Generation of chordoma cell line JHC7 and the identification of Brachyury as a novel molecular target

Abstract

Object: Chordoma is a malignant bone neoplasm hypothesized to arise from notochordal remnants along the length of the neuraxis. Recent genomic investigation of chordomas has identified T (Brachyury) gene duplication as a major susceptibility mutation in familial chordomas. Brachyury plays a vital role during embryonic development of the notochord and has recently been shown to regulate epithelial-to-mesenchymal transition in epithelial-derived cancers. However, current understanding of the role of this transcription factor in chordoma is limited due to the lack of availability of a fully characterized chordoma cell line expressing Brachyury. Thus, the objective of this study was to establish the first fully characterized primary chordoma cell line expressing gain of the T gene locus that readily recapitulates the original parental tumor phenotype in vitro and in vivo.

Methods: Using an intraoperatively obtained tumor sample from a 61-year-old woman with primary sacral chordoma, a chordoma cell line (JHC7, or Johns Hopkins Chordoma Line 7) was established. Molecular characterization of the primary tumor and cell line was conducted using standard immunostaining and Western blotting. Chromosomal aberrations and genomic amplification of the T gene in this cell line were determined. Using this cell line, a xenograft model was established and the histopathological analysis of the tumor was performed. Silencing of Brachyury and changes in gene expression were assessed.

Results: The authors report, for the first time, the successful establishment of a chordoma cell line (JHC7) from a patient with pathologically confirmed sacral chordoma. This cell line readily forms tumors in immunodeficient mice that recapitulate the parental tumor phenotype with conserved histological features consistent with the parental tumor. Furthermore, it is demonstrated for the first time that silencing of Brachyury using short hairpin RNA renders the morphology of chordoma cells to a more differentiated-like state and leads to complete growth arrest and senescence with an inability to be passaged serially in vitro.

Conclusions: This report represents the first xenograft model of a sacral chordoma line described in the literature and the first cell line established with stable Brachyury expression. The authors propose that Brachyury is an attractive therapeutic target in chordoma and that JHC7 will serve as a clinically relevant model for the study of this disease.

Fig. 1

Images characterizing human chordoma tissue. a: Sagittal T1-weighted MR image (left) showing sacral chordoma tissue. Photomicrographs (center and right) showing intraoperatively obtained sacral chordoma tissue with physaliferous phenotype. H & E, original magnification × 10 (center), × 20 (right). b: Photomicrographs showing chordoma tissue positive for S100β (left), cytokeratin (center), and Brachyury (immunohistochemistry; right). Stained with DAB, original magnification × 20. c: Western blot showing expression of Brachyury protein in primary sacral chordoma sample at 49 kD (red arrow).

Fig. 2

Establishment and characterization of human sacral chordoma-derived cell lines. a: Cultured chordoma cells (JHC7) display physaliferous features in phase-contrast images (left and center) and after staining with H & E (right). Original magnification × 10 (left and right), × 40 (center). Nuclear localization of Brachyury expression overlaps with DAPI signal, which counterstains nuclei in overlay images (immunocytochemistry). Original magnification × 20. b: Graph of JHC7 cell growth in vitro (using the MTT assay) demonstrates a doubling time of approximately 5 days. a.u. = arbitrary units. c: Western blot showing Brachyury expression in JHC7 cells harvested from the 4th, 10th, and 15th passages. Immunoreactive bands for Brachyury were prominent at 49 kD, and β-actin expression was probed for equal loading. d: Graph of T gene locus gain in JHC7 cells as demonstrated by an approximately 2-fold change in T gene copy number when compared with human mesenchymal stem cells (MSC). The GAPDH gene served as loading control using DNA PCR. *p < 0.05. e: One representative metaphase for karyotype analysis of G-banded chromosomes identified numerical, structural, and clonal abnormalities in JHC7 cells (for detailed description of analysis, see Results).

Fig. 3

Subcutaneous injection of established chordoma cells results in tumor that recapitulates the parental tumor phenotype. a: Mice injected with 2 million, 1 million, and 500,000 chordoma cells formed tumors within 3–4 months after implantation at the initial site of injection and grossly resembled parental tumor. Tumors pictured were obtained 6 months after injection. b: In a Western blot analysis of the JHC7 chordoma cell line, the original parental tumor (JHC7 Primary Tumor) and chordoma xenograft revealed prominent expression of Brachyury at 49 kD. c: Xenografts revealed a lobulated tumor composed of cells separated by fibrous septa, resembling the parent tumor (JHC7 Primary Tumor/Primary Chordoma). These tumors possessed physaliferous features characteristic of chordoma (H & E). Cells in the xenograft and parental tumor expressed Brachyury that localized to the nucleus (immunohistochemistry). Original magnifications × 4 (first and fourth columns), × 10 (second and fifth columns), and × 40 (third and sixth columns).

Fig. 4

Loss of Brachyury leads to phenotypic changes and loss of proliferation. a: Western blot analysis showing production of Brachyury-knockdown JHC7 cells using shRNA confirmed by loss of Brachyury expression. b: Phase-contrast images of Brachyury-knockdown JHC7 cells exhibiting a loss of physaliferous features and exhibiting a more differentiated-like phenotype with stellate-appearing cells. Original magnification × 10 (left column), × 20 (center column), and × 40 (right column). c: Using an MTT assay, complete loss of proliferation (p < 0.05) is observed in Brachyury knockdown JHC7 cells (Brachyury shRNA) when compared with JHC7 cells with empty vector (control shRNA). d: Using trypan blue exclusion, JHC7 cell viability 20 days after Brachyury knockdown is compared with control shRNA. *p < 0.05. e: Average JHC7 cell diameter of Brachyury shRNA compared with control shRNA cells. *p < 0.05. f: Changes in epithelial and mesenchymal markers in JHC7 cells 10 days after Brachyury knockdown (quantitative reverse transcriptase PCR); only changes in mesenchymal markers were significant (*p < 0.05).