Be 2 c

The human neuroblastoma cell lines BE(2)-C (CRL-2268; ATCC, Manassas, VA, USA), SK-N-DZ (CRL-2149; ATCC), SK-N-F1 (CRL-2141; ATCC), and SHEP1^{44 (link)} cells were cultured in a 1 : 1 mixture of DMEM and Ham's nutrient mixture F12 supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Cells were examined and phase-contrast images captured using an Axio Observer microscope and the software AxioVision (Carl Zeiss MicroImaging, Thornwood, NY, USA).

Drug testing was conducted in vitro on three human NB cell lines: BE(2)-C (purchased in 2011 from ATCC, Manassas, VA), SMS-KCNR (kindly donated by Dr. John Maris in 2004, The Children's Hospital of Philadelphia, PA), and CHLA90 (kindly donated by Patrick Reynolds in 2006, The Children's Hospital of Los Angeles, CA). SMS-KCNR cells were also used for drug testing in vivo. Two of the cell lines tested were MYCN amplified (BE(2)-C and SMS-KCNR) and one was non-amplified (CHLA90). All cell lines were validated by comparing STRs in May 2012 (DNA Diagnostics Center, Fairfield, OH). Cells were cultured in RPMI-1640 with 10% (vol/vol) certified fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Incubation was at 37°C with 5% CO₂. All cell culture reagents were purchased from Life Technologies (Grand Island, NY). Cells were grown in 6-well or 96-well plates and were allowed to grow to 60-70% confluence prior to addition of drugs.

SKOV-3
(ATCC, HTB77), OVCAR-3 (ATCC, HTB-161),
OVCAR-8 (inherited from Laurent Brad’s previous laboratory),
and CAOV-3 (ATCC, HTB75) ovarian cancer cells were grown in complete
DMEM media (Gibco, 11965). IGROV-1 (Sigma, SCC203) and 2008 (kindly
provided by Dr. François X. Claret, University of Texas M.D.
Anderson Cancer Center) ovarian cancer cells were grown in complete
RPMI medium (Gibco, 22400). ES2 (ATCC, CRL-1978) was grown in McCoy’s
5A complete medium (ATCC, 30-2007). BE(2)C (ATCC, CRL-2268), SH-EP1
(ATCC, CRL-2269), SH-SY5Y (ATCC, CRL-2266), KELLY (Sigma, 92110411),
SK-N-AS (Sigma, 94092302), and LAN-5 (COG, http://www.cogcell.org) neuroblastoma
cell-lines were maintained in RPMI1640 media (Gibco, 11875) supplemented
with 10% heat-inactivated FBS. TH-MYCN+/+ cells were derived by mechanical
dissociation of tumors obtained from TH-MYCN homozygous mice^{51 (link)−53 (link)} and were maintained in RPMI1640 media (Gibco, 11875) supplemented
with 20% heat-inactivated FBS, 10–5 mM 2-mercaptoethanol, 1
mM sodium pyruvate, and 1× nonessential amino acids (Gibco, 11140076).

Single drug tests were conducted on the following NB cell lines: BE(2)‐C (ATCC, Manassas, VA), SMS‐KCNR (graciously donated by Dr. John Maris in 2004, The Children's Hospital of Philadelphia, PA), CHLA‐90 (kindly donated by Patrick Reynolds in 2006, The Children's Hospital of Los Angeles, CA), and SH‐SY5Y (ATCC, Manassas, VA). Cell lines were certified by short tandem repeat (STR) analysis (DNA Diagnostics Center, Fairfield, OH). Drug tests were additionally conducted on two Stage 4 relapsed primary NB patient cells MGT‐015‐08 and MGT9‐102‐08 (Helen DeVos Children's Hospital, Grand Rapids, MI). Cells were cultured in RPMI‐1640 with 10% (vol/vol) certified fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Incubation was at 37˚C with 5% CO₂. All cell culture reagents were purchased from Life Technologies (Grand Island, NY). Cells were grown in 6‐well or 96‐well plates. For all experiments using 96‐well plates, BE(2)‐C, CHLA90, and all preliminary cell lines were plated at 3500 cells/well, and SMS‐KCNR and SH‐SY5Y cells were plated at 5000 cells/well. All plated cells were allowed to adhere overnight and grown to 60–70% confluence within the well prior to drug treatment.