UM-Chor1: establishment and characterization of the first validated clival chordoma cell line

Abstract

OBJECTIVE Chordomas are rare malignant tumors thought to arise from remnants of the notochord. They can be located anywhere along the axial skeleton but are most commonly found in the clival and sacrococcygeal regions, where the notochord regresses during fetal development. Chordomas are resistant to many current therapies, leaving surgery as the primary method of treatment. Cancer cell lines have been useful for developing new cancer treatments in a laboratory setting that can then be transferred to the clinic, but there are only 4 validated chordoma cell lines available. The objective of this work was to establish chordoma cell lines from surgical tissue in order to expand the library of lines available for laboratory research. METHODS Chordoma tissue from the clivus was processed and sorted by flow cytometry to obtain an isolated population of chordoma cells. These cells were grown in culture and expanded until enough doublings to consider the line established. Identification of a chordoma cell line was made with known markers for chordoma, and the line was observed for ALDH (aldehyde dehydrogenase) subpopulations and tested in serum-free growth conditions as well as in vivo. RESULTS A fifth chordoma cell line, UM-Chor1, was successfully established. This is the first chordoma cell line originating from the clivus. Validation was confirmed by phenotype and positivity for the chordoma markers CD24 and brachyury. The authors also attempted to identify an ALDH^high cell population in UM-Chor1, UCH1, and UCH2 but did not detect a distinct population. UM-Chor1 cells were able to form spheroids in serum-free culture, were successfully transduced with luciferase, and could be injected parasacrally and grown in NOD/SCID mice. CONCLUSIONS The availability of this novel clival chordoma cell line for in vitro and in vivo research provides an opportunity for developments in treatment against the disease.

Keywords: ALDH = aldehyde dehydrogenase; DAPI = 4′,6-diamidino-2-phenylindole; DEAB = diethylaminobenzaldehyde; EMA = epithelial membrane antigen; FITC = fluorescein isothiocyanate; FSP = fibroblast surface protein; GFP = green fluorescent protein; HBSS = Hank's balanced salt solution; NCAM = neural cell adhesion molecule; PBS = phosphate-buffered saline; PE = phycoerythrin; cell line; chordoma; clivus; oncology; rHIV = recombinant human immunodeficiency virus.

Figure 1

A) The primary clival chordoma exhibited phenotypic characteristics of classical chordoma. B) Small pieces of tumor tissue adhered to a flask coated with collagen I and chordoma cells grew from it. C) UM-Chor1 at passage 35.

Figure 2

A single cell suspension of chordoma cells and fibroblasts was separated by flow cytometry. The unstained cell population (left) was used as a control for the cell population stained with antibody markers for fibroblasts and chordomas (right).

Figure 3

A) UCH1, UCH2, and UM-Chor1 were stained for DAPI, Brachyury, and CD24. Intranuclear staining of brachyury was strong in UM-Chor1. B) Brachyury was also confirmed in UM-Chor1 by flow cytometry, compared to an isotype control antibody.

Figure 4

UCH1, UCH2, UM-Chor1 and fibroblasts from a head and neck patient were analyzed for increased ALDH1 activity by the Aldefluor assay. When compared with a DEAB-inhibited control, UCH1, UCH2, UM-Chor1 registered ALDH^high populations of .48%, .37%, and .17%, respectively. In contrasts, the fibroblasts measured an ALDH^high population of 6.83%.

Figure 5

A) UM-Chor1 cells were plated in a serum-free, low-attachment plate. B) After four days, small clusters of chordospheres were seen. C) Analyzing these chordospheres for ALDH1 against an inhibited control resulted in an ALDH^high population of .24%.

Figure 6

A) UM-Chor1 was successfully transduced with luciferase and 1.0X10⁶ cells were injected parasacrally into three NOD/SCID mice. B) Tumor growth was measured over five weeks by bioluminescence. C) The xenograft resembled the original primary tumor when stained for intranuclear brachyury.