Differential effects of 2-and 3-series E-prostaglandins on in vitro expansion of Lgr5+ colonic stem cells

Arachidonic acid (20:4(5,8,11,14), AA)-derived prostaglandin E-2 (PGE(2)) promotes colon cancer development. In contrast, chemoprotective n-3 polyunsaturated fatty acids supplant AA, thereby decreasing PGE(2) biosynthesis in colonocytes, with eicosapentaenoic acid (20:5(5,8,11,14,17), EPA) in particular being metabolized to a novel 3-series E-prostaglandin (PGE(3)), a putative anti-tumorigenic-cyclooxygenase metabolite. Because transformation of adult stem cells is an extremely important route toward initiating intestinal cancer, we utilized the leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescent protein-internal ribosome entry site (IRES)-creER(T2) knock-in mouse model to isolate and culture colonic organoids, in order to document ex vivo responses to exogenous PGE(2) and PGE(3). Colonic crypts were isolated from transgenic mice and cultured in a Matrigel-based three-dimensional platform. Organoids were treated with exogenous PGE(2), PGE(3) or dimethyl sulfoxide (vehicle control) for 5 days and the number of viable organoids was recorded daily. Subsequently, samples were processed for immunohistochemistry, flow cytometry and real-time PCR analyses. PGE(2) promoted optimal organoid growth and induced significantly higher levels of cell proliferation (P < 0.05) compared with PGE(3) and control. In contrast, the Lgr5-green fluorescent protein-positive stem cell number was uniquely elevated by > 2-fold in PGE(2)-treated cultures compared with PGE(3) and control. This coincided with the upregulation of stem-cell-related Sox9, Axin2 and Cd44 messenger RNAs. Our results demonstrate that relative to AA-derived PGE(2), a known promoter of colon tumorigenesis, EPA-derived PGE(3) has diminished ability to support colonic stem cell expansion in mouse colonic organoids.