Vesículas extracelulares circulantes de pacientes com câncer de mama induzem células dendríticas tolerogênicas modulando GSK3 via miRNA

Abstract

Introduction: Cellular communication through extracellular vesicles (EVs) emerged recently as important modulators of the immune response development and estabilishment of cancer. This study evaluated the hability of EVs from patients with invasive ductal breast cancer to induce phenotypical and functional changes in in vitro differentiation of monocyte-derived dendritic cells (Mo-DC) and T cells activation. The possible mechanisms involved in these changes were also investigated. Methodology: For this, the EVs were isolated, by ultracentrifugation, from plasma of healthy donors (HD) and patients with breast cancer (BC). In vitro differentiation of Mo-DCs was performed with monocytes obtained from healthy donors in the presence of HD or BC EVs (30 μg/mL), at day 0. Mo-DCs were activated with LPS (50 ηg / mL) after five days of culture to induce maturation. After 48 hours of maturation, the Mo-DCs were analyzed phenotypically by flow cytometry. The functional evaluation of generated Mo-DCs and the adaptive immune response profile were performed by allogeneic T lymphocyte proliferation assay (MLR). The intracellular signaling pathways were evaluated after 5 and 30 minutes of LPS-activation that Mo-DCs. To investigate possible mechanisms by which BC EVs were modulating Mo-DCs, we performed the analysis of possible enriched miRNAs in this EVs. In addition, in vivo tests were performed to check the systemic modulation of EVs in the face of an infectious and tumor-associated response. The 4T1 cells were used to induce the tumor in BALB/c mice, since this cancer cell strain can establish in BALB/c and immunosuppressed animals. The infection model was chosen based on a well- characterized Leishmania major infection. Results: DCs differentiated in the presence of EVs from cancer patients showed altered phenotype with decreased expression of HLA-DR (p <0.001), CD80 (p<0,01), CD86 (p <0.001) and CD11c (p <0.001) when compared to DCs differentiated with HD EVs. In addition, we observed that DCs differentiated with EV from cancer patients displayed impaired ability to induce CD4+ and CD8+ T cell proliferation (p<0,05), increased expression of inhibitory receptors and decreased levels of IL-12, IL-6, IL- 4, IL-2 and IFN-gama in the supernatant of the MLR. On the other hand, increased levels of regulatory T lymphocytes (p<0,001) and IL-10 production (p<0,05) were observed. Differentiated Mo-DCs in the presence of BC EVs showed less activation of the mTOR pathway, even after induced stimulation. Besides that, GSK-3β inactivation was not observed, an important pathway for the generation of Mo-DCs with an inflammatory profile. The presence of miR-181b-5p has been identified, only in BC EVs, which regulates GSK3β interaction protein (GSKIP), which has the function of inactivating GSK-3β. The effects of EV was appreciated in an in vivo Leishmania major infection model. In BALB/c animals with tumor induction and treatmented with EVs was possible to identify the modulation of the immune response to the infection, by observing a lower inflammatory infiltrate of cells responsible for mediating local immune response against the parasite, in addition to a strong Th2 response, greater injury, combined with greater tumor growth, when compared to the control group of L. major infection and tumor control group. Conclusion: Specific EVs of breast cancer patients modulate, through miR-181b, the inactivation of GSK-3β in DCs. This modulation promotes phenotypic and functional changes in Mo-DCs, inducing a suppressive state of the adaptive immune response.