Abstract B13: Successful establishment of a novel neurosphere and adherent cell line pair from autopsied pediatric glioblastoma
Authors: Lin Frank Y. , Kogiso Mari , Mao Hua , Wang Yue , Qi Lin , Baxter Patricia , Walter Andrew , Parsons D. Will , Li Xiao-Nan 
Published: May 2015

Abstract
Background: Pediatric glioblastoma (GBM) is a lethal cancer that is generally refractory to conventional treatment strategies. The scarcity of relevant pre-clinical models for this tumor has been a key limitation to the development of novel agents for the treatment of this deadly disease. A growing body of evidence supports the role of tumor progenitor cells in gliomagenesis, with CD133+ cells, a putative marker of neural stem cells and glioma progenitor cells, representing a sub-population that has been implicated in treatment resistance. In order to facilitate basic and pre-clinical studies of pediatric GBM, including high-throughput drug screening and assessment of differential responses to agents screened, we sought to establish a novel primary cell line pair of refractory pediatric GBM consisting of a traditional adherent line paired with a multicellular tumorosphere (neurosphere) line enriched for CD133+ cells.
Methods: Tumor tissue was obtained from an autopsy specimen of a 10 year old pediatric patient with refractory GBM post-XRT and temozolomide and utilized to establish two cell lines: a traditional adherent cell line using serum based media and a neurosphere line grown in serum-free media supplemented with EGF and bFGF. Adherent cells were split over 60 serial passages and neurospheres split over 30 serial passages. Both cell lines were characterized by immunocytochemistry and fluorescence-activated cell sorting (FACS). Tumorigenicity was examined by the intracranial injection of 103, 104, and 105 cells in NOD/SCID mice, and the subsequent assessment of mice survival and tumor histology. Both neurospheres and adherent cells were concurrently subjected to high-throughput in vitro drug screening.
Results: Neurospheres demonstrated greater staining for stem-like marker CD133+ on FACS compared with adherent cells (25% vs. 2%, respectively). Immunofluorescent staining for markers of mature glial (GFAP), neuronal (synaptophysin, NeuN), and oligodendroglial (O4) differentiation were dim for both neurospheres and the adherent cells, while immature neural markers (TuJ1) were maintained. The expression of stem cell associated markers (BMI-1, nestin, OTX2) stained slightly stronger for neurospheres. At tumor doses studied, cells from both lines formed tumors when implanted in mice, and mice injected with 103 neurospheres had significantly decreased survival compared with mice implanted with 103 adherent cells. On high-throughput drug screening, a differential response was observed for multiple targeted agents.
Conclusion: The novel cell line pair, designated BPt-R0315, has been established from a refractory pediatric GBM autopsy sample and comprises a neurosphere line enriched for CD133+ cells paired with a traditional, serum-based adherent cell line. This in vitro model system provides a unique and valuable resource for further pediatric GBM research and allows parallel screening of therapeutic agents against a line enriched for CD133+ cells versus a traditional adherent cell line. As evidence accumulates that tumor progenitor or stem-like cells contribute to gliomagenesis and therapy resistance, the identification of agents effective against CD133+ enriched pediatric GBM offers a promising new strategy against this lethal disease.
Citation Format: Frank Y. Lin, Mari Kogiso, Hua Mao, Yue Wang, Lin Qi, Patricia Baxter, Andrew Walter, D. Will Parsons, Xiao-Nan Li. Successful establishment of a novel neurosphere and adherent cell line pair from autopsied pediatric glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B13.