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Overview ideogram of CGH data of all chordoma cell lines. Every cell line corresponds to one color. Lines on the left side are losses, and on the right side are gains of chromosomal material. Arrows mark the CDK6 region on 7q and p16 region on 9p.

Detailed CGH karyograms of the chordoma cell lines as shown in the ideogram in Supplementary Figure 1 (according to ISCN: International System for Human Cytogentic Nomenclature).

Results of the single tandem repeats analysis of the cell lines U-CH1, U-CH2, UCH3, UCH6, UCH7, U-CH10, U-CH11 and UCH12. The results of the sex typing marker AMEL and the detected alleles for the markers D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, D22S1045 are given.

Expression data of mRNA as shown by box plot graphs for brachyury (A), collagen 2A1 (B) and CD24 (C).

Unsupervised cluster analysis of mRNA expression data of all chordoma cell lines. The T-gene (i.e., brachyury) is overexpressed (red). Cluster Analysis of chordoma cell lines with chondrocyte, liposarcoma and sarcoma expression data from GEO and own data from a cell line with cartilaginous differentiation and from tissue of a vertebral disc.

Effect of Abemaciclib (LY2835219) on the growth of chordoma lines U-CH1, U-CH6, CAL51 (negative control), and MCF7 (positive control). Cells were grown in 96-well-plates were incubated for 3 days at 37{degree sign}C with increasing concentrations of Abemaciclib (3, 6, 10, 30, 60, 100, 300, 600, 1000 nM) in the medium or left untreated for control. The amount of viable cells was determined by using the MTS cell viability test (GraphPad Prism software, Version 6).

Western blot analyses of 8 chordoma cell lines showing expression of brachyury at 47 kDa.

Clinical data of chordoma patients. Every sample was positive for brachyury. Asterisks (*) mark the parental chordomas of the established chordoma cell lines.

Proliferation assay using Alamar Blue assay for U-CH2, 3, 6 and 10. Increasing concentrations of Palbociclib lead to an increase of growth inhibition of chordoma cells.

Inversion microscopy views of the published chordoma cell lines U-CH1, U-CH2, U-CH 3, 6, 7, 10, 11, and 12. The cells showing the typical physaliphorous cytology; bars=100µm.

Supplementary Figure Legend

Immunocytology of U-CH1 control vs inhibited cells with 1000 nM Palbociclib for 3 days. Ki-67 decreases from 20% down to

Data Supplement from Preclinical Characterization of Novel Chordoma Cell Systems and Their Targeting by Pharmocological Inhibitors of the CDK4/6 Cell-Cycle Pathway

Expression data of mRNA as shown by box plot graphs for brachyury (A), collagen 2A1 (B) and CD24 (C).

List of up and down regulated genes on expression level with a p-value of 10-4 a FC of 10.

FISH results regarding the CDKN2A status in chordoma cell lines and the corresponding parental tumor.

Western blot analysis for cleaved PARP and cleaved caspase 3 of all chordoma cell lines as compared to HEK-293 cells (+/- stimulation with TNFα and Bortezomib for 18h). No corresponding bands are apparent in the cell lines after inhibition with 1000 nM Palbociclib for 3 days. U-CH 1-6 (A) and U-CH 7-12 (A).

To establish a chordoma tissue cohort (n = 43) and to correlate localization, size, metastasis, residual disease (R-status), recurrences, histological subtype, matrix content, and Ki-67 proliferation index with patients’ overall survival (OS). We used routine histopathology supplemented by immunohistochemistry. In our patient cohort (median age 69 years, range 17 to 84 years) the median OS was 8.25 years. 24 chordomas were localized in the sacrum, 6 in lumbar vertebrae, 7 in thoracic and cervical vertebrae, 5 were limited to the clivus, and one was localized in the nasal septum. Ten patients had metastases, with pulmonary, nodal, and hepatic involvement. 23 patients had recurrent disease. 23 chordomas were classified as ‘not otherwise specified’ (NOS). Besides NOS, we found the following differentiation patterns: renal cell cancer like in six cases, chondroid in four cases, hepatoid differentiation in three cases, and anaplastic morphology in six cases. Ki-67 index of ≥10 %, presence of metastasis, and the low content of extracellular matrix were statistically linked to poor OS (p

Chordomas are tumors that arise at vertebral bodies and the base of the skull. Although rare in incidence, they are deadly owing to slow growth and a lack of effective therapeutic options. In this study, we addressed the need for chordoma cell systems that can be used to identify therapeutic targets and empower testing of candidate pharmacologic drugs. Eight human chordoma cell lines that we established exhibited cytology, genomics, mRNA, and protein profiles that were characteristic of primary chordomas. Candidate responder profiles were identified through an immunohistochemical analysis of a chordoma tissue bank of 43 patients. Genomic, mRNA, and protein expression analyses confirmed that the new cell systems were highly representative of chordoma tissues. Notably, all cells exhibited a loss of CDKN2A and p16, resulting in universal activation of the CDK4/6 and Rb pathways. Therefore, we investigated the CDK4/6 pathway and responses to the CDK4/6–specific inhibitor palbociclib. In the newly validated system, palbociclib treatment efficiently inhibited tumor cell growth in vitro and a drug responder versus nonresponder molecular signature was defined on the basis of immunohistochemical expression of CDK4/6/pRb (S780). Overall, our work offers a valuable new tool for chordoma studies including the development of novel biomarkers and molecular targeting strategies. Cancer Res; 75(18); 3823–31. ©2015 AACR.