SAT-590 Role of Adipocyte Hyperplasia and Hypertrophy in the Release and Charge of Exosomes in Human Fat Cells

Abstract In obesity, the increase in the number and / or size of adipocytes leads to chronic low-grade systemic inflammation that conditions the development and evolution of a series of pathologies such as coronary heart disease or cancer. The change from hyperplasia to hypertrophy has been characterized by the expression of early and late adipogenic biomarkers, but little is known about the communications between cells. Exosomes are small extracellular vesicles (30-100 nm in diameter) released by exocytosis in almost all cell types. Due to its wide distribution and absorption by different cell types, exosomes have been considered an attractive tool for diagnosis, therapy and response evaluation. Objective: To characterize the exosomes released by human fat cells during differentiation and the effect of hyperplasia on the function of these. Material and methods: Human cells (SW-872) were cultured for 24h in DMEM-F12- with FBS serum (free of steroids and exosomes), differentiated with adipogenic cocktail for 7 days, and hypertrophy for further 24h G1 agonist treatment. Exosomes were isolated from conditioned medium of SW872 cells by ultracentrifugation and characterized by immunoblotting against exosomal markers, (Nanoparticle Tracking Analysis (NTA) and transmission electron microscopy (TEM). Cancer initiating cells (CICs) were isolated from HeyA8 ovarian cancer cells using culture selecting conditions. CICs were 24h treated with exosomes (1x1011 particles/mL) and then seeded over matrigel to carry out 3Dmigration assays. Results: SW872 cells showed the morphological characteristics described for this cell line and MR expression was observed. Successful isolation of SW872-derived exosomes was confirmed by assessing the particle size distribution by NTA, the morphology by TEM and the presence of exosome markers (Alix, HSP70, TSG101, and CD36) by immunoblotting. Preadipocyte differentiation showed a significant decrease in the exosome concentration (pre,1.3x1011 particles/ml vs adipo, 1.5x1010 particles/ml p <0.0001) and in the size of these nanovesicles (102.2 ±3.1nm vs 69.8 ±20.1nm p = 0.05). When the differentiated cells become hydrophobic the concentration of exosomes released showed a significant increase in the concentration (1.5x1010 part/ml vs 5.5x1010 part/ml p <0.0001) and no changes in size were observed (139.5±15.2 nm vs 119.9±14.3). A functional 3D analysis shows that exosomes from hypertrophic adipocytes induce an increase on the migration of HEYA8 o-derived CICs.Conclusions: These preliminary results show that during the differentiation of the adipocyte the position of exosomes probably change as a reflection of cell specialization. Hypertrophic cells-derived exosome can modulate the migratory capacity of CICs a reflex of change of exosome content during differentiation. Further analysis will analyze the exosome cargo (i.e. miRNA) during this process.