The mean fluorescence in the tumors was not decreased by RT but decreased 38-44% in tumors treated with SU or SU+RT. The control tumors grew to a mean of 1378 mm(3) after 12 days. SU alone or RT alone delayed tumor growth by 56% and 41%, respectively, but maximal growth inhibition (71%) was observed with the combination therapy. SU target effects were confirmed by loss of target receptor phosphorylation and alterations in SU-related gene expression. Cancer cell proliferation was decreased and apoptosis increased in the SU and RT groups, with a synergistic effect on apoptosis observed in the SU+RT group. RT had a minimal effect on the tumor microvessel density and endothelial cell-specific apoptosis, but SU alone or SU+RT decreased the microvessel density by >66% and induced significant endothelial cell apoptosis. CONCLUSION: SU inhibited STS growth by effects on both cancer cells and tumor vasculature. SU also augmented the efficacy of RT, suggesting that this combination strategy could improve local control of STS.

Gamma-irradiation modulates vascular smooth muscle cell and extracellular matrix function: Implications for neointimal development.

OBJECTIVE: Migration of vascular smooth muscle cells (SMCs) into the subintimal space, and their proliferation and resultant deposition of extracellular matrix are key processes in the development of intimal hyperplasia, leading to vascular recurrent stenosis. The purpose of this study was to investigate the effects of clinically administered doses of gamma-radiation on SMCs and extracellular matrix proteins in vitro, to better understand how it impinges on cellular and extracellular components of recurrent stenosis. METHODS: The effects of gamma-irradiation (10, 20 Gy) on SMC migration into three-dimensional collagen matrix gels was quantitated by calibrated light microscopy, and the release of metalloproteinases into conditioned media was investigated with an enzyme-linked immunosorbent assay and zymography. Collagen production was assayed with [(3)H]-proline incorporation, and SMC phenotype changes with confocal microscopy with a fluorescent alpha-actin antibody. The effect of gamma-irradiation on extracellular matrix was investigated by quantitating untreated SMC proliferation ((3)H-thymidine incorporation) on irradiated endothelial cell-derived matrix and by assessing structural collagen matrix changes with sodium dodecylsulfate polyacrylamide gel electrophoresis. All groups were compared with nonirradiated control groups. RESULTS: SMC vertical migration was significantly decreased by gamma-irradiation (48% and 55%, respectively; P <.0001). Irradiation did not generate measurable matrix protein crosslinks, nor did it alter the production of metalloproteinases or collagen synthesis. However, gamma-irradiation decreased the ability of extracellular matrix to induce nonirradiated SMC proliferation (15% reduction; P =.0028). Moreover, gamma-irradiation reversed the secretory phenotype of cultured SMCs to a contractile type. CONCLUSIONS: The gamma-irradiation-induced reduction of cellular migration, changes in SMC phenotype, and functional activity of matrix-bound factors, and no measurable effects on the production of extracellular matrix proteins, may in part explain the diverse effects of gamma-irradiation on the restenotic response.

Mechanisms of reduced human vascular cell migration after photodynamic therapy.

Restenosis results from intimal hyperplasia and constrictive remodeling following cardiovascular interventions. Photodynamic therapy (PDT) has been shown to inhibit intimal hyperplasia in vivo by preventing neointimal repopulation of the treated vessel. This study was undertaken in an attempt to further dissect the mechanisms by which PDT acts on secreted and extracellular matrix proteins to inhibit migration of cultured human vascular cells. PDT of three-dimensional collagen gels inhibited invasive human smooth muscle cell (SMC) migration, whereas cell-derived matrix metalloproteinase production remained unaltered. Additionally, PDT generated cross-links in the collagen gels, a result substantiated in an ex vivo model whereby PDT rendered the treated vessels resistant to pepsin digestion and inhibited invasive migration of SMC and fibroblasts. These data support the premise that by inducing matrix protein cross-links, rendering the vessel resistant to degradation, in vivo PDT inhibits repopulation of the vessel and therefore intimal hyperplasia.