Este trabalho foi financiado pelo NIH concede R01 CA107469, CA125577 R01 e U01CA154224 para CG Kleer, a Universidade do Centro de Câncer de Apoio de Michigan.

1. Preparação de Materiais e Meios de Cultura <ol><li> Fator de crescimento Thaw reduzida (TFG) Matrigel a 4 ° CO / N. </li><li> Aqueça mídia completas para linha celular necessário a 37 ° C. MCF10A-ATCC especificado media; Meio F-12 de Ham-HME suplementado com 5% de soro fetal bovino, ml, 5 ug / ml de insulina, factor de 1 ug / hidrocortisona de 10 ng / mL de crescimento epidérmico, e 100 ng de toxina da cólera ml /; F-12 media de SUM149-Ham suplementado com FBS a 5%, 2 ug / ml de hidrocortisona e de 5 ug / ml de insulina; e MDA-MB-231-10% de FBS-DMEM </li><li> Precool um slide de câmara de 8 poços no gelo. </li><li> Prepare cultura de tecidos capô e suprimentos. </li></ol> 2. Três Cultura Dimensional em 8 poços Deslize Câmara <ol><li> Casaco de cada poço a 8 poços corrediça com 40 ul de GFR Matrigel. Adicionar o Matrigel no centro da lâmina e, em seguida, espalhada com uma pipeta de ponta P200. Tente espalhar o mais uniformemente possível, evitando bolhas e espalhando que pode levar aformação do menisco. </li><li> Incubar as lâminas a 37 ° C sob 5% de CO 2. Enquanto o Matrigel está solidificando, trypsinize as células, recolher e girar a 150 xg em cultura de tecidos centrifugar por 4 min. </li><li> Contar as células e dilui-se a 12.500 células / mL em meios completos da linha celular respectivos. Adicionar TFG Matrigel a 2% e adicionar 400 ul a cada poço de uma lâmina de câmara de pré-revestidas de Matrigel. </li><li> Incubar as lâminas na incubadora de CO 2. Dar novas mudanças de mídia completas a cada 4 º dia até 15 dias. </li><li> O tratamento com inibidores farmacológicos: Para estudos de inibidor de adicionar os inibidores de moléculas pequenas para o meio completo no momento do plaqueamento seguido por mudanças de meio fresco suplementado com os inibidores de três em três dias, enquanto o crescimento das células em TFG Matrigel. </li><li> O tratamento com a proteína purificada: <ol><li> Placa as células seguintes passos 2,1-2,3. Permitir que as culturas a crescer durante 4 dias, seguido por starvati soropor 16 h. </li><li> No dia 5, tratar as células com uma concentração apropriada de proteína purificada em 0,1% de FBS suplementado com os meios de células em jejum de soro. Dê mudança meio fresco com proteína purificada após 2 dias. </li><li> No dia 10 de substituir a meios condicionados com a mídia HME completos. Permitir que as culturas a crescer durante mais 5 dias. </li></ol></li><li> Coloração por imunofluorescência de estruturas acinares cultivadas em Matrigel: <ol><li> Prepare a 2% de paraformaldeído, 0,5% de Triton X-100 e 100 mM de glicina em 1x PBS, pH 7,4. Preparar tampão de imunofluorescência (IF tampão), que compreende de 1 x PBS contendo 0,1% de albumina de soro bovino, 0,2% de triton X-100, 0,05% de Tween-20. </li><li> Use uma pipeta de ponta P200 para aspirar cuidadosamente a meios condicionados. </li><li> Fixar as estruturas acinares com paraformaldeído a 2% preparada de fresco, à temperatura ambiente (RT) durante 20 min. </li><li> Permeabilizar as células com 0,5% de Triton X-100 durante 10 min a 4 ° C. </li><li> Lava-se a culturas três vezes com 100 mM glicina, 10-15 minutos para cada lavagem. </li><li> Bloquear as células com 10% de soro de cabra em tampão IF, chamado bloco primário, durante 1 hora a RT. </li><li> Incubar as culturas com 20 ng / ml de cabra anti-rato F (ab &#39;) 2 em tampão fragmento bloco primário durante 30 min. Esta etapa é importante para bloquear a espécie de IgG de ratinho imunorreactivas em Matrigel. </li><li> Incubar com anticorpo primário em que a segunda solução de bloqueio (bloco primário 20 ug / ml de cabra anti-rato F (ab &#39;) 2 fragment) O / N a 4 ° C. </li><li> Lavar três vezes com tampão IF por 10-15 min cada um com balanço suave. </li><li> Incube com anticorpos secundários conjugados fluorescentes em tampão IF para 45 min. </li><li> Lavar 3x por 10 minutos cada com tampão IF com balanço suave. </li><li> Montar as lâminas com o reagente anti-fade contendo DAPI para visualizar os núcleos. </li><li> Permitir que as lâminas secar O / N à temperatura ambiente. </li><li> A aquisição de imagens: Adquirir imagens confocal nas milhas da colôniadsection de células cultivadas em cima de Matrigel. Para mais informações adquirir uma série de secções ópticas ao longo de todo o comprimento da estrutura acinar por Z empilhamento. </li></ol></li></ol> 3. Três Cultura Dimensional em 2-bem Câmara Slides para Immunoblotting <ol><li> Precool um slide câmara de 2-bem no gelo. Siga passos 2,1-2,4 intensificação os reagentes para a câmara de 2-bem corrediça por exemplo revestimento com 160 mL de Matrigel e adicionar 1.6 ml de células / Matrigel misturar a cada poço da 2-bem corrediça. </li><li> Colher as estruturas acinares de Matrigel, utilizando o estojo de colheita de células de cultura 3D disponível comercialmente seguindo as instruções do fabricante. </li><li> Diluir a lavagem das células e colheita de células buffers para 1x e pré-resfriamento a 4 ° C. Lavar e deslocamento de Matrigel deve ser levada a cabo em gelo. </li><li> Lavar as estruturas acinares 3x utilizando o tampão de lavagem celular. </li><li> Recolher o ácinos em 15 ml tubos de centrífuga usando bu colheita de célulasffer seguido por 30 min de incubação num agitador rotativo a 4 ° C. Misture bem a suspensão com uma pipeta de 1 ml. Isto vai quebrar o Matrigel em pequenos pedaços e libertar a maior parte das células do tampão de colheita de células. </li><li> Agregar as células a 200 xg numa centrifugadora de cultura. Retire a camada flutuante de hidrogel a partir de células peletizadas com uma pipeta de ponta P200. </li><li> Ressuspender as lisados ​​em tampão de amostra de SDS fornecidos com o kit. Tampão de lise RIPA também pode ser usado. </li></ol> 4. Quantificação de vs maligno Fenótipo benigna da mama <ol><li> Cultivar células em triplicatas para cada conjunto de tratamento seguindo os passos 2,1-2,4. </li><li> Faça imagens de contraste de fase de estruturas acinares de cada poço em 5X ou 10X resolução usando um microscópio de contraste de fase ligada a um shifter de slides e um computador. Tome as imagens movendo o cursor de um quadro para outro e, assim, cobrindo todo o bem do slide compartimentado. </li><li> Contar o número total de estruturas acinareso número de ácinos único plana redonda e do número de estruturas multiacinar ou estruturas esmo crescente falta de organização e aos processos de ramificação que dele emanam. </li><li> Calcule a porcentagem de estruturas acinares plana redondas individuais versus estruturas malignas e enredo como gráfico de colunas. Calcula-se a significância pelo teste t de Student. </li></ol>

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Os autores declaram não haver interesses financeiros concorrentes.

Nós descrevemos aqui a cultura tridimensional de células epiteliais mamárias de uma membrana basal reconstituída e a utilidade deste sistema para diferenciar entre versus maligna fenótipo benigna da mama. Este sistema também pode ser utilizado para cultura de diferentes tipos de células de outros tecidos glandulares e tem sido relatada como tendo sido utilizados com sucesso para a cultura epitelial da próstata, do epitélio brônquico, e as células epiteliais da tiróide, para citar alguns 12-14. A i...

Cultura tridimensional (3D) de células epiteliais de mama na membrana basal reconstituída é um importante sistema modelo para estudar o fenótipo complexo e sinalização associada da mama normal e câncer de mama 2,3. A unidade funcional de mama lobular é a unidade terminal de conduta (TDLU) que consiste de uma pequena conduta que se ramifica em ácinos. Os ácinos são estruturas altamente organizadas, compostas por duas camadas de células, células epiteliais e mioepiteliais, que estão rodeados por uma membrana basal 5. As características mais distintivas de ácinos normais são polarização celular, o apego à membrana basal subjacente e contatos célula-célula especializados. Essa organização complexa é interrompido em carcinomas invasivos. As culturas 2D amplamente utilizados, em que as células são crescidas como monocamadas, não permitem a formação de ácinos. Assim, a cultura em monocamada é falta de fornecimento de um sistema para capturar a intrincada relação entre as células epiteliaisem mama normal e sua desregulamentação em câncer de mama. Culturas epiteliais monotípicas funcionais de células da mama foram desenvolvidas em vários laboratórios de 2,3. Cultura 3D envolve o crescimento de células da mama em Matrigel, que é um extracto solubilizado derivado de Engelbreth-Holm-Swarm células de sarcoma de ratinho e está disponível comercialmente. Outros substratos 3D como o colágeno também são usados. As células da mama podem ser cultivadas em 3D pelo ensaio incorporado 3D, em que as células são cultivadas incorporado em Matrigel 2. Alternativamente, as células podem ser cultivadas por 3D em cima ensaio, também chamado de teste de sobreposição 3D, que é rentável, pois requer menor volume de Matrigel, e facilita o monitoramento do lapso de tempo de formação de colônias por imagem de contraste de fase e é ideal para imagens em situ 2 -3. O 3D no topo ensaio tem sido utilizado para definir a correlação entre o fenótipo acinar e do perfil de expressão do gene 7. Cultura 3D pode ser effectively usado para distinguir células normais e benignos do carcinoma invasivo. Células mamárias normais e benignos formar crescimento preso estruturas polarizadas com um lúmen bem definidas no centro em Matrigel enquanto que as células de carcinoma invasivo crescer prolífica e ao acaso, sem compensação da luz. E6/E7 imortalizada de células epiteliais mamárias humanas e uma linha celular MCF10A, que é uma linha celular imortalizada espontaneamente, isolado a partir de uma paciente de 36 anos de idade com alterações fibrocística, têm sido utilizados com sucesso para recuperar o fenótipo benigna da mama em 3D 3,8 e serviram um sistema-modelo para estudar a função supressora de tumores oncogénicos e de várias moléculas de 8,9. Estas células benignas podem ser modificados para manipular a gene (s) de interesse com a introdução de lentivírus / retrovírus. Se o gene (s) de interesse é um supressor de tumor, knockdown shRNA mediada conduzirá a uma transformação maligna que, quando o gene de interesse é uma sobre-expressão de oncogenes em células benignasdeve mostrar um fenótipo maligno. Em ambos os casos, as estruturas acinares formados em 3D pode captar a transformação maligna. Células individuais benignos banhado em 3D começam a proliferar e formar rodada estruturas esféricas. Por dia 5-8 agregado esférico de células irá diferenciar em uma camada circundante de células polarizadas que está em contacto com o Matrigel e uma massa interna de células polarizadas menos, que carecem de contacto com o Matrigel. Nos próximos 10-15 dias, as células interiores vão começar a morrer por apoptose formando um lúmen claro. As células malignas vai crescer desordenadamente perder sua polaridade. Células altamente malignas geralmente formar estruturas de ramificação. Estas diferenças no fenótipo pode ser capturada por tempo fase lapso de imagem contraste e por in situ imunomarcação com marcadores moleculares relevantes. Os marcadores mais vulgarmente utilizados são alfa 6-integrina, um marcador de polaridade basal, em associação com o marcador da proliferação de fosfo-histona 3 e o apoptotmarcador ic caspase-3. Esta última é importante para determinar se existe a formação de lumen no centro dos ácinos, uma característica de células normais e benignas da mama. Outros polaridade e de contato célula-célula marcadores incluem GM130, laminina, ERM, E-caderina. Alternativamente as linhas de células de cancro da mama podem ser modificados para manipular o gene de interesse. Neste caso, a reversão para um crescimento mais preso fenótipo benigno pode ser usado como um lido para distinguir do fenótipo do cancro. Cultura 3D também pode ser usada com cautela para delinear as vias de sinalização envolvidas na transformação maligna 10-11. Usando o acima mencionado limite lidos, inibidores farmacológicos, os anticorpos de bloqueio ou proteínas recombinantes podem ser usadas se localizar na sinalização molecular conduzindo a uma transformação maligna. Com esforços contínuos de vários laboratórios, é possível extrair as células de 3D para isolar RNA e também preparar lisados ​​por immunoblotting e imunizaçãonoprecipitation. Estas estratégias são descritas em um passo a passo e forma detalhada no protocolo.

<table><tbody><tr><td>Growth Factor Reduced Matrigel</td><td>BD Biosciences</td><td>354230</td><td>Avoid repeat freeze thaw&amp;nbsp;</td></tr><tr><td>Lab-Tek glass hamber slides 8-well</td><td>Thermo Fisher Scientific</td><td>177402</td><td>Precool on ice&amp;nbsp;</td></tr><tr><td>Lab-Tek glass chamber slides 2-well&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>177380</td><td>Precool on ice&amp;nbsp;</td></tr><tr><td>Goat anti mouse F(ab&amp;acute;)&lt;sub&gt;2 &lt;/sub&gt;fragment</td><td>Jackson Immunoresearch</td><td>115-006-006</td><td></td></tr><tr><td>Normal goat serum</td><td>Jackson Immunoresearch</td><td>005-000-121</td><td></td></tr><tr><td>Fluorescent conjugated Alexa antibodies</td><td>Molecular Probes</td><td></td><td>Please look at molecular probes website</td></tr><tr><td>Prolong gold antifade reagent with DAPI</td><td>Molecular Probes</td><td>P36935</td><td></td></tr><tr><td>3D Culture Cell Harvesting kit</td><td>Trevigen</td><td>3448-020-k</td><td></td></tr><tr><td>Anti-Apha-6-integrin</td><td>Fisher Scientific</td><td>MAB1378</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-GM130</td><td>BD Biosciences</td><td>610822</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-phospho-histone 3 (Ser10)FITC Conjugated</td><td>Fisher Scientific</td><td>16-222</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-Cleaved caspase 3 (Asp 175)</td><td>Cell Signaling</td><td>9661s</td><td>1:50 dilution for IF</td></tr><tr><td>Anti-E-cadherin</td><td>BD Biosciences</td><td>610181</td><td>1:200 dilution for IF</td></tr></tbody></table>

three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells

Carcinomas da mama invasivos são um grupo de tumores epiteliais malignos caracterizada pela invasão de tecidos adjacentes e propensão para metástase. A interação de sinais entre as células cancerosas e seu microambiente exerce uma poderosa influência sobre o crescimento do câncer de mama e comportamento biológico 1. No entanto, a maioria destes sinais a partir da matriz extracelular são perdidas ou a sua relevância é subinvestigada quando as células são cultivadas em cultura de duas dimensões (2D) como uma monocamada. Nos últimos anos, a cultura tridimensional (3D) com uma membrana basal reconstituída tem surgido como um método de escolha para recapitular a arquitectura dos tecidos de células mamárias benignas e malignas. As células cultivadas em 3D reter as pistas importantes da matriz extracelular e fornecem um sistema ex vivo 2,3 fisiologicamente relevante. De notar que há cada vez mais evidências sugerindo que as células se comportam de forma diferente quando cultivada em 3D, em comparação com 4 2D. Cultura 3Dpode ser eficazmente utilizada como um meio para diferenciar o fenótipo maligno do fenótipo benigna da mama e para cimentar a sinalização celular e molecular envolvida 3. Uma das características distintas de células epiteliais benignas é que eles são polarizados de modo a que o citoplasma é apical para o lúmen e o citoplasma basal repousa sobre a membrana basal. Esta polaridade apico-basal é perdida em carcinomas invasivos da mama, que são caracterizadas por desorganização celular e formação da anastomose e ramificação túbulos que esmo infiltrados do estroma circundante. Estas diferenças histopatológicas entre glândula benigna e carcinoma invasivo pode ser reproduzido em 6,7 3D. Usando os Read-outs apropriadas como a quantificação de estruturas individuais acinares redondos, ou a expressão diferencial de marcadores moleculares validados para a proliferação celular, a polaridade e a apoptose, em combinação com outras técnicas de biologia molecular e celular, cultur 3De pode fornecer uma ferramenta importante para compreender melhor as alterações celulares durante a transformação maligna e para delinear a sinalização responsável.

Cultura 3D pode ser efetivamente usado para diferenciar fenótipo maligno das células epiteliais mamárias humanas do peito benigno. Células benignas Solteiro banhado em Matrigel crescer como estruturas esféricas organizados, que podem facilmente ser fotografada como único ácinos plana rodada em um avião usando imagens de contraste de fase. As células malignas único banhado em Matrigel crescer de forma anárquica, mostrar muito elevado índice de refração e são difíceis de imagem em um plano. Como mostrado n...

Watch this Scientific Journal Video about Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells at JoVE.com

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Três cultura tridimensional de células epiteliais mamárias de uma membrana basal reconstituída é um método útil para recapitular a arquitectura in vivo da mama benigna, e para diferenciar o fenótipo maligno do fenótipo da mama benigna. Importante, este sistema pode ser aplicado para estudar os carcinomas invasivos em outros tecidos.

Três Culturas dimensionais: Uma ferramenta para o estudo normal Acináceo Arquitetura vs transformação maligna das células de mama

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

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JoVE Journal

Medicine

19.8K visualizações.  University of Michigan Comprehensive Cancer Center.  Três cultura tridimensional de células epiteliais mamárias de uma membrana basal reconstituída é um método útil para recapitular a arquitectura in vivo da mama benigna, e para diferenciar o fenótipo maligno do fenótipo da mama benigna. Importante, este sistema pode ser aplicado para estudar os carcinomas invasivos em outros tecidos. 

Vídeo: Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Analisando a comunicação entre monócitos e células de câncer primário da mama em uma matriz Extracellular extrair (ECME)-sistema tridimensional baseado em

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Pesquisa do câncer

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Ensaio esferóide para TGF-β Medida induzida Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Medicina

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

Imitando e manipulando Metaplasia acinares-para-Ductal pancreático na cultura de pilha 3-dimensional

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Avaliação In Vitro da Transformação Oncogênica em Células Epiteliais Mamárias Humanas

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Cultura de Tecidos modelo tridimensional para estudar primária da medula óssea humana e suas Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Mediadora fosfolipídida induzida transformação em culturas tridimensionais

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantificação de cancro da mama invasiva das células usando um modelo (3D) tridimensional

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Elaboração e análise de<em&gt; In Vitro</em&gt; Três mama Dimensional Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Bioengenharia

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Geração de Organóides Mammary mosaicos por Trypsinização Diferencial

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Biologia do Desenvolvimento

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolamento de células epiteliais mamárias de tridimensional Mixed-célula esferoidal co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...