Ewing's Sarcoma Family Tumors (ESFT)
Preclinical Testing Laboratory

Recognizing the need for preclinical testing in ESFT, beyond what is provided in the NCI PPTP, a group of Children's Oncology Group (COG) investigators sought to have established a laboratory devoted to ESFT preclinical testing. These investigators, which included Poul Sorensen, MD PhD, Mark Bernstein, MD, and Richard Gorlick, MD, approached a generous donor, The Charles and Dana Nearburg Foundation, who via the National Childhood Cancer Foundation (NCCF), is providing funding for an ESFT preclinical testing laboratory. From applications submitted in response to an RFA from the COG, a grant review committee selected the proposal from the USC-CHLA Institute for Pediatric Clinical Research (IPCR). The funding for the ESFT Preclinical Testing Lab (www.ESFTlab.org) is provided in memory of Rett Nearburg (www.rett.org).

The ESFT Preclinical Testing Lab is under the direction of C Patrick Reynolds, MD PhD. Co-investigators are Nino Keshelava, MD and William May, MD.

Methods employed:

The primary cytotoxicity assay for the lab is the DIMSCAN 96 well assay. DIMSCAN employs fluorescein diacetate (FDA) to identify viable cells, with quenching of fluorescence by eosin Y, and viable cells quantified using a DIMSCAN digital image microscopy system. (1,2). The DIMSCAN assay has a 4 log dynamic range when testing solid tumor cell lines (3, 4), including ESFT (5). Testing is carried out under standard culture conditions (20% O2), but drugs shown to be active in initial testing are also assessed in bone marrow-level oxygen tension (5 % O2) and in 2% O2, which approximates the oxygen levels found in many tumors (6). Assessing drug interactions (additivity, synergy, antagonism) is done employing fixed-ratios of drug concentrations and the combination index approach (4,7) All cell lines used are human, have been tested and shown to be free of mycoplasma, and have been demonstrated to be unique using short tandem repeats (8).

Xenograft testing is carried out using cell lines established as xenograft tumors. Primary testing employs standard subcutaneous tumor models in nu/nu mice (9). Testing against disseminated disease models in SCID mice (created by tail vein injection) is also employed (9). Bone metastases in disseminated disease models are monitored using a Faxitron radiograph system (9).

ESFT Cell Lines

SK-N-MC
CHP-100
TC-32
TC-71
CHLA-9
CHLA-10
CHLA-258

ESFT Xenografts

TC-71
SK-N-MC
CHLA-10
CHLA-258

Laboratory Funding

Laboratory Publications

Bibliography

1. Proffitt RT, Tran JV, Reynolds CP: A fluorescence digital image microscopy system for quantitating relative cell numbers in tissue culture plates. Cytometry 24:204-213,1996.
2. Keshelava N, Frgala T, .Krejsa J, Kalous O, Reynolds, CP: DIMSCAN: a microcomputer fluorescence-based cytotoxicity assay suitable for pre-clinical testing of combination chemotherapy. Methods in Molecular Medicine Chemosensitivity Vol 1 ed. Blumenthal RD, Totowa: Humana Press pp 139-154, 2005.
3. Keshelava N, Seeger RC, Groshen S, Reynolds CP: Drug resistance patterns of human neuroblastoma cell lines derived from patients at different phases of therapy. Cancer Research 58:5396-5405, 1998.
4. Maurer BJ, Cabot MC, Reynolds CP: Syngergism of N-(4-hydroxypheynl)retinamide cytotoxcity by modulators of ceramide metabolism in solid tumor cell lines. J Natl Cancer Inst 92:1897-1908, 2000.
5. Batra S, Reynolds CP, Maurer BJ: Fenretinide cytotoxicity for Ewing's sarcoma (ES) and primitive neuroectodermal Tumor (PNET) cell lines is decreased by hypoxia and synergistically enhanced by ceramide modulators. Cancer Research 64:5415-5424, 2004.
6. Grigoryan R, Keshelava N, Anderson C, Reynolds, CP: In vitro testing of chemosensitivity in physiological hypoxia. Methods in Molecular Medicine Chemosensitivity Vol 1 ed. Blumenthal RD, Totowa: Humana Press pp 87-100, 2005.
7. Reynolds, CP, Maurer BJ: Assessing response to anti-neoplastic drug combinations in tissue culture models. Methods in Molecular Medicine Chemosensitivity Vol 1 ed. Blumenthal RD, Totowa: Humana Press pp 173-184, 2005.
8. Cabral DJ, Feaman HV, Sheard MA, Reynolds CP: Short tandem repeat genotyping using Identifiler^TM is a reproducible and cost-effective method for establishing and monitoring identity of pediatric cancer cell lines. Proc Amer Assoc Cancer Res 48:96, 2007.
9. Reynolds, CP, Sun BC, DeClerck YA, Moats RA: Assessing growth and response to therapy in murine tumor models. Methods in Molecular Medicine Chemosensitivity Vol 2 ed. Blumenthal RD, Totowa: Humana Press pp 335-350, 2005.