Characterization and Drug Resistance Patterns of Ewing's Sarcoma Family Tumor Cell Lines

William A May,Rita S Grigoryan,Laura L Christensen,Daniel J Cabral,Jasmine Jenabi,Elizabeth R Lawlor,Nino Keshelava,Lingyun Ji,Timothy J Triche,C Patrick Reynolds,Javier S Castresana

doi:10.1371/journal.pone.0080060

Abstract

Despite intensive treatment with chemotherapy, radiotherapy and surgery, over 70% of patients with metastatic Ewing's Sarcoma Family of Tumors (EFT) will die of their disease. We hypothesize that properly characterized laboratory models reflecting the drug resistance of clinical tumors will facilitate the application of new therapeutic agents to EFT. To determine resistance patterns, we studied newly established EFT cell lines derived from different points in therapy: two established at diagnosis (CHLA-9, CHLA-32), two after chemotherapy and progressive disease (CHLA-10, CHLA-25), and two at relapse after myeloablative therapy and autologous bone marrow transplantation (post-ABMT) (CHLA-258, COG-E-352). The new lines were compared to widely studied EFT lines TC-71, TC-32, SK-N-MC, and A-673. These lines were extensively characterized with regard to identity (short tandem repeat (STR) analysis), p53, p16/14 status, and EWS/ETS breakpoint and target gene expression profile. The DIMSCAN cytotoxicity assay was used to assess in vitro drug sensitivity to standard chemotherapy agents. No association was found between drug resistance and the expression of EWS/ETS regulated genes in the EFT cell lines. No consistent association was observed between drug sensitivity and p53 functionality or between drug sensitivity and p16/14 functionality across the cell lines. Exposure to chemotherapy prior to cell line initiation correlated with drug resistance of EFT cell lines in 5/8 tested agents at clinically achievable concentrations (CAC) or the lower tested concentration (LTC): (cyclophosphamide (as 4-HC) and doxorubicin at CAC, etoposide, irinotecan (as SN-38) and melphalan at LTC; P<0.1 for one agent, and P<0.05 for four agents. This panel of well-characterized drug-sensitive and drug-resistant cell lines will facilitate in vitro preclinical testing of new agents for EFT.

Highlights

Ewing’s Family of Tumors (EFT) (Ewing’s sarcoma (ES) and peripheral primitive neuroectodermal tumors (PNET)) are aggressive malignancies occurring in the childhood through adolescent/ young adult years [1]
These oncogenic fusion transcription factors have been shown to alter the expression of a number of tumor promoting target genes, though none has yet been shown to correlate with clinical outcome [5,6]
These include both patients treated with first-line chemotherapy and patients relapsing after myeloablative chemotherapy +/2 radiotherapy supported by autologous stem cell transplant

Summary

Introduction

Ewing’s Family of Tumors (EFT) (Ewing’s sarcoma (ES) and peripheral primitive neuroectodermal tumors (PNET)) are aggressive malignancies occurring in the childhood through adolescent/ young adult years [1]. Despite advances in the treatment of EFT that have led to survival rates of approximately 65–75% for localized disease, outcomes for patients with metastatic or recurrent EFT remain poor [1,2,3]. A distinguishing feature of EFT is the universal presence of EWS/FLI1 (and related EWS/ETS) fusion transcription factors [4]. These oncogenic fusion transcription factors have been shown to alter the expression of a number of tumor promoting target genes, though none has yet been shown to correlate with clinical outcome [5,6]. Resistance to chemotherapy may be an acquired phenomenon, reflecting a selection of the tumor cell population best able to survive exposure to cytotoxic agents

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