Eferocitose de células de carcinoma espinocelular de cabeça e pescoço por macrófagos: influência da galanina, dos produtos solúveis secretados por células tumorais e do fenótipo dos macrófagos
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Data
2018-03-28
Autores
González Maldonado, Laura Andrea
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Universidade Estadual Paulista (Unesp)
Resumo
Os macrófagos representam uma das principais pontes entre imunidade inata e adaptativa e estão presentes em grande número tanto no estroma circundando tumores sólidos quanto no interior da massa tumoral (TAMs, tumor-associated macrophages), onde podem representar até 50% da massa da lesão. Nos carcinomas espinocelulares de cabeça e pescoço (HNSCC), TAMs apresentam predominantemente o perfil M2 (pró-tumoral), e a quantidade de TAMs é inversamente relacionada ao prognóstico. A efetividade do tratamento não-cirúrgico (quimio/radioterapia ou imunoterapia) dos tumores sólidos é diretamente relacionada à indução de morte das células neoplásicas. No processo de reparo, as células apoptóticas são removidas por fagocitose por outros tipos celulares, num processo denominado eferocitose. Embora seja um processo importante para o reparo e homeostasia tecidual, a eferocitose pode afetar o fenótipo dos macrófagos, favorecendo a polarização para o perfil M2, associado à progressão de HNSCC. A galanina é um peptídeo de 29 aminoácidos amplamente distribuída no organismo e com amplo espectro de efeitos biológicos. A expressão constitutiva de galanina por células de OSCC está associada à maior agressividade do tumor e tem sido estudado como marcador prognóstico de agressividade do tumor e também como um possível alvo terapêutico de HNSCC. O objetivo do presente trabalho é determinar a influência dos produtos secretados pelas células de carcinoma espinocelular de cabeça e pescoço, bem como da galanina isoladamente, no fenótipo de macrófagos e atividade de eferocitose. Para determinar a influência dos produtos secretados no perfil fenotípico dos macrófagos por células tumorais (independentemente do contato direto célula:célula) e da galanina, as células foram tratadas com meio condicionado (CM) contendo os produtos secretados das linhagens tumorais UM-SCC-1 e UM-SCC-22B. Adicionalmente, para avaliar efeito do contato célula:célula e dos produtos secretados sobre a expressão gênica e na eferocitose dos macrófagos ‘pré-polarizados’ em M1 ou M2, foi realizada a co-cultura com as linhagens celulares tumorais apoptóticas UM-SCC-1 e UM-SCC-22B. O perfil fenotípico (M1/M2) foi determinado por RT-qPCR e a modulação na eferocitose dos macrófagos pela citometria de fluxo e microscopia de fluorescência. Os produtos secretados pelas células tumorais, assim como o contato direto célula:célula influenciam a expressão de TNF e IL10 em macrófagos, com aumento significativo da expressão global de TNF e IL10 em co-culturas de macrófagos com as linhagens tumorais UM-SCC-1 e UM-SCC-22B (P<0.05). Por outro lado, o pré-condicionamento dos macrófagos com produtos solúveis das células tumorais antes da co-cultura (contato célula:célula) levou à uma diminuição da expressão de TNF e IL10 (sem diferença significativa com os macrófagos M0). Na eferocitose, os macrófagos polarizados para o perfil M2 apresentaram uma maior atividade da eferocitose de células de carcinoma espinocelular de cabeça e pescoço do que macrófagos polarizados para o perfil M1 (31.38% de macrófagos M2 eferocitóticos versus 15,90% para macrófagos M1em co-cultura com çélulas UM-SCC-1, P<0.05). Adicionalmente, verificou-se que a eferocitose de células tumorais influencia o fenótipo dos macrófagos.
Macrophages are a major link between innate and adaptive immunity and are present both in the surrounding stroma and within various solid tumors (when they are denominated tumor-associated macrophages, TAMs), where these cells may represent up to 50% of the tumor mass. In head and neck squamous cell carcinomas (HNSCC), TAMs are predominantly of the M2 phenotype and the prognosis is inversely correlated with their abundance. The effectiveness of non-surgical treatment of solid tumors (chemotherapy/radiotherapy) is directly related to the induction of neoplasic cell death. In the repair process, apoptotic/dead cells must be removed by phagocytosis by other cell types (particularly macrophages), in a process called efferocytosis. Although this is a critical step in the repair and reestablishment of tissue homeostasis, efferocytosis itself may affect the phenotype of macrophages, skewing the cells towards an M2 phenotype, which is deemed as a 'pro-tumoral' phenotype. Galanin is a 29 aminoacid peptide of 29 produced by various cell types and with a broad spectrum of biological effects. The constitutive expression of galanin by head and neck cancer cells is associated with the increased of tumor aggressiveness and has been proposed as a biomarker of tumor aggressiveness and also as a possible therapeutic target in HNSCC. Thus, the goal of the present study was to determine the role of secreted products from head and neck cancer cells, and of galanin independently, on macrophage phenotype and efferocytosis. In order to determine the influence of secreted products from tumor cells and of galanin in the phenotypic profile of the macrophages (independent of cell-to-cell contact), the cells were treated with conditioned medium (CM) prepared from tumor cell lines UM-SCC- 1 and UM-SCC-22B. Furthermore, to evaluate the effect of cell-to-cell contact and secreted products on the gene expression and efferocytosis of 'pre-polarized' M1 or M2 macrophages, co-cultures with tumor cell lines UM-SCC-1 and UM-SCC-22B with UV-induced apoptosis were performed. The phenotypic profile (M1 / M2) was determined by RT-qPCR and eferocytosis was assessed by flow cytometry and fluorescence microscopy. Products secreted by tumor cells, as well as direct cell:cell contact influence the expression of TNF and IL10 in macrophages. Our results shows an increased expression of TNF and IL10 when macrophages were co-cultured with UM-SCC-1 and UM-SCC-22B tumor cells (P <0.05). However, when macrophages were pre-conditioned with soluble products of these tumor cell lines, there was a decrease in gene expression of TNF and IL10 (without significant difference versus M0 macrophages). M2 macrophages (31.38%) were more effective in the efferocytosis of head and neck squamous cell carcinoma cells than M1 macrophages (31.38% of efferocytotic M2 macrophages versus 15,90% of M1 macrophages for the UM-SCC-1 cell line, P< 0.05). In addition, it was observed that the efferocytosis of tumor cells influences the phenotype of the macrophages.
Macrophages are a major link between innate and adaptive immunity and are present both in the surrounding stroma and within various solid tumors (when they are denominated tumor-associated macrophages, TAMs), where these cells may represent up to 50% of the tumor mass. In head and neck squamous cell carcinomas (HNSCC), TAMs are predominantly of the M2 phenotype and the prognosis is inversely correlated with their abundance. The effectiveness of non-surgical treatment of solid tumors (chemotherapy/radiotherapy) is directly related to the induction of neoplasic cell death. In the repair process, apoptotic/dead cells must be removed by phagocytosis by other cell types (particularly macrophages), in a process called efferocytosis. Although this is a critical step in the repair and reestablishment of tissue homeostasis, efferocytosis itself may affect the phenotype of macrophages, skewing the cells towards an M2 phenotype, which is deemed as a 'pro-tumoral' phenotype. Galanin is a 29 aminoacid peptide of 29 produced by various cell types and with a broad spectrum of biological effects. The constitutive expression of galanin by head and neck cancer cells is associated with the increased of tumor aggressiveness and has been proposed as a biomarker of tumor aggressiveness and also as a possible therapeutic target in HNSCC. Thus, the goal of the present study was to determine the role of secreted products from head and neck cancer cells, and of galanin independently, on macrophage phenotype and efferocytosis. In order to determine the influence of secreted products from tumor cells and of galanin in the phenotypic profile of the macrophages (independent of cell-to-cell contact), the cells were treated with conditioned medium (CM) prepared from tumor cell lines UM-SCC- 1 and UM-SCC-22B. Furthermore, to evaluate the effect of cell-to-cell contact and secreted products on the gene expression and efferocytosis of 'pre-polarized' M1 or M2 macrophages, co-cultures with tumor cell lines UM-SCC-1 and UM-SCC-22B with UV-induced apoptosis were performed. The phenotypic profile (M1 / M2) was determined by RT-qPCR and eferocytosis was assessed by flow cytometry and fluorescence microscopy. Products secreted by tumor cells, as well as direct cell:cell contact influence the expression of TNF and IL10 in macrophages. Our results shows an increased expression of TNF and IL10 when macrophages were co-cultured with UM-SCC-1 and UM-SCC-22B tumor cells (P <0.05). However, when macrophages were pre-conditioned with soluble products of these tumor cell lines, there was a decrease in gene expression of TNF and IL10 (without significant difference versus M0 macrophages). M2 macrophages (31.38%) were more effective in the efferocytosis of head and neck squamous cell carcinoma cells than M1 macrophages (31.38% of efferocytotic M2 macrophages versus 15,90% of M1 macrophages for the UM-SCC-1 cell line, P< 0.05). In addition, it was observed that the efferocytosis of tumor cells influences the phenotype of the macrophages.
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Palavras-chave
Neoplasias bucais, Macrófagos, Fagocitose, Galanina, Mouth neoplasms, Macrophages, Phagocytosis, Galanin