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Establishment of pancreatic cancer zebrafish xenografts for personalized medicine in oncology practice
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O cancro do pâncreas é das malignidades mais agressivas e mortais. Esta doença raramente é diagnosticada num estadio em que a resseção cirúrgica é viável. A maioria dos doentes, a quando do diagnóstico, encontram-se num estadio avançado onde as opções terapêuticas são limitadas. Para além disso, as características peculiares do microambiente tumoral do cancro do pâncreas, com um estroma fibrótico extremamente denso, compromete a distribuição eficaz dos fármacos anticancerígenos. A quimioterapia sistémica é a única opção terapêutica para doentes com cancro do pâncreas avançado – FOLFIRINOX ou gemcitabina+nab-paclitaxel. No entanto, ainda não existe na clínica marcadores eficazes com valor preditivo que permitem identificar qual a melhor terapêutica para cada doente. Consequentemente, os doentes são submetidos a múltiplas rondas de tratamento e toxicidades desnecessárias, até encontrar a terapia que seja mais eficaz. A imunoterapia também tem sido explorada como terapia complementar para o tratamento do cancro do pâncreas, incluindo inibidores de checkpoint imunológicos. Contudo, o microambiente tumoral rico em fibroblastos e células imunes com atividade imunossupressora, constitui um obstáculo significativo. Além disso, muitos dos doentes não são elegíveis para este tipo de terapia e portanto estratégias mais personalizadas estão a ser a investigadas em ensaios clínicos. Desta forma, um teste capaz de prever as respostas de cada doente antes do tratamento, seria de grande valor para o tratamento personalizado do cancro do pâncreas. O principal objetivo deste projeto de investigação foi testar as principais opções terapêuticas para o cancro do pâncreas em estadio avançado - FOLFIRINOX e gemcitabina+nab-paclitaxel - utilizando o modelo xenógrafo de peixe-zebra. Com este objetivo, xénografos de peixe-zebra foram gerados utilizando linhas celulares humanas de cancro do pâncreas (Panc-1 e MIA PaCa-2), e várias características tumorais foram analisadas por microscopia confocal, incluindo dinâmica tumoral – proliferação e morte celular – e composição do microambiente tumoral. Os efeitos citotóxicos do nivolumab em monoterapia e em combinação com gemcitabina+nabpaclitaxel (ensaio clínico a decorrer) também foram avaliados. Os resultados demonstraram que as linhas celulares de cancro do pâncreas apresentam diferentes capacidades de implantação no modelo de xénografos de peixe-zebra. Relativamente às terapias anticancerígenas, os nossos resultados demonstraram que os xénografos de peixe-zebra são capazes de revelar respostas tumorais ao FOLFIRINOX e gemcitabina+nab-paclitaxel, incluindo comprometimento da proliferação celular e indução da apoptose. Neste projeto, testámos também a imunoterapia com o anticorpo anti-PD-1- nivolumab. Surpreendentemente os xénografos de peixe-zebra submetidos ao nivolumab em monoterapia e em combinação com gemcitabina+nab-paclitaxel também revelaram sensibilidade celular, com indução significativa da apoptose e redução do tamanho tumoral. De seguida, decidimos caracterizar o microambiente tumoral em particular o infiltrado de neutrófilos e macrófagos. Aos 4 dias pós-injeção, a percentagem de neutrófilos aumentou em relação ao primeiro dia, e os macrófagos do tipo M2 (atividade pró-tumoral) passaram a dominar o microambiente tumoral. Para estudar o papel destes infiltrados na tumorigénese, gerámos xenógrafos em mutantes hipomórficos. A redução de neutrófilos, levou a um aumento do tamanho tumoral, enquanto que a redução de macrófagos, levou a um efeito contrário – diminuição do tamanho tumoral. Estes dados sugerem que os neutrófilos e macrófagos têm um papel antagónico, os neutrófilos com um papel anti-tumoral e os macrófagos pró-tumoral. Sumariamente, os nossos resultados realçam a viabilidade de usar xénografos de peixe-zebra como um modelo in vivo para o screening de respostas tumorais às opções terapêuticas do cancro do pâncreas, e para o estudo da complexidade do microambiente tumoral.
In the modern era of cancer research, pancreatic cancer has proven to be one of the most aggressive and lethal malignancies. Pancreatic cancer is rarely diagnosed at a time when surgical resection is feasible. Therefore, most of the patients present with an advanced disease, at the time of diagnosis, in which treatment options are limited. In addition, the pancreatic cancer microenvironment has peculiar characteristics with a thick layer of stroma, which builds up around the tumor and compromises an efficient drug delivery. Systemic chemotherapy remains the only treatment option for patients with advanced pancreatic cancer – FOLFIRINOX or gemcitabine+nab-paclitaxel. However, effective biomarkers to help predict treatment responses for each patient are still lacking. Consequently, patients go through several trial-and-error approaches and subjected to unnecessary side effects, until the best therapy is found. Immunotherapy has also been explored as a complementary therapy for the treatment of pancreatic cancer, including immune checkpoint inhibitors. But, the tumor microenvironment enriched in fibroblasts and immune cells with immunosuppressive activity poses a major obstacle. Besides, many patients are not eligible for this type of treatment, and therefore more personalized regimens are being investigated in clinical trials. In this way, a test able to predict individual responses before treatment would be of great value for personalized pancreatic cancer treatment. The ultimate goal of this research project was to screen the major therapeutic options for PC treatment, FOLFINIROX and gemcitabine+nab-paclitaxel, using the zebrafish xenograft model. Additionally, the cytotoxic effects of nivolumab as a monotherapy, and in combination with gemcitabine+nab-paclitaxel (clinical trial ongoing) were also evaluated. To address this, zebrafish xenografts were generated with established human pancreatic cancer cell lines (Panc-1 and MIA PaCa-2), and several cancer hallmarks were analyzed through confocal microscopy, including tumoral dynamics – proliferation and cell death – and composition of the tumor microenvironment. Data revealed that pancreatic cancer cell lines have different capacities to engraft in the zebrafish xenograft model. Regarding anticancer therapies, results showed that zebrafish xenografts are able to reveal anti-tumor responses to both FOLFIRINOX and gemcitabine+nab-paclitaxel regimens, leading to impaired cell proliferation and induction of apoptosis. In this project, we also tested anti-PD-1-nivolumab immunotherapy. Surprisingly, zebrafish xenografts subjected to nivolumab and nivolumab in combination with gemcitabine-nab-paclitaxel also revealed cellular sensitivity, with significant induction of apoptosis and tumor size shrinkage. Next, we decided to characterize the tumor microenvironment, in particular neutrophil and macrophage populations. At 4 days post-injection, the percentage of neutrophils increased in comparison with the first day, and M2-like macrophages (protumoral activity) started to dominate the tumor microenvironment. To study the role of both populations in tumorigenesis, zebrafish xenografts were generated using hippomorphic mutants as hosts. Reduction of neutrophils induced an increase in the tumor size, while reduction of macrophages induced an opposite effect – decrease of the tumor size. These results suggest that neutrophils and macrophages are playing opposing roles, neutrophils as anti-tumoral and macrophages as pro-tumoral. Altogether, and most importantly, our results highlight the feasibility of using the zebrafish xenograft model as an in vivo screening platform for pancreatic cancer therapy, and to study the complexity of the tumor microenvironment.
In the modern era of cancer research, pancreatic cancer has proven to be one of the most aggressive and lethal malignancies. Pancreatic cancer is rarely diagnosed at a time when surgical resection is feasible. Therefore, most of the patients present with an advanced disease, at the time of diagnosis, in which treatment options are limited. In addition, the pancreatic cancer microenvironment has peculiar characteristics with a thick layer of stroma, which builds up around the tumor and compromises an efficient drug delivery. Systemic chemotherapy remains the only treatment option for patients with advanced pancreatic cancer – FOLFIRINOX or gemcitabine+nab-paclitaxel. However, effective biomarkers to help predict treatment responses for each patient are still lacking. Consequently, patients go through several trial-and-error approaches and subjected to unnecessary side effects, until the best therapy is found. Immunotherapy has also been explored as a complementary therapy for the treatment of pancreatic cancer, including immune checkpoint inhibitors. But, the tumor microenvironment enriched in fibroblasts and immune cells with immunosuppressive activity poses a major obstacle. Besides, many patients are not eligible for this type of treatment, and therefore more personalized regimens are being investigated in clinical trials. In this way, a test able to predict individual responses before treatment would be of great value for personalized pancreatic cancer treatment. The ultimate goal of this research project was to screen the major therapeutic options for PC treatment, FOLFINIROX and gemcitabine+nab-paclitaxel, using the zebrafish xenograft model. Additionally, the cytotoxic effects of nivolumab as a monotherapy, and in combination with gemcitabine+nab-paclitaxel (clinical trial ongoing) were also evaluated. To address this, zebrafish xenografts were generated with established human pancreatic cancer cell lines (Panc-1 and MIA PaCa-2), and several cancer hallmarks were analyzed through confocal microscopy, including tumoral dynamics – proliferation and cell death – and composition of the tumor microenvironment. Data revealed that pancreatic cancer cell lines have different capacities to engraft in the zebrafish xenograft model. Regarding anticancer therapies, results showed that zebrafish xenografts are able to reveal anti-tumor responses to both FOLFIRINOX and gemcitabine+nab-paclitaxel regimens, leading to impaired cell proliferation and induction of apoptosis. In this project, we also tested anti-PD-1-nivolumab immunotherapy. Surprisingly, zebrafish xenografts subjected to nivolumab and nivolumab in combination with gemcitabine-nab-paclitaxel also revealed cellular sensitivity, with significant induction of apoptosis and tumor size shrinkage. Next, we decided to characterize the tumor microenvironment, in particular neutrophil and macrophage populations. At 4 days post-injection, the percentage of neutrophils increased in comparison with the first day, and M2-like macrophages (protumoral activity) started to dominate the tumor microenvironment. To study the role of both populations in tumorigenesis, zebrafish xenografts were generated using hippomorphic mutants as hosts. Reduction of neutrophils induced an increase in the tumor size, while reduction of macrophages induced an opposite effect – decrease of the tumor size. These results suggest that neutrophils and macrophages are playing opposing roles, neutrophils as anti-tumoral and macrophages as pro-tumoral. Altogether, and most importantly, our results highlight the feasibility of using the zebrafish xenograft model as an in vivo screening platform for pancreatic cancer therapy, and to study the complexity of the tumor microenvironment.
Descrição
Tese de mestrado, Oncobiologia, Universidade de Lisboa, Faculdade de Medicina, 2021
Palavras-chave
Cancro do pâncreas Xenógrafos de peixe-zebra Quimioterapia FOLFIRINOX Gemcitabina+nab-paclitaxel Nivolumab Sistema imunitário inato Teses de mestrado - 2021