Characteristics of human Ewing/PNET sarcoma models

Abstract

Ewing/PNET (peripheral neuroepithelioma) tumors are rare aggressive bone sarcomas occurring in young people. Rare-disease clinical trials can require global collaborations and many years. In vivo models that as accurately as possible reflect the clinical disease are helpful in selecting therapeutics with the most promise of positive clinical impact. Human Ewing/PNET sarcoma cell lines developed over the past 45 years are described. Several of these have undergone genetic analysis and have been confirmed to be those of Ewing/PNET sarcoma. The A673 Ewing sarcoma line has proven to be particularly useful in understanding the biology of this disease in the mouse. The chromosomal translocation producing the EWS/FLI1 fusion transcript characterizes clinical Ewing sarcoma. Cell lines that express this genetic profile are confirmed to be those of Ewing sarcoma. The A673 Ewing sarcoma line grows in culture and as a xenograft in immunodeficient mice. The A673 model has been used to study Ewing sarcoma angiogenesis and response to antiangiogenic agents. Many Ewing sarcoma clinical specimens express the cell surface protein endosialin. Several Ewing sarcoma cell lines, including the A673 line, also express cell surface endosialin when grown as subcutaneous tumor nodules and as disseminated disease; thus the A673 is a useful model for the study of endosialin biology and endosialin-directed therapies. With the advent of tools that allow characterization of clinical disease to facilitate optimal treatment, it becomes imperative, especially for rare tumors, to develop preclinical models reflecting disease subsets. Ewing PNET sarcomas are a rare disease where models are available.

Figure 1a

Representative complex karyotype of the A673 cell line showing multiple rearrangements, including a chromosome 11 and 22 fusion, b: Fluorescence in situ hybridization (FISH) analysis for EWSR1 in A673 Ewing sarcoma cells.

Figure 2a

Cytokine profiling of human A673 Ewing sarcoma cell-conditioned medium. b: Angiogenic growth factor profiling of human A673 Ewing sarcoma cell-conditioned medium.

Figure 3

Growth delay of subcutaneously implanted A673 human Ewing sarcoma xenografts. Mice were treated with intraperitoneal injections of bevacizumab (10, 18.6 or 37.2 mg/kg) twice weekly. The lower-dose treatments (10 and 18.6 mg/kg) were initiated when tumors were 100 mm³, and the high-dose treatment (37.2 mg/kg) was initiated when the tumors were 300 mm³.

Figure 4

Immunohistochemical staining of human A673 human tumor xenografts after treatment with bevacizumab or control buffer is shown. The staining for CD31 to visualize endothelial cells is green, and the staining for NG2 to visualize pericytes is red.

Figure 5a

Histologic and immunohistochemical features of Ewing sarcoma. Classic Ewing sarcoma appears as sheet of monotonous round cells. The cells have little cytoplasm and round nuclei. The cells show strong plasma-membrane staining for endosialin. There is no staining with the isotype control antibody, b: Endosialin staining intensities in 9 human Ewing sarcoma clinical specimens. Each dotted line is a clinical specimen showing the endosialin staining intensity at 1+, 2+ and 3+ levels. The solid line is the mean staining intensity for the 9 clinical specimens at each level (19).

Figure 6a

Endosialin cell surface protein expression in 5 human Ewing sarcoma cell lines and Hek293 cells transfected to express high levels of endosialin. Four of the 5 human Ewing sarcoma cell lines express endosialin. b: Human A673 Ewing sarcoma cells immunostained with an antibody to endosialin showing a cell surface staining pattern. c. The mean fluorescence intensity from flow cytometric histograms for SK-ES-1 and A673 human Ewing sarcoma cells showing the relative fluorescence intensity for expression of CD146.

Figure 7

Immunohistochemical staining for endosialin in tissue from immunodeficient mice with disseminated A672 Ewing sarcoma xenografts, along with the scoring of the intensity of endosialin tumor staining for each specimen, is shown.