Molecular microevolution in cat mammary tumor lines: genomic and cytogenomic cell models
Ficheiros
Data
2017
Autores
Título da revista
ISSN da revista
Título do Volume
Editora
Resumo
Os tumores de mama espontâneos em gata têm-se revelado ao longo do tempo como
excelentes modelos para o estudo e compreensão de como se inicia e desenvolve o
processo tumoral, bem como para melhorar as ferramentas de diagnóstico ou mesmo
para o desenvolvimento e aumento da eficiência de terapias bem-sucedidas para o
Homem. As semelhanças entre os tumores mamários da gata e da mulher vão desde os
genes e pathways envolvidos na tumorigénese, até às características histológicas, entre
outras.
No entanto, a investigação oncológica apenas com recurso a animais é praticamente
inviável e levanta uma série de questões importantes, nomeadamente éticas. Nesse
cenário, surgem os modelos celulares: linhas celulares derivadas de tecidos ou células
tumorais que são um recurso extremamente valioso como modelos in vitro para a
investigação oncológica. A grande vantagem dos modelos celulares depende da sua
disponibilidade e facilidade na manipulação, mas depende grandemente da sua
adequada caracterização. O presente trabalho teve dois objetivos principais, o
estabelecimento de várias linhas celulares de tumores mamários de gata, sendo que
uma delas, a linha celular FkMTp cresceu sem interrupção durante aproximadamente
dois anos e a sua caracterização detalhada aos níveis da análise fenotípica,
citogenómica e genética. Em conjunto, este trabalho teve como grande finalidade a de
criar uma ferramenta única e distinta, bem como um recurso para a investigação
fundamental e aplicada do cancro. Para atingir estes objetivos, foi realizada uma
abordagem diferente na metodologia durante o estabelecimento da linha celular, o que
permitiu a criação de um "banco" preservado de diferentes “momentos” do tumor in vitro
ao longo do tempo. Este "banco" estende-se desde a cultura primária até a passagem
154, e inclui: linhas celulares criogenicamente preservadas, preparações
cromossómicas fixadas, DNA genómico, RNA e células (para isolamento de proteínas),
preservados a cada seis passagens da linha celular (durante todo o processo de
estabelecimento). De seguida e de forma a validar este modelo celular, foram
caracterizadas, a diferentes níveis, até 18 passagens da linha celular FkMTp, desde a
análise morfológica das células, até à análise das alterações dinâmicas na estrutura do
citoesqueleto, das suas capacidades de crescimento sem ancoragem, de cicatrização,
invasão e migração, até o nível citogenético, onde as alterações não clonais, transitórias
e cromossómicas clonais (incluindo breakpoints) foram identificadas, permitindo revelar
o padrão de evolução cariotípico, bem como os perfis de expressão de genes
importantes implicados no início e na progressão tumoral.
Os resultados obtidos durante este trabalho são demonstrativos de que a linha celular
FkMTP é um excelente modelo para o estudo dos tumores mamários da mulher e
especialmente, o subtipo triplo negativo e um modelo ideal para compreender as várias
vias envolvidas na tumorigénese, desde os primeiros eventos até a progressão do
tumor.
Spontaneous cat mammary tumors have been emerging over the time as excellent models to study and understand how tumorigenesis begins and evolve, as well as to improve the diagnostic tools or even for the development and enhancement of successful therapies for the human counterpart. The similarities between cat and human mammary tumors goes from the genes and pathways involved in cancer initiation and progression until the histological features, among others. However, animal based-research alone is almost unfeasible and raise a number of important and ethical issues. In this scenario, cellular models arise: the cell lines derived from tumor tissues or cells are an extremely valuable resource as in vitro models to cancer research. The great advantage of cellular models relies in their readily availability and easiness of manipulation, but depends on their suitable characterization. The present work had two major goals, the establishment of several cat mammary cancer cell lines, one of them, the FkMTp cell line that grew without interruption for almost two years, and its detailed characterization at the phenotypic, cytogenomic and gene analysis levels. Together, this work aimed to create a unique and distinct tool, as well as a resource for the cancer fundamental and applied research. To accomplish those goals, a distinctive approach in the methodology was used during the cell line establishment, which allowed the generation of a preserved “bank” of different “moments” of tumor progression along the time. These “bank” extends from the primary culture until passage 160, and it includes cryogenically preserved cell lines, fixed chromosome preparations, genomic DNA, RNA and cells (for protein isolation), preserved every six passages of the cancer cell line (during all the establishment process). Following and to validate this cell model for cancer research, up to 18 passages of the FkMTp cell line were characterized at different levels, from the cells’ morphology, to the analysis of the dynamic changes on the cytoskeleton structure, their abilities to grow without anchorage, to wound-healing, to perform invasion and migration, until the cytogenetic level, where the non-clonal, the transitional and the clonal chromosome alterations (including the breakpoints disclosure) were identified, allowing to uncover the pattern of karyotypic evolution and expression profiles of important genes implicated in tumor initiation and progression. The results obtained during this work prove that the cell line FkMTP is an excellent cellular model to study human breast cancer, and especially, the triple negative subtype and an optimal model to understand the several pathways involved in tumorigenesis, from the early events towards tumor progression.
Spontaneous cat mammary tumors have been emerging over the time as excellent models to study and understand how tumorigenesis begins and evolve, as well as to improve the diagnostic tools or even for the development and enhancement of successful therapies for the human counterpart. The similarities between cat and human mammary tumors goes from the genes and pathways involved in cancer initiation and progression until the histological features, among others. However, animal based-research alone is almost unfeasible and raise a number of important and ethical issues. In this scenario, cellular models arise: the cell lines derived from tumor tissues or cells are an extremely valuable resource as in vitro models to cancer research. The great advantage of cellular models relies in their readily availability and easiness of manipulation, but depends on their suitable characterization. The present work had two major goals, the establishment of several cat mammary cancer cell lines, one of them, the FkMTp cell line that grew without interruption for almost two years, and its detailed characterization at the phenotypic, cytogenomic and gene analysis levels. Together, this work aimed to create a unique and distinct tool, as well as a resource for the cancer fundamental and applied research. To accomplish those goals, a distinctive approach in the methodology was used during the cell line establishment, which allowed the generation of a preserved “bank” of different “moments” of tumor progression along the time. These “bank” extends from the primary culture until passage 160, and it includes cryogenically preserved cell lines, fixed chromosome preparations, genomic DNA, RNA and cells (for protein isolation), preserved every six passages of the cancer cell line (during all the establishment process). Following and to validate this cell model for cancer research, up to 18 passages of the FkMTp cell line were characterized at different levels, from the cells’ morphology, to the analysis of the dynamic changes on the cytoskeleton structure, their abilities to grow without anchorage, to wound-healing, to perform invasion and migration, until the cytogenetic level, where the non-clonal, the transitional and the clonal chromosome alterations (including the breakpoints disclosure) were identified, allowing to uncover the pattern of karyotypic evolution and expression profiles of important genes implicated in tumor initiation and progression. The results obtained during this work prove that the cell line FkMTP is an excellent cellular model to study human breast cancer, and especially, the triple negative subtype and an optimal model to understand the several pathways involved in tumorigenesis, from the early events towards tumor progression.
Descrição
Tese de Doutoramento em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Gato , Modelo animal , Linhas celulares , Carcinogénese , Microevolução , Rearranjos cromossómicos , Genes críticos do cancer