Abstract 5199: Primary culture of human midgut carcinoid cell lines and isolation of a putative carcinoid stem cell population
Authors: Eric Sceusi , Rashid Asif , Zhou Yunfei , Samuel Shaija , Fan Fan , Xia Ling , Ye Xiang-Cang , Jia Lu , Tozzi Federico , Puja Gaur , Jason Fleming , Eddie Abdalla , Curley Steven , Vauthey Jean-Nicolas , Zweidler-McKay Patrick , Raymond Meyn , James Yao , Ellis Lee M. 
Published: June 2012

Abstract
Background: Research in carcinoid and neuroendocrine tumors (NETs) of midgut origin is limited by a lack of cell lines. The existing foregut NET lines, BON and H727, are phenotypically distinct from midgut NETs highlighting the importance of establishing midgut derived NET cell lines. Therapeutic options are limited for midgut NETs. NET cell lines are difficult to culture for long periods (thus few cell lines). We are developing methods to improve primary and prolonged NET cell culture in an attempt to identify novel therapeutic approaches. Along these lines, we are conducting studies to identify a putative cancer stem cell population in human NET specimens to provide new insights regarding potential targets in NETs.
Methods: Sterile human midgut NET surgical specimens were digested to a single cell suspension and fibroblast depleted with magnetic beads. The NET cells were maintained on collagen I coated plates for primary culture. A portion of the primary cell culture was used for FACS by the Aldefluor assay to isolate potential cancer stem cells (CSCs). Sorted cells were grown in serum-free media on low attachment plates to assess sphere formation and preserved for RNA isolation and subsequent gene array. Subcutaneous xenografts in nude and NOD/SCID gamma mice using fresh tumor implants and NET cell suspensions were performed as potential means to further propagate NET cells.
Results: The NET markers, somatostatin receptor 2 and chromogranin A, were detectable in cultures from a midgut carcinoid liver metastasis at passage 3 and 5, as well as in one mouse xenograft. While retention of NET markers from xenograft implants is promising, tumor growth remains limited and slow, with tumors in 5/59 mice over 4-10 months. We are now studying tumor xenografts in more immunodeficient NOD/SCID gamma mice with current patient samples to potentially enhance xenograft growth. FACS sorting identified an ALDH+ population of NET cells, which formed spheres (a characteristic of CSCs) more frequently than ALDH(−) negative cells. A FACS sorted midgut sample plated at 10k cells/well in sphere forming media produced spheres as early as 8 days in the ALDH+ population, while the ALDH- cells did not form spheres (sham sorted cells formed rare spheres), which was also observed in other samples where viable cells were obtained. Additionally, the ALDH+ cells from this population were cutlured on plastic and remained viable for 4 weeks, while the ALDH- population was not viable in culture. RNA isolation for gene array is underway for the ALDH+ population to characterize these cells in midgut carcinoids.
Conclusions: We have developed a system allowing for NET primary culture for up to 4 weeks. We have also identified a putative CSC population in NETs. These techniques are the first step in obtaining cells for in vitro studies and in identifying novel therapeutic targets for NETs.
Supported by the The Raymond and Beverly Sackler Foundation.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2011-5199