The PAF receptor-mediated signaling as limiting factor of antitumor chemotherapy: assessment of immune response and the tumor microenvironment

Abstract

The PAF receptor (PAF-R) is a G protein-coupled receptor present in leukocytes, endothelial cells and some types of tumor. Besides pro-inflammatory effects, PAF-R activation induces suppressor phenotype in leukocytes and systemic immunosuppression. Further, several authors showed that PAF-R promotes tumor growth and suggest that this is due to suppression of immune response and promotion of tumor microenvironment formation. Studies have shown that PAF-R antagonists inhibited tumor growth and that association of antagonists with chemotherapy potentiated this inhibition. Other study showed that treatment of tumors with chemotherapy drugs induced PAF-R ligands and this resulted in increased growth of a second tumor in the mice. Moreover, other studies suggest that activation of PAF-R present in tumor cells also contributes to tumor growth. In one of them, we showed that treatment of tumor cells with chemotherapy drug induced increase in PAF-R expression and that its activation decreased the cell susceptibility to cytotoxic effect of the chemotherapy drug. These results suggest that PAF-R contributes to tumor growth when expressed either in tumor or normal cells, and that its activation during chemotherapy may be a limiting factor of this treatment. However, the mechanisms by which PAF-R expressed in tumor or normal cells acts are not known. In this project, we intend to evaluate the role of PAF-R on tumor growth and chemotherapy, as well as on immunological and microenvironmental factors when the receptor is expressed by tumor or normal cells separately. For that, 4T1 cells (breast cancer cell line) will be transduced with PAF-R or empty plasmid and will be injected in BALB/c WT and PAF-R KO mice. When the tumors reach about 0.5 cm3, the mice will be treated with doxorubicin and PAF-R agonist or different antagonists, and the tumor volume assessed each three days. At the end of the tumor growth experiments, metastatic sites will be analyzed in the lungs. Moreover, tumors will be analyzed for detection of changes in parameters involved with immune response and tumor microenvironment, such as expression of immunomodulatory and pro-inflammatory cytokines (TGF-beta, IL-10, IFN-gamma and IL-6), pro-angiogenic factors (VEGF, EGF, FGF and microvessels), immune cells (cytotoxic T cells, regulatory T cells, myeloid-derived suppressor cells, M2 macrophages and inflammatory cells) and tumor repopulation-related factors (COX-2 and caspase 3). Furthermore, studies in vitro with PAF-R+ and PAF-R- 4T1 cells will be done to evaluate whether PAF-R blockage or activation alters cell response to cytotoxic effects of chemotherapy drugs and whether these drugs induce PAF-R ligands and increase in PAF-R expression. For that, 4T1 cells will be treated with PAF-R agonist or antagonists and doxorubicin, the cell death/viability, levels of PAF-R expression and PAF-R ligands will be assessed. We believe this project will contribute to understand the mechanisms by which PAF-R acts on tumor growth and its participation as a limiting factor of chemotherapy. (AU)