S2022 A Most Valuable Immunohistochemical Panel for Intestinal and Pancreatobiliary Classification in Carcinoma of the Ampulla of Vater

Abstract

Introduction: Epidemiologic and experimental studies have shown that dietary intake of n3 polyunsaturated fatty acid (PUFA) reduces cancer risk. We have previously reported that in contrast to the n-6 PUFA arachidonic acid (AA) the n-3 PUFA eicosapentaenoic acid (EPA) decreased pancreatic cancer (PaCa) growth. However, the observed effects using standard two dimensional (2D) culture conditions were only modest. It has become increasingly recognized that the phenotype of cultured cancer cells changes if cells are allowed to grow in more physiologically relevant three-dimensional (3D) assays. The aim of the present study was therefore to assess the effects of AA and EPA on PaCa cell growth using 3D growth assays. Methods and results: The human PaCa cell lines MIA PaCa-2 with (MP2+COX2) or without (MP2-COX2) stable COX-2 expression, and BxPC-3, which express wildtype COX2, were used for our studies. Culture dishes were coated with 50% Matrigel (diluted 1:1 with serum-free culture medium). Cells were seeded on top and overlaid with serum-free culture medium. Fatty acids were present in the Matrigel and the culture medium on top. Colony formation and cell growth were measured after 5-7 days by counting and BrdU incorporation. For comparison, standard 2D growth was evaluated using MTT assays. Exposure of MP2+COX2 cells to AA (5 μM) in the 3D assay increased the growth by more than 10-fold (count) and 2.8-fold (BrdU incorporation), while it enhanced growth only by 1.5fold in the 2D assay (MTT). EPA (200 μM) decreased growth in 3D assays by more than 10-fold but only by 1.6-fold in 2D assays. Similar effects were seen in the COX-2 expressing BxPC-3 cells. In MP2-COX2 cells, AA had only a marginal growth-enhancing effect, which was still more pronounced in 3D assays (2.5-fold increase in cell count vs. 1.3-fold increase in MTT). EPA also markedly decreased cell growth in MP2-COX2 cells with a more than 10-fold reduction in 3D assays but only a 3.6-fold decrease in 2D assays. Conclusion: In pancreatic cancer cells the growth enhancing effects of the n-6 PUFA AA as well as the growth inhibitory effects of the n-3 PUFA EPA were significantly more pronounced in 3D culture assays compared to standard 2D assays. AA significantly increased the growth of COX-2 positive pancreatic cancer cells, while EPA decreased the growth of COX-2 positive and -negative pancreatic cancer cells. This suggests that the effects of EPA on pancreatic cancer cell growth are at least in part mediated by COX-2 independent mechanisms. Our data suggest that the physiologically more relevant 3D culture system should be the preferred model to evaluate drug efficiency In Vitro.