Os efeitos da interação do tecido adiposo de indivíduos com obesidade sobre fibroblastos pulmonares e seu papel no câncer de mama

Abstract

Obesity is related to the genesis and/or worsening of several diseases, such as breast cancer. The development of metastatic disease in this neoplasm is related to a worse prognosis. However, it remains to be better understood whether and how adipose tissue from individuals with obesity (OAT) can modulate stromal cells in secondary sites, such as fibroblasts, and thus contribute to the spread of malignant cells and metastasis. Here, we investigated whether soluble factors and small extracellular vesicles (sEVs) derived from OAT (sEVsOb) could influence functional changes in fibroblasts, as well as whether these possible changes could increase the migration of triple-negative breast tumor cells MDA-MB-231 in vitro. Adipose tissue was obtained from eutrophic and obese volunteers and cultivated in culture medium. The supernatant was collected and ultracentrifuged to obtain media depleted of sEVs from the TA of eutrophic (MdEu) and obese (MdOb) individuals and the sEVsOb. Isolated sEVs were characterized by dynamic light scattering (DLS) and transmission electron microscopy. Wi-38 lung fibroblasts were treated with MdEu, MdOb or sEVsOb and sEVs internalization, viability, proliferation, colony formation and in vitro wound healing assays were performed. Furthermore, soluble pro-inflammatory mediators were investigated in the supernatant of treated fibroblasts, in addition to gene expression of genes associated with fibroblast activation. The treated fibroblasts were also cocultured with MDA-MB-231 cells, using Transwell insert, to verify tumor cell migration. We isolated sEVs with an average hydrodynamic diameter of 122.4 nm and dish morphology typical of sEVs. Furthermore, these nanovesicles were successfully internalized by lung fibroblasts. None of the treatments changed cell proliferation and viability assessed by the trypan blue exclusion method. However, metabolic viability, assessed by MTT, was increased in cells treated with MdEu and decreased in cells treated with MdOb, after 48 hours of treatment. Furthermore, sEVsOb decreased metabolic viability 24 and 48h after treatment. We observed that MdEu, but not MdOb, favored colony formation after 14 days of treatment. Only MdEu increased collective migration in the in vitro healing assay 24 hours after treatment. Furthermore, we observed an increase in the soluble mediators Ang1, IL1-β, IL-6, RANTES, MIF and TNF-α in fibroblasts treated with MdOb 24 and 48 hours after treatment. sEVsOb only induced Ang1 secretion by lung fibroblasts. We did not observe any difference in the expression of the S100A4 and CCL2 genes between the different groups. Finally, we did not observe increased migration of MDA-MB-231 cells co-cultured with treated fibroblasts. Lung fibroblasts undergo functional changes in the context of obesity, but under these conditions, in vitro, they are not involved in increased tumor cell migration. However, further investigations are needed to verify whether endocrine communication between the OAT and fibroblasts may be important for other pathophysiology of the respiratory system, as well as for the chemoattraction and activation of leukocytes which, together, may potentiate pro-metastatic responses in breast cancer.