Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture

Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype). Next, associations with molecular features were realized through (i) differential analysis within each morphological cluster, and (ii) regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPARγ has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPARγ has been validated through two supporting biological assays

Significant Correlation between Chromosomal Aberration and Nuclear Morphology in Urothelial Carcinoma

We aimed to identify whether there is any correlation between chromosomal/genetic changes, nuclear morphology and the histological grade of urothelial carcinomas of the urinary bladder. Morphometry and multicolour fluorescence in situ hybridisation (FISH) techniques were applied to 250 cells in five low-grade cases and 350 cells in seven high-grade cases of urothelial carcinoma. Compared with low-grade carcinomas, most high-grade cases showed larger and more variable nuclear size, more frequent polysomy of centromere enumeration probes (CEPs) 3, 7 and 17, and the loss of the 9p21 locus. The number of CEP signals in cells was increased as the nuclear area of the cells became larger. Cells with gains in two or more types of CEP had significantly larger nuclei than cells with normal FISH signal patterns. In conclusion, the present study indicates that there was a correlation between nuclear morphology and chromosomal/genetic changes which were related to histological grading. Thus, we show that differences in the chromosomal/genetic aberrations present in low- and high-grade tumours can affect not only nuclear morphology but also the histopathological and clinical behaviour of urothelial carcinomas

Choosing the right cell line for breast cancer research

Breast cancer is a complex and heterogeneous disease. Gene expression profiling has contributed significantly to our understanding of this heterogeneity at a molecular level, refining taxonomy based on simple measures such as histological type, tumour grade, lymph node status and the presence of predictive markers like oestrogen receptor and human epidermal growth factor receptor 2 (HER2) to a more sophisticated classification comprising luminal A, luminal B, basal-like, HER2-positive and normal subgroups. In the laboratory, breast cancer is often modelled using established cell lines. In the present review we discuss some of the issues surrounding the use of breast cancer cell lines as experimental models, in light of these revised clinical classifications, and put forward suggestions for improving their use in translational breast cancer research

Modelos de cancro da mama: do in vitro para o in vivo

A incidência e mortalidade de cancro da mama têm vindo a aumentar ao longo dos anos. Esta doença foi descrita pela primeira vez no ano 3000 a.C. por Edwin Smith Papyrus como uma doença grave sem tratamento conhecido. Atualmente, continua a ser necessário o delineamento de estratégias de prevenção, deteção e tratamento do cancro da mama. Para tal, é extremamente importante a utilização de modelos alternativos ao Homem. A utilização de animais para fins experimentais iniciou-se há muitos séculos (2000 anos a.C.), com os Babilónios e os Assírios a utilizarem animais para a realização de cirurgias. Os modelos in vitro são relativamente recentes em comparação com os modelos in vivo, uma vez que a primeira linha celular de cancro da mama (BT-20) foi descoberta apenas em 1958 por Lasfargues e Ozzello. Os modelos in vivo são os mais utilizados pois mimetizam quase na sua totalidade o comportamento da neoplasia no organismo. Em 1965, Howell realizou o primeiro estudo de carcinogénese mamária quimicamente induzida em ratos. Posteriormente, surgiu a necessidade da criação de animais geneticamente modificados para reduzir ao máximo as diferenças nos mecanismos tumorais. Mesmo assim, não é possível mimetizar a 100% o processo de carcinogénese humano nestes animais devido à sua elevada complexidade e número limitado de genes. Atualmente existe uma diversidade de modelos in vitro e in vivo para o estudo do cancro da mama. A escolha do modelo animal mais adequado é um dos passos mais importantes no delineamento experimental. Os objetivos do trabalho e o tipo de dados que poderão ser obtidos do modelo animal são aspetos fundamentais a ter em consideração. Todos os modelos apresentam vantagens e desvantagens e a sua seleção merece uma reflexão cuidada à luz dos objetivos do trabalho

Sparse multitask regression for identifying common mechanism of response to therapeutic targets

Motivation: Molecular association of phenotypic responses is an important step in hypothesis generation and for initiating design of new experiments. Current practices for associating gene expression data with multidimensional phenotypic data are typically (i) performed one-to-one, i.e. each gene is examined independently with a phenotypic index and (ii) tested with one stress condition at a time, i.e. different perturbations are analyzed separately. As a result, the complex coordination among the genes responsible for a phenotypic profile is potentially lost. More importantly, univariate analysis can potentially hide new insights into common mechanism of response

Ezrin phosphorylation on tyrosine 477 regulates invasion and metastasis of breast cancer cells

Background The membrane cytoskeletal crosslinker, ezrin, a member of the ERM family of proteins, is frequently over-expressed in human breast cancers, and is required for motility and invasion of epithelial cells. Our group previously showed that ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in deregulation of cell-cell contacts and scattering of epithelial cells. In particular, ezrin phosphorylation on Y477 by Src is specific to ezrin within the ERM family, and is required for HGF-induced scattering of epithelial cells. We therefore sought to examine the role of Y477 phosphorylation in ezrin on tumor progression. Methods Using a highly metastatic mouse mammary carcinoma cell line (AC2M2), we tested the effect of over-expressing a non-phosphorylatable form of ezrin (Y477F) on invasive colony growth in 3-dimensional Matrigel cultures, and on local invasion and metastasis in an orthotopic engraftment model. Results AC2M2 cells over-expressing Y477F ezrin exhibited delayed migration in vitro, and cohesive round colonies in 3-dimensional Matrigel cultures, compared to control cells that formed invasive colonies with branching chains of cells and numerous actin-rich protrusions. Moreover, over-expression of Y477F ezrin inhibits local tumor invasion in vivo. Whereas orthotopically injected wild type AC2M2 tumor cells were found to infiltrate into the abdominal wall and visceral organs within three weeks, tumors expressing Y477F ezrin remained circumscribed, with little invasion into the surrounding stroma and abdominal wall. Additionally, Y477F ezrin reduces the number of lung metastatic lesions. Conclusions Our study implicates a role of Y477 ezrin, which is phosphorylated by Src, in regulating local invasion and metastasis of breast carcinoma cells, and provides a clinically relevant model for assessing the Src/ezrin pathway as a potential prognostic/predictive marker or treatment target for invasive human breast cancer.Canadian Breast Cancer Research Alliance (BEE, 017374)Canadian Institutes of Health Research (BEE, 102644)Physicians Society Inc.Association pour le développement de la recherche sur le cancer (France

A cultura de células em 3 dimensões e a sua aplicação em estudos relacionados a formação do lúmen

A cultura celular é caracterizada por permitir a manutenção de células vivas em laboratório independente do organismo que a originou. A utilização desta técnica possibilitou a melhor compreensão dos mecanismos moleculares da célula permitindo importantes avanços científicos no que se refere, por exemplo, a produção de vacinas e a biologia da célula tumoral. A cultura de células em 3-dimensões (3D) derivou-se inicialmente da cultura de células comumente utilizadas (células em monocamada). O diferencial da cultura 3D é permitir que as células explorem as 3-dimensões do espaço, aumentando assim as interações com o ambiente e entre as células. Quando crescidas neste sistema, as células formam estruturas denominadas de esferóides multicelulares. Estes esferóides apresentam em seu interior uma heterogeneidade celular, formação de microambiente e exposição diferencial a diversos fatores como nutrientes e oxigênio. Pelo fato destas características se mostrarem muito semelhantes a tumores avasculares in vivo, a cultura de células 3D avançou em diversas linhas de pesquisa tornando-se um modelo bastante utilizado em ensaios radiológicos e de quimioterápicos. Em estudos relacionados à biologia do câncer de mama, vem ganhando espaço a utilização de esferóides para estudos que visam à compreensão da morfogênese do espaço lumina

The scaffold protein p140Cap limits ERBB2-mediated breast cancer progression interfering with Rac GTPase-controlled circuitries.

The docking protein p140Cap negatively regulates tumour cell features. Its relevance on breast cancer patient survival, as well as its ability to counteract relevant cancer signalling pathways, are not fully understood. Here we report that in patients with ERBB2-amplified breast cancer, a p140Cap-positive status associates with a significantly lower probability of developing a distant event, and a clear difference in survival. p140Cap dampens ERBB2- positive tumour cell progression, impairing tumour onset and growth in the NeuT mouse model, and counteracting epithelial mesenchymal transition, resulting in decreased metastasis formation. One major mechanism is the ability of p140Cap to interfere with ERBB2- dependent activation of Rac GTPase-controlled circuitries. Our findings point to a specific role of p140Cap in curbing the aggressiveness of ERBB2-amplified breast cancers and suggest that, due to its ability to impinge on specific molecular pathways, p140Cap may represent a predictive biomarker of response to targeted anti-ERBB2 therapies

Acidic Pericellular Ph: Effects On Proteolysis And Gene Expression As Determined In 3d Models Of Breast Carcinoma

Among the non-cellular microenvironmental factors that contribute to malignancy of solid tumors is an acidic peritumoral pH. The first objective was to determine if an acidic extracellular pH observed in vivo (i.e., pHe 6.8) affects the activity of proteases, such as cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional cultures. At pHe 6.8 there were increases in pericellular active cysteine cathepsins and in degradation of DQ-collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e. type IV collagen, in an acidic peritumoral environment. The second objective was to use a microarray approach to identify significantly altered genes that may be of importance in the response to acidic pH in ductal carcinoma in situ (DCIS). We have found that STAT1-directed signaling is up-regulated in response to acidic pH extracellularly. Its role in altering glycolytic pathways suggests an important role for this transcription factor in the development of an acidic extracellular pH