Methods

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 (2). 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 (3). Assessing drug interactions (additivity, synergy, antagonism) is done employing fixed-ratios of drug concentrations and the combination index approach (4). 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.

Xenograft testing is carried out using EFT cell lines established as xenograft tumors. Primary testing employs standard subcutaneous tumor models in nu/nu mice (5). Testing against disseminated disease models in SCID mice (created by tail vein injection) is also employed (5). Pulmonary metastases in disseminated disease models are monitored using a Faxitron radiograph system (5).

All cel llines and xenografts employed in the EFT preclincal testing lab have been characterized for response to standardly employed cytotxic agents, validated by molecular markers to be EFT in origin, and using short tandem repeat assay (STR) to match the origial patient, or (if no tissue from the patient is availalbe to validate the line) to have a unique STR signature (1).

References:
1.  Grigoryan R, Keshelava N, Cabral DJ, May W, Reynolds CP: Drug resistance patterns of Ewings sarcoma family tumor cell lines derived from patients at different phases of therapy. Proc Amer Assoc Cancer Research 49: 3756, 2008.

The cell lines and xenografts employed in the EFT Preclinical Testing Lab are available via the Children's Oncology Group Cell Culture and Xenograft repository at www.COGcell.org.

EFT Cell Lines

  • SK-N-MC
  • CHP-100
  • TC-32
  • TC-71
  • CHLA-9
  • CHLA-10
  • CHLA-25
  • CHLA-32
  • CHLA-258
  • CHLA-352

EFT Xenografts

  • TC-71
  • SK-N-MC
  • CHLA-10
  • CHLA-258
  • CHLA-352

Bibliography

  • 1. 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.
  • 2. Frgala T, Kalous O, Proffitt RT, Reynolds CP: A novel cytotoxicity assay with a 4 log dynamic range that identifies synergistic drug combinations. Molecular Cancer Therapeutics (in press, 2006)
  • 3. 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.
  • 4. 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.
  • 5. 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.