Anchorage-independent growth of Ewing sarcoma cells under serum-free conditions is not associated with stem-cell like phenotype and function

Abstract

Novel treatment strategies for Ewing sarcoma aim to eliminate residual tumor cells that have maintained the capacity to reinitiate tumor growth after intensive conventional therapy. Preclinical models that more closely mimic in vivo tumor growth than standard monolayer cultures are needed. Sphere formation under anchorage-independent, serum-free conditions has been proposed to enrich for cells with tumor-initiating, stem cell-like properties in various solid cancers. In the present study, we assessed the phenotype and functional stem cell characteristics of Ewing sarcoma spheres. Spheres were generated under serum-free culture conditions from four Ewing sarcoma cell lines and four relapse tumor biopsies. Standard monolayer cultures were established as controls. Median levels of surface expression of the Ewing sarcoma marker CD99 as well as the supposed stem cell marker CD133 and the neural crest marker CD57 were comparable between spheres and monolayers. Ewing sarcoma spheres from individual tumors failed to continuously self-renew by secondary sphere formation. They contained variable proportions of side populations (SPs). Sphere culture did not enhance the in vivo tumorigenicity of Ewing sarcoma cells in a murine xenograft model. We conclude that sphere formation under serum-free conditions is not a reliable tool to enrich for cells with stem cell characteristics in Ewing sarcoma. By mimicking the anchorage-independent, multicellular growth of Ewing sarcoma micrometastases, in vitro sphere growth may still add value as a preclinical tool to evaluate the efficacy of novel therapeutics.

The CXCR4 antagonist plerixafor (AMD3100) promotes proliferation of Ewing sarcoma cell lines in vitro and activates receptor tyrosine kinase signaling

“…Ewing sarcoma cell lines A673, TC-32, and TC-71 were originally received from the cell line bank at Children’s Hospital Los Angeles; CADO-ES1 from DSMZ (Braunschweig, Germany); and VH-64 from F van Valen (Institute of Experimental Musculoskeletal Medicine, University Hospital Münster). The low-passage cell culture DC-ES-6 was established in our laboratory and previously described [ 22 ]. LAN-5 neuroblastoma cells were originally provided by R Seeger (Los Angeles, CA) and HL-60 acute myeloid leukemia cells were purchased from ATCC (Manassas, VA).…”

Pediatric sarcomas (Review)

“…Interestingly, only partial overlap was observed among CD57 high and CD133 + populations of cells, suggesting that CD57 identify different population of Ewing's sarcoma cells with CSC phenotype. Previously, Leuchte et al ( 34 ) argued against a role of CD133 and CD57 as markers of CSCs in Ewing's sarcoma.…”

Pharmacological mTOR targeting enhances the antineoplastic effects of selective PI3Kα inhibition in medulloblastoma

“…Next we sought to test the effects of combined PI3Kα and mTOR inhibition on stem-like cancer cells grown in 3-D. Ewing sarcoma spheroids may not represent a reliable system to study stem-like cancer cell biology 42 . By contrast, we and others have shown that medulloblastoma cells grown under cancer stem cell conditions form 3-D neurospheres that adopt cancer stem cell characteristics 26,43–45 .…”