Epigenetic reprogramming and re-differentiation of a Ewing sarcoma cell line

Abstract

Developmental reprogramming techniques have been used to generate induced pluripotent stem (iPS) cells from both normal and malignant cells. The derivation of iPS cells from cancer has the potential to provide a unique scientific tool to overcome challenges associated with the establishment of cell lines from primary patient samples and a readily expandable source of cells that may be used to model the initial disease. In the current study we developmentally reprogrammed a metastatic Ewing sarcoma (EWS) cell line to a meta-stable embryonic stem (ES)-like state sharing molecular and phenotypic features with previously established ES and iPS cell lines. EWS-iPS cells exhibited a pronounced drug resistant phenotype despite persistent expression of the oncogenic EWS-FLI1 fusion transcript. This included resistance to compounds that specifically target downstream effector pathways of EWS-FLI1, such as MAPK/ERK and PI3K/AKT, which play an important role in EWS pathogenesis. EWS-iPS cells displayed tumor initiation abilities in vivo and formed tumors exhibiting characteristic Ewing histopathology. In parallel, EWS-iPS cells re-differentiated in vitro recovered sensitivity to molecularly targeted chemotherapeutic agents, which reiterated pathophysiological features of the cells from which they were derived. These data suggest that EWS-iPS cells may provide an expandable disease model that could be used to investigate processes modulating oncogenesis, metastasis, and chemotherapeutic resistance in EWS.

Keywords: Ewing sarcoma; drug resistance; epigenetics; induced pluripotent stem cells; reprogramming.

Figure 1

Generation of iPS cells from the Ewing cell line, CHLA-10. (A) Cell reprogramming schematic and timeline. (B) EWS-iPS cell colonies (A–E) enriched for the expression of SOX2 via puromycin selection are discernible by GFP fluorescence. Parent (HEK293T and CHLA-10), as well as HEK293T iPS cell controls are shown (scale = 200 micron).

Figure 2

EWS-iPS cell characterization. (A) Immunofluorescence microscopic detection of stem cell specific antigens (SSEA3, SSEA4, and TRA-1-60) in EWS-iPS clones. Brightfield, FITC, and DAPI nuclear stained cells are presented. Mouse IgM and rat IgM negative controls are shown. Scale = 200 micron. (B) PCR analysis of the EBNA1/oriP-based transgenic reprogramming factors in EWS-iPS clone B and clone C cells (right panel). Purified pEP4-EO2S-EN2K and empty pEP4 reprogramming plasmid controls are shown (left panel). (C) RT-PCR analysis of total ES cell-specific transcripts. Reverse transcriptase minus (-RT) control reactions are shown. (D) Alkaline phosphatase staining of parent (left column) and derived iPS cells (right column). (E) In vitro embryoid body (EB) formation assays. CHLA-10 parent, EWS-iPS B and C, and control HEK293T-iPS EBs. Scale = 200 micron. (F) Teratoma formation assays. H&E sections of control CHLA-10 (top panel), EWS-iPS cell clone B (middle panels), and EWS-iPS cell clone C (bottom panels) xenografts are shown. Astericks (^*) denote undifferentiated Ewing cells and yellow arrows denote myoblasts. Magnified images are depicted in yellow boxes. Scale = 200 micron.

Figure 3

Comprehensive analysis of DNA methylation. (A,B) Dendograms depicting unsupervised hierarchical clustering based on differential methylation at 7687 differentially methylated CpGs. In the accompanying heatmaps, green indicates a CpG methylation of less than 50% and red of more than 50%. Unsupervised hierarchical clustering based on differential methylation at (C) promoter regions of endogenous ES pluripotency genes and (D) stem cell specific DMRs. Heatmaps depict background subtracted β-values. TSS and 5′UTR denote probes located at transcriptional start sites and 5′untranslated regions, respectively. Methylation values for comparative cell lines (25 ES, 6 differentiated ES, 29 iPS, 6 differentiated iPS, and 5 hydatidiform mole samples) were obtained from the NCBI GEO database.

Figure 4

EWS-iPS cells demonstrate preservation of oncogene expression and acquired resistance to EWS-FLI1 targeted agents. (A) Dendograms depicting the methylation status of CpG sites spanning the EWS promoter of CHLA-10 parent, TC32 Ewing (grown in monolayer or as spheriods), EWS-iPS (clones B and C), and control iPS (iPS201B7_1312 and HDF51IPS_592) cell lines. Green indicates a CpG methylation of less than 50% and red of more than 50%. TSS and 5′UTR denote probes located at transcriptional start sites and 5′untranslated regions, respectively. (B) EWS-FLI1 gene expression in CHLA-10 parent (n = 4) and EWS-iPS (n = 4) (clones B and C) cells. Standard error of the mean is reported. p = 0.80 (iPS B vs. CHLA-10) and p = 0.64 (iPS C vs. CHLA-10). (C) Drug sensitivity assays. CHLA-10 parent (n = 6), iPS B (n = 3), or iPS C (n = 3) cell lines were exposed to chemotherapeutic agents or DMSO vehicle (untreated) in iPS base media containing 10 ng/ml human bFGF for a period of 48 h. Following treatment, MTS assays were performed and relative cell viabilities determined (relative cell viability = 490 nm absorbance treated/490 nm absorbance untreated). Standard error of the mean is reported. ^*p < 0.0001 (iPS vs. CHLA-10).

Figure 5

EWS-iPS cells exhibit tumor initiating properties. (A) EWS-iPS cells differentiate into Ewing cells that morphologically resemble CHLA-10 parental cell lines in vitro. EWS-iPS (clones B and C) were dissociated into individual cell suspensions (middle panels) and differentiated by plating in Ewing media [IMDM, 20% FBS, 0.2% L-Glut, 0.1% ITS] for a period of 48 h (bottom panels). Red boxes denote magnified fields. Brightfield or SOX2-dependent GFP marker channels are shown (scale = 200 micron). (B) In vivo differentiated EWS-iPS (n = 5) and parental (n = 9) cell lines possess comparable in vivo growth rates (p = 0.28; iPS vs. CHLA-10) and tumor latencies (p = 0.41; iPS vs. CHLA-10). Standard error of the mean is reported. (C) H&E stained sections of control CHLA-10 (left panel), EWS-iPS B (middle panel), and EWS-iPS C (right panel) tumor xenografts are shown. Astericks (^*) denote blood vessels. Scale = 200 micron.

Figure 6

Re-differentiated EWS-iPS cells recover sensitivity to chemotherapeutic agents targeting oncogenic pathways downstream of EWS-FLI1. CHLA-10 parent (n = 6) and (A) iPS (clones B and C; n = 3) or (B) differentiated iPS (iPS diff. clones B and C; n = 3) were exposed to chemotherapeutic agents or DMSO vehicle (untreated) in iPS cell base media containing 10 ng/ml human bFGF for a period of 48 h. Following treatment, MTS assays were performed and relative cell viabilities determined (relative cell viability = 490 nm absorbance treated/490 nm absorbance untreated). Standard error of the mean is reported. ^*p < 0.005 (iPS vs. CHLA-10).