A significant epidemiological association between obesity and pancreatic ductal adenocarcinoma (PDAC) has previously been described, as well as a correlation between the degree of pancreatic steatosis, PDAC risk and prognosis. The underlying mechanisms are still not completely known.After co-culture of 3T3-L1 adipocytes and MiaPaCa2 with an in vitro transwell system we observed the appearance of fibroblast-like cells, along with a decrease in number and size of remaining adipocytes. RT-PCR analyses of 3T3-L1 adipocytes in co-culture showed a decrease in gene expression of typical markers of mature adipocytes, in parallel with an increased expression of fibroblast-specific and reprogramming genes. We found an increased WNT5a gene and protein expression early in MiaPaCa2 cells in co-culture. Additionally, EMSA of c-Jun and AP1 in 3T3-L1 demonstrated an increased activation in adipocytes after co-culture. Treatment with WNT5a neutralizing antibody completely reverted the activation of c-Jun and AP1 observed in co-cultured adipocytes.Increasing doses of recombinant SFRP-5, a competitive inhibitor for WNT5a receptor, added to the co-culture medium, were able to block the dedifferentiation of adipocytes in co-culture.These data support a WNT5a-mediated dedifferentiation process with adipocytes reprogramming toward fibroblast-like cells that might profoundly influence cancer microenvironment.

These data allow us to hypothesize a dedifferentiation process of adipocytes toward fibroblast-like cells, which can promote migration of melanoma cells through activation of Wnt5a and the intracellular pathways of β-catenin and LEF-1.

Recent experimental data seem to suggest a relevant role for 1,25[OH]2cholecalciferol (1,25[OH]2D3) in adipocyte physiology and pathophysiology, with some studies showing adipogenic and pro-inflammatory properties, and others lipolytic and anti-inflammatory functions. Moreover, to our knowledge, the role of cholecalciferol (D3) in adipocytes function is still not known. Therefore, the aim of this study was to investigate in vitro the effects of 1,25[OH]2D3, as well as of D3, in 3T3-L1 adipocytes in basal and inflammatory conditions, testing the effects of different calcium concentrations in adipocytes culture medium. In 3T3-L1 adipocytes, CYP27A1 and CYP27B1 mRNA were detected in basal conditions and induced after D3 treatment. Pre-treatment of 3T3-L1 adipocytes not only with 1,25[OH]2D3, but also with D3 before inflammatory stimulation, significantly prevented the increase in gene expression and protein secretion of IL-6 and TNF-α, and significantly increased IL-10 mRNA and protein production compared with adipocytes treated only with lipopolysaccharide (LPS). Biological effects of D3 were still present after inhibition of P450 activity with ketokonazole. LPS determined a decrease in cell area compared with controls, paralleled by a significant increase in optical density (OD) of lipid droplets, whereas 1,25[OH]2D3 and D3 alone significantly increased adipocytes area and decreased OD. Pretreatment with both forms of vitamin D preserved cells from the reduction in their area observed after LPS treatment. LPS decreased more the area of cells grown in a high calcium medium than of adipocytes grown in a low calcium medium. In the presence of a high calcium medium, 1,25(OH)2D3 treatment preserved cell area, maintaining its anti-inflammatory and adipogenic properties. In conclusion our results show that D3, besides 1,25[OH]2D3, presents anti-inflammatory effects on 3T3-L1, as well as that adipocytes have the enzymatic pathways necessary to locally regulate the production of active forms of vitamin D, capable of influencing adipocyte phenotype and function.

This study reveals important morphological and functional changes in the AT surrounding the tumor as an increase in lipolysis and in adiponectin-producing adipocytes, preferentially infiltrated by a macrophage subset, with prevalent reparative inflammatory response.

These data, while assessing previously reported results, highlighted new evidence about the time-dependency in LPS-mediated adipokine production, thus contributing to the comprehension of the inflammatory response of adipocyte.