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O efeito da eHSP90 na ativação de macrófagos no microambiente de cancro do ovário
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As proteínas de choque térmico (heat-shock proteins, HSPs) desempenham importantes funções nos processos de crescimento tumoral, metastização e evasão à apoptose. A HSP90, em particular, tem-se destacado como promissor alvo terapêutico devido à sua capacidade de, intracelularmente, estabilizar proteínas oncogénicas e, extracelularmente, contribuir para a invasão tumoral. No nosso laboratório exploramos, atualmente, os efeitos de um novo reagente bloqueador da atividade da forma extracellular (e) da HSP90, o anticorpo anti-eHSP90, após termos demonstrado a sua capacidade de bloquear a migração e invasão celular em linhas tumorais humanas eHSP90+. Considerando o envolvimento de outras HSPs na complexa sinalização que regula os macrófagos associados ao tumor (tumour-associated macrophages, TAMs) em fenótipos pró- (M1) ou anti-inflamatórios (M2), estudámos os efeitos imunomoduladores da eHSP90, que poderão ser benéficos no contexto do tratamento do cancro. Neste trabalho procurámos avaliar a ação da eHSP90 na imunomodulação de TAMs M1 e M2 num microambiente de cancro do ovário (as neoplasias do ovário têm elevados rácios M2/M1, relacionados com a progressão tumoral e com piores taxas de sobrevivência) através de um ensaio de co-cultura de macrófagos humanos com OVCAR-8, uma linha celular humana de cancro do ovário que expressa eHSP90 (eHSP90+). Sumariamente, monócitos do sangue periférico foram diferenciados em macrófagos (M0) com GM-CSF, durante 7 dias em cultura. Nas 48h seguintes, os macrófagos foram polarizados nos fenótipos M1 ou M2, usando LPS + INF-γ ou IL-4 + IL-13, respetivamente. Para testar as potenciais funções imunomoduladoras da eHSP90 no microambiente tumoral, macrófagos M0 foram colocados em co-cultura com anti-eHSP90 e células OVCAR-8, em meio promotor da diferenciação M1 ou M2. A análise fenotípica foi realizada por citometria de fluxo usando marcadores de superfície celular, CD14, CD91 (recetor HSP), CD64 e CD1a, e um kit de citocinas inflamatórias para medir a produção de IL-8, IL-1b, IL-6, IL-10, TNF e IL-12p70. Os nossos resultados revelaram a existência de papel imunomodulatório da eHSP90 no contexto do “microambiente tumoral de cancro do ovário” aqui simulado, em particular na modulação da atividade dos macrófagos M1, verificando-se uma redução significativa na expressão das citocinas pró-inflamatórias TNF e IL-12p70 quando é inibida a atividade da eHSP90. Dado o potencial clinico da eHSP90 enquanto alvo terapêutico anti-tumoral, as funções imonumodulatórias descritas neste trabalho deverão ser tidas em conta ao usar o anticorpo anti-eHSP90 em ensaios clínicos com doentes oncológicos.
Heat-shock proteins (HSP) play important roles in tumour cell growth, metastasis and apoptosis evasion. Particularly HSP90 has gained interest as a promising anticancer drug target, due to its intracellular roles in stabilising “client” key oncogenic proteins and extracellular actions in tumour cell invasiveness. In our laboratory we are currently studying the effects of a new blocking reagent for the activity of the extracellular (e) form of HSP90, an antibody against eHSP90, having demonstrated its capacity to block cell migration and invasion in human eHSP90+ cancer cell lines. Considering the involvement of other HSP family members in complex checkpoints signals that regulate pro- (M1) and anti-inflammatory (M2) tumour-associated macrophages (TAMs), we decided to explore additional immunomodulatory roles of eHSP90 that may be beneficial in the context of cancer treatment. With our work we aim to evaluate the effects of eHSP90 in the immunomodulation of M1 and M2 TAMs in ovarian tumour microenvironment context (ovarian tumours have high M2/ M1 ratio, correlated with cancer progression and poor patient survival). For that, we have simulated the microenvironment of ovarian cancer using a co-culture assay of human macrophages with OVCAR-8, a human eHSP90+ ovarian cell line, in M1 or M2 promoting context. Briefly, peripheral blood monocytes were differentiated into macrophages (M0) with GMCSF for 7 days in culture. In the following 48h, macrophages were further polarized either into M1 or M2 phenotypes using LPS + INF-γ or IL-4 + IL-13, respectively. To test the immunomodulatory functions of eHSP90 in a tumour microenvironment, M0 macrophages were then co-cultured with anti-eHSP90 and OVCAR8 cells in M1 or M2-medium. Phenotypic analysis was performed by flow cytometry using cell surface markers, CD14, CD91 (HSP receptor), CD64 and CD1a, and an inflammatory cytokines kit to measure the production of IL-8, IL-1b, IL-6, IL-10, TNF, and IL-12p70. Our results revealed an immunomodulatory role for eHSP90’s in the context of a simulated ovarian cancer tumour microenvironment, in particular in the modulation of M1 macrophages activity, with a significant reduction in the expression of the proinflammatory cytokines TNF and IL-12p70 when the activity of eHSP90 is inhibited. Given the clinical potential of eHSP90 as an antitumour therapeutic target, the immunomodulatory functions described in this work should be taken into account when using the anti-eHSP90 antibody in clinical trials with cancer patients.
Heat-shock proteins (HSP) play important roles in tumour cell growth, metastasis and apoptosis evasion. Particularly HSP90 has gained interest as a promising anticancer drug target, due to its intracellular roles in stabilising “client” key oncogenic proteins and extracellular actions in tumour cell invasiveness. In our laboratory we are currently studying the effects of a new blocking reagent for the activity of the extracellular (e) form of HSP90, an antibody against eHSP90, having demonstrated its capacity to block cell migration and invasion in human eHSP90+ cancer cell lines. Considering the involvement of other HSP family members in complex checkpoints signals that regulate pro- (M1) and anti-inflammatory (M2) tumour-associated macrophages (TAMs), we decided to explore additional immunomodulatory roles of eHSP90 that may be beneficial in the context of cancer treatment. With our work we aim to evaluate the effects of eHSP90 in the immunomodulation of M1 and M2 TAMs in ovarian tumour microenvironment context (ovarian tumours have high M2/ M1 ratio, correlated with cancer progression and poor patient survival). For that, we have simulated the microenvironment of ovarian cancer using a co-culture assay of human macrophages with OVCAR-8, a human eHSP90+ ovarian cell line, in M1 or M2 promoting context. Briefly, peripheral blood monocytes were differentiated into macrophages (M0) with GMCSF for 7 days in culture. In the following 48h, macrophages were further polarized either into M1 or M2 phenotypes using LPS + INF-γ or IL-4 + IL-13, respectively. To test the immunomodulatory functions of eHSP90 in a tumour microenvironment, M0 macrophages were then co-cultured with anti-eHSP90 and OVCAR8 cells in M1 or M2-medium. Phenotypic analysis was performed by flow cytometry using cell surface markers, CD14, CD91 (HSP receptor), CD64 and CD1a, and an inflammatory cytokines kit to measure the production of IL-8, IL-1b, IL-6, IL-10, TNF, and IL-12p70. Our results revealed an immunomodulatory role for eHSP90’s in the context of a simulated ovarian cancer tumour microenvironment, in particular in the modulation of M1 macrophages activity, with a significant reduction in the expression of the proinflammatory cytokines TNF and IL-12p70 when the activity of eHSP90 is inhibited. Given the clinical potential of eHSP90 as an antitumour therapeutic target, the immunomodulatory functions described in this work should be taken into account when using the anti-eHSP90 antibody in clinical trials with cancer patients.
Descrição
Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2019
Palavras-chave
Proteínas de choque térmico Microambiente tumoral Macrófagos associados ao tumor Vigilância imunológica tumoral Invasão tumoral Histologia