Abstract 4106: Methods for the development of carcinoid cell lines and carcinoid associated fibroblasts

Abstract

Abstract Background: Research in carcinoid and neuroendocrine tumors (NETs) is limited by the lack of existing midgut derived cell lines. The two existing NET cell lines, BON and H727, are foregut derived. Foregut NETs are phenotypically distinct from midgut NETs, thus the development of a midgut derived NET cell line is of critical importance. Human NETs contain areas of stroma that likely effect carcinoid cell growth. In other types of cancer, cancer associated fibroblasts (CAFs) have been shown to secrete factors that mediate tumor cell growth. In our preliminary studies, cultured human NET cells showed better attachment to plates when they were in close association with fibroblasts. Unfortunately, the relatively slow growth of NET cells allows fibroblast overgrowth and ultimately detachment of the cancer cells. We have developed a novel approach to processing and culturing NET cells through the isolation of autologous CAFs and their use as irradiated feeder layers. Methods: Human NET surgical specimens are procured under sterile conditions and processed in our lab. The tissue is digested to a single cell suspension. Anti-fibroblast labeled beads (Miltenyi Biotec) are used to separate CAFs from tumor cells. CAFs are then cultured and irradiated to produce feeder layers for the tumor cells. The NET cells are initially maintained on extracellular matrix protein coated plates and then plated on top of the CAF monolayer. Results: CAFs in culture can survive 40 Gy of irradiation and will not proliferate after this treatment. These cells were viable at 12 days, at which point they were split. These irradiated fibroblasts were able to reattach to standard culture flasks and were viable, but did not proliferate after splitting, while the non-irradiated CAF controls had reached confluence by day 12 of this second passage. We currently have 5 patient NET samples that have been processed and plated in vitro on autologous CAFs. NET cells can then be separated from the irradiated fibroblasts using standard trypsinization followed by fibroblast separation using anti-fibroblast beads as described above. The tumor cells can then be used for analysis or further culturing. Early observations shows that the cancer cells appear more elongated and flattened when adjacent to fibroblasts, possibly indicating better attachment and survival. Conclusions: While NET cells remain difficult to culture, we have developed a system that utilizes autologous CAFs in our efforts to potentially establish new NET cell lines for in vitro and in vivo studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4106.