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Xenógrafos de peixe-zebra como modelo para avaliação de quimio e radiossensibilidade de tumores com mutação BRCA2
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Introdução: O cancro da mama é o cancro com maior incidência e maior mortalidade nas mulheres a nível mundial. Um dos subtipos de cancro da mama está associado a mutação BRCA1/2. Este representa 5% de todos os cancros da mama, e 20% dos cancros da mama familiares. A mutação destes genes está também associada a cancro do ovário, próstata e pâncreas. O advento de uma família de fármacos designada de inibidores da PARP originou uma mudança na abordagem terapêutica a estes cancros. No entanto, tem sido relatado que nem todos os doentes com cancros associados a mutações BRCA1/2 respondem a esta terapêutica. Por outro lado, existem doentes sem mutação que apresentam melhoria quando submetidos à mesma. Torna-se assim evidente a necessidade de um teste funcional que permita caracterizar, em tempo útil, a resposta de diferentes tumores ao tratamento com inibidores da PARP, independentemente do seu perfil genético ou molecular.
Objectivo: Testar se o modelo de xenógrafos de peixe-zebra tem capacidade de diferenciar respostas funcionais de tumores com e sem mutação BRCA2 quando tratados com Olaparib, um inibidor da PARP, num curto período de tempo.
Materiais e métodos: Larvas de peixe-zebra foram injectadas com células das linhas isogénicas VC8-B2 (WT) e VC8 (mutBRCA2). 24h pós-injecção, os xenógrafos foram distribuídos de forma aleatória pelas diferentes condições de tratamento – Controlo, Olaparib, radiação ionizante (25Gy) e radiação ionizante (25Gy) seguida de Olaparib. Os xenógrafos foram fixados em formaldeído 4dpi e 5dpi, e analisados por imunofluorescência de confocal.
Resultados: Na ausência de tratamento (controlos) conseguimos detectar claras diferenças de comportamento das linhas isógenicas que apenas diferem na presença (VC8-B2) ou ausência (VC8) do gene BRCA2. Aos 4dpi, os tumores de VC8 (mutBRCA2) apresentaram uma menor capacidade de implantação, uma maior taxa de morte celular e um menor tamanho que os tumores de VC8-B2 (WT). No entanto, apresentaram uma maior expressão de Ki67. Para além disso, foi possível detectar que, na ausência da proteína BRCA2, os tumores apresentaram uma menor capacidade neo-angiogénica. Quando tratados com Olaparib, verificou-se um aumento significativo da morte celular dos xenógrafos de VC8 (mutBRCA2) (P<0,0001), mas o mesmo não se verifica nos xenógrafos de VC8-B2 (WT). O aumento de Caspase3 nos xenógrafos de VC8 (mutBRCA2) é também visível nos restantes regimes terapêuticos testados, existindo um efeito sinérgico aquando do tratamento com o regime combinatório de radiação ionizante e Olaparib.
Discussão: O peixe-zebra apresenta-se como um modelo capaz de distinguir diferentes comportamentos basais de linhas celulares com diferentes status BRCA2 no que toca à sua capacidade de implantação, proliferação, apoptose e potencial angiogénico. De forma mais importante, este modelo é capaz de diferenciar o impacto do tratamento com Olaparib sobre a indução de apoptose celular em tumores com e sem mutação BRCA2, bem como a resposta destes a tratamento com radiação ionizante e com um regime terapêutico combinatório.
Introduction: Breast cancer is the cancer with the highest incidence and mortality in women worldwide. A particular subtype of breast cancer is the one associated with BRCA1/2 mutations – it represents 5% of all breast cancers and 20% of familial breast cancers. These mutations are also associated to some types of ovarian, prostate and pancreatic cancer. With the development of PARP inhibitors, the therapeutic approach of these cancers suffered a change. However, it has been reported that not all patients with a BRCA1/2 mutation respond to this treatment, and that some patients with a preserved BRCA function do. Therefore, the need for a functional test that allows a rapid characterization of the response of different tumors to the treatment with a PARP inhibitor, regardless of their genetic or molecular profile, becomes evident. Objective: To test if the zebrafish xenografts model has the ability to differentiate, over a short period of time, functional responses to PARP inhibitors in tumors with and without BRCA2 mutations. Materials and methods: Zebrafish larvae were injected with the isogenic cell lines VC8-B2 (WT) and VC8 (mutBRCA2). 24h post-injection, the xenografts were randomly distributed through different treatment conditions – Control, Olaparib, ionizing radiation (25Gy) and ionizing radiation (25Gy) followed by treatment with Olaparib. Xenografts were fixed in formaldehyde at 4dpi and 5dpi, and analysed by immunofluorescence confocal microscopy. Results: When no treatment was applied (Controls) it was possible to observe clear differences in the behaviour of the isogenic cell lines, which only differ between themselves in regards to the presence (VC8-B2) or absence (VC8) of a functional BRCA2 gene. At 4dpi, VC8 (mutBRCA2) xenografts presented a lower rate of engraftment, a higher rate of cell death and a smaller size of tumors than VC8-B2 (mutBRCA2) xenografts. However, they displayed a higher expression of Ki67. Besides, it was possible to observe that, in the absence of a functional BRCA2 protein, the tumors presented a lower angiogenic potential. When treated with Olaparib, VC8 (mutBRCA2) xenografts presented a significant increase in the expression of Caspase3 (P<0,0001). The same effect does not occur in VC8-B2 (mutBRCA2) xenografts. Capase3 expression in VC8 (mutBRCA2) xenografts is also significantly increased in every other treatment condition, and a synergistic effect is visible when xenografts were treated with the combinatorial regimen. Discussion: Zebrafish presents as a model with the ability to distinguish different basal behaviours of cell lines with different BRCA2 status in regards to their engraftment, proliferation, apoptosis and angiogenic potential. Besides, this model can differentiate the impact of Olaparib on Caspase3 fold induction in tumors with and without a BRCA2 mutation, as well as their response to treatment with ionizing radiation and a combinatorial regimen.
Introduction: Breast cancer is the cancer with the highest incidence and mortality in women worldwide. A particular subtype of breast cancer is the one associated with BRCA1/2 mutations – it represents 5% of all breast cancers and 20% of familial breast cancers. These mutations are also associated to some types of ovarian, prostate and pancreatic cancer. With the development of PARP inhibitors, the therapeutic approach of these cancers suffered a change. However, it has been reported that not all patients with a BRCA1/2 mutation respond to this treatment, and that some patients with a preserved BRCA function do. Therefore, the need for a functional test that allows a rapid characterization of the response of different tumors to the treatment with a PARP inhibitor, regardless of their genetic or molecular profile, becomes evident. Objective: To test if the zebrafish xenografts model has the ability to differentiate, over a short period of time, functional responses to PARP inhibitors in tumors with and without BRCA2 mutations. Materials and methods: Zebrafish larvae were injected with the isogenic cell lines VC8-B2 (WT) and VC8 (mutBRCA2). 24h post-injection, the xenografts were randomly distributed through different treatment conditions – Control, Olaparib, ionizing radiation (25Gy) and ionizing radiation (25Gy) followed by treatment with Olaparib. Xenografts were fixed in formaldehyde at 4dpi and 5dpi, and analysed by immunofluorescence confocal microscopy. Results: When no treatment was applied (Controls) it was possible to observe clear differences in the behaviour of the isogenic cell lines, which only differ between themselves in regards to the presence (VC8-B2) or absence (VC8) of a functional BRCA2 gene. At 4dpi, VC8 (mutBRCA2) xenografts presented a lower rate of engraftment, a higher rate of cell death and a smaller size of tumors than VC8-B2 (mutBRCA2) xenografts. However, they displayed a higher expression of Ki67. Besides, it was possible to observe that, in the absence of a functional BRCA2 protein, the tumors presented a lower angiogenic potential. When treated with Olaparib, VC8 (mutBRCA2) xenografts presented a significant increase in the expression of Caspase3 (P<0,0001). The same effect does not occur in VC8-B2 (mutBRCA2) xenografts. Capase3 expression in VC8 (mutBRCA2) xenografts is also significantly increased in every other treatment condition, and a synergistic effect is visible when xenografts were treated with the combinatorial regimen. Discussion: Zebrafish presents as a model with the ability to distinguish different basal behaviours of cell lines with different BRCA2 status in regards to their engraftment, proliferation, apoptosis and angiogenic potential. Besides, this model can differentiate the impact of Olaparib on Caspase3 fold induction in tumors with and without a BRCA2 mutation, as well as their response to treatment with ionizing radiation and a combinatorial regimen.
Descrição
Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2018
Palavras-chave
Cancro da mama Mutação BRCA1/2 Xenógrafos de peixe-zebra Oncobiologia