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Role of soluble products secreted by oral squamous cell carcinoma cells and of galanin on macrophage phenotype and efferocytosis

Grant number: 16/12139-1
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): February 01, 2017
Effective date (End): December 31, 2017
Field of knowledge:Health Sciences - Dentistry
Principal Investigator:Carlos Rossa Junior
Grantee:Natalie Aparecida Rodrigues Fernandes
Home Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

Macrophages are the prototypical antigen-presenting cell and are characterized by their varying phenotype ultimately determined by cues from the microenvironment, which may promote a classic/pro-inflammatory (M1) phenotype or an alternative/anti-inflammatory (M2) phenotype. Macrophages represent one of the major links between innate and adaptive immunity and are one of the most abundant cell types in solid tumors, representing up to 50% of the tumor mass, in which they are denominated tumor-associated macrophages (TAMs). In oral squamous cell carcinoma, TAMs are predominantly of the M2 phenotype and the abundance of TAMs is inversely correlated to the prognosis. The efficacy of non-surgical treatment (chemo/radiotherapy and/or immunotherapy) is directly related with their capacity of killing the neoplasic cells. In the repair process, the dead/apoptotic cells are removed by phagocytosis by other cell types (particularly macrophages), in a process called efferocytosis. Although this is an important process for the repair and re-establishment of tissue homeostasis, efferocytosis is influenced by the phenotype of macrophages, which in the tumor microenvironment is largely determined by soluble products secreted by the tumor cells. Among these secreted products, increased production of galanin (a neuropeptide of 29 aminoacid residues) is considered a marker of tumor aggressiveness. Previous studies indicate that oral squamous cell carcinoma and galanin have immunosuppressive effects, possibly favouring polarization towards the M2 phenotype, which is considered of 'pro-tumoral' activity. The purpose of this study is to assess the influence of cancer cell-secreted products and also of galanin on the phenotype and efferocytosis by macrophages. We will use a murine macrophage cell line (RAW264.7), which will be primed with soluble products from two oral squamous cell carcinoma cell lines (with high and low constitutive production of galanin) or treated directly with galanin. The phenotype of the macrophages will be assessed by RT-qPCR. After induction of apoptosis on these neoplasic cell lines by exposure to UV light and by treatment with Cobalt chloride, the macrophages primed in the different experimental conditions will be added in co-culture experiments to assess efferocytosis by flow cytometry and fluorescence microscopy. (AU)