Neuroblastoma Alkylator Modulator Testing Laboratory

Director: Co-Investigator:	C Patrick Reynolds, MD PhD Nino Keshelava, MD
Trainees:	Rita Grigoryan, MD Terri Harned, MD

This laboratory is a part of a project funded by an NCI RO1 grant focused on identifying mechanisms of resistance to alkylating agents in neuroblastoma and also identifying modulators of such resistance in preclinical studies that can then be tested in clinical trials.

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). 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 (5). Assessing drug interactions (additivity, synergy, antagonism) is done employing fixed-ratios of drug concentrations and the combination index approach (4,6) 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 (7).

Xenograft testing is carried out using cell lines established as xenograft tumors. Primary testing employs standard subcutaneous tumor models in nu/nu mice (8). Testing against disseminated disease models in SCID mice (created by tail vein injection) is also employed (8). Bone metastases in disseminated disease models are monitored using a Faxitron radiograph system (8).

Drug Sensitive Neuroblastoma Cell lines

SMS-KCNR
SMS-SAN
SMS-KAN
SMS-KCNR
SMS-LHN
SK-N-BE(1)
CHLA-122
CHLA-15

Modestly Drug Resistant Neuroblastoma Cell Lines

CHLA-20
LA-N-6

Multi-Drug Resistant Neuroblastoma Cell Lines

SK-N-BE(2)
CHLA-79
CHLA-90
CHLA-119
CHLA-140
CHLA-136
CHLA-171
CHLA-291

Drug Sensitive Neuroblastoma Xenografts

SMS-KCNR
SMS-SAN (both as subcutaneous and disseminated disease)
SMS-LHN

Modestly Drug Resistant Neuroblastoma Xenografts

CHLA-255 (subcutaneous and disseminated disease)

Multi-Drug Resistant Neuroblastoma Xenografts

CHLA-90
CHLA-119
CHLA-136 (subcutaneous and disseminated disease)
CHLA-140

Clinical Trials Enabled by this Laboratory

• N2002-01: A Phase I Study of High-Dose Pyrazoloacridine (PZA) (NSC 366140) Supported with Autologous Hematopoietic Stem Cell Rescue in Children with Recurrent or Resistant Neuroblastoma.
• N99-02: Modulation of Intensive Melphalan (L-PAM) by Buthionine Sulfoximine (BSO) (NSC-326321) and Autologous Stem Cell Support For Recurrent High-Risk Neuroblastoma (NCI 68).

Laboratory Funding

NCI RO1 CA82830

The USC-CHLA Institute for Pediatric Clinical Research

Laboratory Publications

1. Anderson CP, Seeger RC, Satake N, Meek WE, Keshelava N, Bailey HH, Monforte-Munoz HL, Reynolds CP: Buthionine sulfoximine and myeloablative concentrations of melphalan overcome resistance in a melphalan-resistant neuroblastoma cell line. J Pediat Hematol Oncol 23:500-505, 2001.
2. Anderson C, Reynolds CP: Cytotoxicity of buthionine sulfoximine (BSO) and melphalan/BSO in combination for neuroblastoma cell lines derived after myeloablative therapy. Bone Marrow Transplantation 30:135-140, 2002.
3. Yang B, Keshelava N, Anderson CP, Reynolds CP: Antagonism of buthionine sulfoximine cytotoxicity for human neuroblastoma cell lines by hypoxia is reversed by the bioreductive agent tirapazamine. Cancer Research 63:1520-1526, 2003.
4. Keshelava N, Tsao-Wei D, Reynolds CP: Pyrazoloacridine is active in multi-drug-resistant neuroblastoma cell lines with non-functional p53. Clinical Cancer Research 9:3492-3502, 2003.
5. Yang B, Reynolds CP: Tirapazamine cytotoxicity for neuroblastoma is p53-dependent. Clinical Cancer Research 11:2774-2780, 2005.

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. 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.
6. 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.
7. In preparation
8. 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.