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.

The molecular aspects of chordoma

“…Brachyury has been silenced in the JHC7 primary sacral chordoma cell line by shRNA in vitro, which arrested growth [42]. Nelson and associates searched for brachyury target genes with an integrated functional genomics approach, and brachyury was determined to activate transcription.…”

Molecular characterization of chordoma xenografts generated from a novel primary chordoma cell source and two chordoma cell lines

“…11,12,14,20 To date, very few chordoma cell lines have been reported, namely U-CH1 17 and U-CH2b, 2 along with many more putative chordoma cell lines. Only a few of these have been shown to generate tumors in established mouse xenograft models and to retain molecular characteristics of the parent tumor and expanded cells.…”

Chordoma and its embryonic determinants

“…The CH1 and CH2 cells are no different in that respect. It can be hypothesized that if these cells were to regain brachyury expression, their proliferative capacity would increase since it has been shown that loss of brachyury in chordoma cultures induces growth arrest and senescence (21)(22)(23) Finally, assessing the difference in proliferation between cultured chordoma cells with enriched DMEM and enriched DMEM containing 33% FCM showed no apparent visual difference. However, in order to reliably conclude that the FCM supplementation does indeed have no effect, the proliferation pattern of both cultures needs to be assessed over longer time.…”