Abstract 2786: Establishing validated pediatric cancer cell lines and direct xenografts from post-mortem samples.
Authors: Woodburn Tito , Hall Heather A. , White Kerri , Calderon Ruben , Blaydes Rachel , Farooqi Ahsan , Hogarty Michael , Mosse Yael , Maris John , Erdreich-Epstein Anat , Miller Bradley , Reynolds C. Patrick 
Published: August 2014

Abstract
Background: Cell lines and direct xenografts (XGs) provide important laboratory models for cancer research. The COG Cell Line & Xenograft Repository (www.COGcell.org) establishes, characterizes, banks, and distributes cell lines and XGs from a wide-range of pediatric cancers. We explored the use of post-mortem (PM) samples, in particular readily-obtainable blood samples with circulating tumor cells, to initiate cell lines in vitro and direct-to-mouse XGs.
Methods: Tumor was minced and blood, bone marrow (BM), or pleural fluid mononuclear cells were separated by a ficoll gradient. Cells were cultured in IMDM + 20% FBS + 4mM L-Glutamine + ITS at 37°C in 20% O2 / 5% CO2. A subset of samples was also cultured in BM level hypoxia (5% O2) and/or tumor level hypoxia (2% O2). Another subset of samples was cultured in a serum-free formulation (Neurobasal-A) in addition to the standard IMDM formulation; 15 samples were also injected directly into NOD/SCID, nu/nu, or NSG mice. Neuroblastoma (NB) origin was confirmed by expression (RT-PCR) of tyrosine hydroxylase (TH), Ewings Family of Tumors (EFT) line origin by detection of the EWS-FLI1 translocation, and lymphoma by B-cell markers via flow cytometry. All lines were validated for identity by 16 loci short tandem repeat (STR) analysis (compared to original patient sample) and all tumor lines were tested for the lack of the Epstein-Barr Virus (EBV) genome by PCR.
Results: We received 33 PM samples (brain tumor = 1, EFT = 1, lymphoma = 3, NB = 26, RMS = 2) and established 24 continuous cell lines: 1 brain tumor, 1 EFT, 3 lymphoma, 17 NB, and 2 RMS; 4/9 from tumor, 15/18 from blood, 4/5 from BM, 1/1 from pleural fluid. Ten direct XGs were established (9 with matching cell lines): 3 lymphoma and 7 neuroblastoma; 8/9 from blood, 1/1 from BM, and 2/3 from tumor). The overall take rate for PM cell lines was 76%. Tumor was the least successful at 44% while PM blood samples were the most successful with an 89% take rate. Cell lines were also established for all 15 samples cultured in hypoxia and 5 of 6 in serum-free media. All cell lines and XGs were validated to original patient tissue or blood using STR analysis. All NB lines expressed TH and the EFT line has a EWS/FLI1 translocation. Blood or marrow mononuclear cells matching 9 cell lines and xenografts were transformed with EBV to generate lymphoblastoid cell lines as a source of germ line DNA.
Conclusions: PM samples enable establishing cell lines and xenografts from heavily-treated patients with progressive disease. These PM cell lines and XGs should manifest therapeutic resistance and thus closely reflect the tumor biology of subjects enrolled in early-phase trials. Physicians, patients, and parents should be made aware of the value of obtaining PM samples for research. These cell lines and XGs from PM samples will provide a renewable resource for cancer genomic, biological, and preclinical therapeutic studies of therapy-refractory pediatric cancers.
Citation Format: Tito Woodburn, Heather A. Hall, Kerri White, Ruben Calderon, Rachel Blaydes, Ahsan Farooqi, Michael Hogarty, Yael Mosse, John Maris, Anat Erdreich-Epstein, Bradley Miller, C. Patrick Reynolds. Establishing validated pediatric cancer cell lines and direct xenografts from post-mortem samples. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2786. doi:10.1158/1538-7445.AM2013-2786