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Deciphering melanoma stroma to improve immunotherapy efficacy
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Resumo(s)
O melanoma é um cancro de pele que afeta a população mundial. Apesar dos vários esforços no combate desta doença, a mortalidade é dificilmente evitável nos estadios mais avançados, denotando-se a necessidade da descoberta de tratamentos life-saving. Os efeitos secundários severos, a resistência adquirida e a baixa taxa de resposta limitam as opções terapêuticas. Uma das razões associadas a esta baixa taxa de resposta prende-se com o microambiente imunossupressor do melanoma, que leva à polarização fenotípica pró-tumoral ou estados anérgicos das células imunológicas, dificultando a resposta às imunoterapias.
Assim, o propósito deste trabalho consiste no estudo duma abordagem capaz de reverter este microambiente imunossupressor. O conhecimento dos reguladores e promotores da geração/ recrutamento de mediadores imunossupressores/ progressão tumoral a montante pode ser a chave para desbloquear o inquestionável papel da imunoterapia. Guiados pela literatura, focámos a investigação na FAK, uma tirosina cinase, regulada e hiperativada em vários tipos de tumores, estando ligada a vias promotoras de cancro. Daí surgiu o interesse em inibidores da FAK (por exemplo, Defactinib), que impedem a fosforilação da FAK e consequentemente a sua ativação.
Assim, primeiramente, foi feita uma avaliação da expressão da FAK e FAK fosforilada na linha celular murina de melanoma, B16F10. Como esperado, a FAK revelou-se sobreexpressa (>99%) e hiperactivada (>74%). A pequena molécula inibidora Defactinib foi utilizada com o intuito de prevenir esta hiperactivação. A viabilidade celular de B16F10, incubada com várias concentrações de Defactinib foi averiguada: diminuindo consideravelmente com concentrações superiores a 1 μM, concentração muito superior à sua concentração inibitória (IC50=0,6nM), mostrando-se concentração-dependente. Isto sugeria que concentrações terapêuticas só modulariam a via, não precipitando a citotoxicidade. Finalmente, para compreender o papel da FAK na modulação do microambiente, realizamos estudos de co-cultura ex vivo de esplenócitos e células tumorais isoladas de ratinhos não imunizados ou imunizados com antigénio de melanoma MUT30, para identificar que regime seria frutífero. Diferentes tratamentos combinados (MUT30/ Defactinib / Combo) também foram usados para avaliar o seu impacto nas populações celulares. As diferenças significativas deveram-se sobretudo ao tratamento combo que em cocultura aumentou a frequência das células T CD4+ e CD8+ que expressavam TNFα.
Melanoma is a worldwide skin cancer. Despite the multiple efforts in fighting this disease, mortality at the late stages is hardly avoided, which demands for the discovery of life-saving treatments. Severe side effects, acquired resistance, and low response rate limit the treatment options. One of the underlying reasons for this outcome is the melanoma immunosuppressive microenvironment, being responsible for the polarization into pro-tumoral phenotype or anergic states of immune system cells, that dampen the response to immunotherapy. Bearing this in mind, this work aims to reverse the melanoma immunosuppressive environment. Knowledge of the upstream regulators and promoters of the generation and recruitment of immunosuppressive/ tumor progression mediators may be the key to unlock the unquestionable role of immunotherapy in cancer treatment. Guided by literature, we focused our investigation on FAK, a tyrosine kinase, which is reported to been upregulated and hyperactivated in several tumor types, being connected to several cancer-promoter pathways. From this arose our interest on FAK inhibitors (e.g.; Defactinib), which prevent FAK phosphorylation and consequently its activation. This said, firstly evaluation of FAK and phosphorylated FAK expression in murine melanoma cell line, B16F10 was performed. As expected, FAK was found overexpressed (>99%) and hiperactivated (>74%). To prevent FAK hyperactivation, the small-molecule inhibitor Defactinib was evaluated. Firstly, cell viability of B16F10, incubated with several concentrations of Defactinib was accomplished: the viability decreased considerably with concentrations higher than 1 µM, much higher concentration than its half maximal inhibitory concentration (IC50 = 0.6 nM). Loss viability was concentration dependent. This suggested that therapeutic concentrations would only modulate the pathway without signs of cytotoxicity. Finally, to understand the role of FAK modulation on melanoma microenvironment, we performed ex vivo co-culture studies of splenocytes and tumor cells isolated from non-immunized mice or mice immunized with melanoma antigen MUT30, to distinguish which regimen would be fruitful. Different combination treatments (MUT30/ Defactinib / Combo) were also used to evaluate their impact on cell populations. Significant differences were mainly observed in combo treatments which in co-culture enhanced CD4+ and CD8+ T cells expressing TNFα.
Melanoma is a worldwide skin cancer. Despite the multiple efforts in fighting this disease, mortality at the late stages is hardly avoided, which demands for the discovery of life-saving treatments. Severe side effects, acquired resistance, and low response rate limit the treatment options. One of the underlying reasons for this outcome is the melanoma immunosuppressive microenvironment, being responsible for the polarization into pro-tumoral phenotype or anergic states of immune system cells, that dampen the response to immunotherapy. Bearing this in mind, this work aims to reverse the melanoma immunosuppressive environment. Knowledge of the upstream regulators and promoters of the generation and recruitment of immunosuppressive/ tumor progression mediators may be the key to unlock the unquestionable role of immunotherapy in cancer treatment. Guided by literature, we focused our investigation on FAK, a tyrosine kinase, which is reported to been upregulated and hyperactivated in several tumor types, being connected to several cancer-promoter pathways. From this arose our interest on FAK inhibitors (e.g.; Defactinib), which prevent FAK phosphorylation and consequently its activation. This said, firstly evaluation of FAK and phosphorylated FAK expression in murine melanoma cell line, B16F10 was performed. As expected, FAK was found overexpressed (>99%) and hiperactivated (>74%). To prevent FAK hyperactivation, the small-molecule inhibitor Defactinib was evaluated. Firstly, cell viability of B16F10, incubated with several concentrations of Defactinib was accomplished: the viability decreased considerably with concentrations higher than 1 µM, much higher concentration than its half maximal inhibitory concentration (IC50 = 0.6 nM). Loss viability was concentration dependent. This suggested that therapeutic concentrations would only modulate the pathway without signs of cytotoxicity. Finally, to understand the role of FAK modulation on melanoma microenvironment, we performed ex vivo co-culture studies of splenocytes and tumor cells isolated from non-immunized mice or mice immunized with melanoma antigen MUT30, to distinguish which regimen would be fruitful. Different combination treatments (MUT30/ Defactinib / Combo) were also used to evaluate their impact on cell populations. Significant differences were mainly observed in combo treatments which in co-culture enhanced CD4+ and CD8+ T cells expressing TNFα.
Descrição
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2022, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
Melanoma Tumor microenvironment FAK Defactinib Immunotherapy Mestrado integrado - 2022