We thank Simon Hayward (Vanderbilt University Medical Center) for the BPH-1 cells; and Thomas Cox and Janine Erler (Biotech Research &amp; Innovation Centre, University of Copenhagen) for their assistance with rheological measurements. MR-T was funded by Worldwide Cancer Research, formerly The Association of International Cancer Research (Grant 08-0803 to JS), The British Embassy Montevideo and Agencia Nacional de Investigacion e Innovacion (UK_RH_2015_1_2 to MR-T). MC was supported by Prostate Cancer UK (Grant S14-017 to JS and GS). KW was funded by The China Scholarship Council. MAM was funded by The Saudi Arabian Cultural Bureau.

<ol>
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The authors have nothing to disclose.

Um protocolo para a geração de ácinos glandular 3D a partir de CME em géis RBM puros 6 foi modificada em um estudo anterior pela adição de 4 mg / ml tipo I colágeno para matriz RBM. A adição de colagénio resultou no módulo de elasticidade do gel RBM aumento de 175 ± 37-1589 ± 380 Pascais. Este aumento de 9,1 vezes na rigidez modulado o crescimento, sobrevivência, migração e diferenciação das CMEs 21. O protocolo foi modificado de novo através da inclusão de um passo de tratamento com D - (-) - ribose para promover a reticulação não enzimática do colagénio do tipo I, que tinha sido adicionado ao gel RBM. O resultante aumento de 15 vezes na rigidez foi encontrado para cooperar com a transformação oncogénica de MECs para promover o seu comportamento invasivo 22. A abordagem experimental do tipo adicionando I colágeno para géis RBM facilita a interação direta do CME com fibras de colágeno, o que só ocorre no tecido humano após a barreira física entre o estromae epitélio fornecida pela BM sofre degradação proteolítica. Ao gerar ácinos glandular 3D a partir de PECs em géis RBM puros pré-tratados com GLA, o protocolo atual abre o caminho para estudar como BM rigidez per se pode acionar o seu comportamento invasivo (Figura 3). Os níveis de BM rigidez induzidas neste protocolo tem relevância fisiológica. A incubação com GLA 50 mM durante 6 h e 14 h, respectivamente, aumentou os módulos elásticos do gel RBM pura para 175 ± 90 e 322 ± 160 em comparação com 122 ± 55 Pascais em géis RBM tratados com PBS (Tabela 1). Este aumento de 1,7 a 3,2 vezes em RBM rigidez recapitula a 2,5 a 3,4 vezes aumento na rigidez observada em comparação com maligno da próstata normal ou o tecido de BPH 23-26. Conforme descrito em uma publicação recente 13 as alterações morfológicas induzidas pela acumulação de AGE e RBM rigidez no PEC ácinos pode ser quantificada para uma mudança estatisticamente significante de arundada de forma poligonal, diminuição luminal / área total acinar, e as células salientes migram do acina no-AGE rica RBM (Figura 3). Imunotransferência também pode ser usado para avaliar os marcadores de EMT (por exemplo, perda de E-caderina 13) e o comportamento contráctil (por exemplo leve da miosina de cadeia fosforilada-2, pMLC2 13) em PEC cultivadas em normais versus RBM rígida (Figura 3). A avaliação subsequente por meio da coloração de imunofluorescência e microscopia confocal podem ser aplicadas para visualizar o BM (por exemplo, a laminina, colagénio IV e acumulação de AGE 13), a polaridade apical-para-basal celular (por exemplo, localização apical EEA1: antigénio endossomal precoce 1; e GM130: 130 kDa cis-Golgi marcador 13) e padrões de moléculas de adesão celular (por exemplo, a e-caderina de localização para junções célula-célula 13) (Figura 3). <img alt = &quot;Figura 3&quot; src = &quot;/ files / ftp_upload / 54230 / 54230fig3.jpg&quot; /&gt; Figura 3:. Visão geral dos diferentes protocolos aqui apresentados O diagrama mostra como preparar e endurecer a membrana basal reconstituída (RBM) com glicoaldeído (reação de Maillard), como propagar células para a RBM rígida, como analisar o RBM rígido ( extensão da reacção de Maillard) e procedimentos que podem ser utilizados para analisar as alterações celulares e moleculares induzidos por RBM rica em produtos FGA. AGE, produtos finais da glicação avançada; BM, membrana basal; DAPI, 4 &#39;, 6-diamidino-2-fenilindol; EEA1, cedo antigénio endossomal 1; GAPDH, gliceraldeído-3-fosfato desidrogenase; GLA, glicoaldeído; GEE, éster etílico de glicina; GM130, 130 kDa marcadores cis-Golgi; p-MLC2 (Thr18 / Ser19), miosina de cadeia leve 2 fosforilada em locais treonina 18 e serina 19; RBM, membrana basal reconstituída; SDS-PAGE, electroforese em dodecil sulfato de gel de poliacrilamida de sódio. Para bar RWPE1 ácinos Escala = 10 mm; para células de tumor PC3barra de escala = esferóides 100 um. Esta figura foi modificado a partir da referência 13. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> solução de problemas etapas será necessário se D - (-) - ribose é escolhido como o agente de ligação cruzada para RBM. Durante o desenvolvimento do protocolo, verificou-se que o tratamento com 1 M D - (-) - ribose durante 72 horas, como descrito anteriormente para géis RBM / colágeno 22, resultou na desidratação e encolhimento dos géis RBM. A avaliação de concentrações mais baixas de D - (-) - ribose e tempos de tratamento mais curtos pode ajudar a ultrapassar esta limitação. Uma limitação potencial em futuras aplicações do protocolo pode ser encontrado em níveis mais elevados de RBM rigidez são desejados. Se os tempos de incubação mais longos e maiores concentrações de GLA são utilizados para induzir níveis mais elevados de rigidez de gel RBM será necessário<html> avaliar se estas condições de tratamento ter um impacto sobre a sobrevivência e proliferação celular, tal como previamente descrito 13. Deve também notar-se que a incubação de células RWPE-1 com soro induz uma transição EMT-fenotípica e como a exposição a soro ou materiais contendo soro deve ser evitada. Por exemplo, se experiências envolvem a transfecção de ARN interferente curto oligonucleótidos (siARN), o procedimento deve ser optimizada utilizando células RWPE-1 cultivadas em KSFM, sem mudar as células para meio de transfecção baixo soro. Este inconveniente pode comprometer o nível de silenciamento de genes obtidos quando utilizando siRNA transiente abordagens no modelo. Para algumas proteínas alvo, seria aconselhável empregar vectores shRNA induzíveis para o silenciamento do gene ajustável ea redução desejada em níveis de proteína. Adaptações que incorporam reticulação enzimática por células do estroma ou células de tumor associado lisil-oxidase (LOX) 17 também poderia ser incorporada modelos futuros. jove_content &quot;&gt; Este protocolo facilitará o futuro estudo de mecanismos pró-invasivos desencadeadas por idade-dependente BM rigidez nas PECs (RWPE-1, BPH-1) e avaliação de metas anti-metastáticos em PTCs invasivos (PC3). Dado que BPH é considerado como sendo uma desordem metabólica 27, este protocolo também abre o caminho para a melhor compreensão da relação entre distúrbios metabólicos e aumento do risco de cancro da próstata. Dado que a BM rigidez induzida por sua exposição a produtos FGA pode ser um gatilho para a invasividade noutros cancro tipos, que será de interesse para usar o protocolo de criar modelos similares que incorporam células epiteliais normais e células tumorais de outros órgãos (por exemplo, mama, cólon, ovário, pâncreas). As etapas críticas no âmbito do protocolo, juntamente com os seus horários, estão resumidos na Figura 4. Durante o passo inicial é essencial para manter a solução estoque de RBM a 4 ° C, enquanto que derrete para evitar a sua polymerization. Pontas de pipeta não deve ser colocado na solução estoque rM até que tenham sido arrefecida a 4 ° C. Para o próximo passo, é também importante para garantir que as lâminas de câmaras foram equilibrados a 4 ° C antes de serem revestidos com a solução RBM. Assim que a temperatura da solução é aumentada RBM acima de 4 ° C, ele irá sofrer polimerização irreversível para formar um gel. É essencial que o RBM não é perturbada durante a fase de polimerização para garantir que ela forma uma superfície plana apropriada para a cultura de células e análise microscópica. A duração da incubação com GLA com ou sem inibidores da reacção de Maillard (cianoboro-hidreto de sódio e amingoguanidine) irá determinar qual a rigidez do gel torna-se RBM. Recomenda-se a utilização de uma incubação de 6 h com GLA se forem necessárias condições semi-rígidas, e 14 h de incubação, se as condições rígidas são necessárias (Tabela 1). tempos de incubação alternativo ou concentrações de GLA pode ser usado se diferentes níveisde rigidez são desejados. Neste caso análise reológica dos géis RBM precisa de ser incorporado como um passo essencial. A seguir ao passo de têmpera da reacção de Maillard, por incubação com Gee e os passos de lavagem subsequentes com PBS, os géis RBM podem ser utilizadas imediatamente ou armazenadas a 4 ° C durante até 48 h antes de seu uso para cultura de células. Uma vez que as culturas de células são configurados é importante mudar o meio de cultura (incluindo quaisquer tratamentos) a cada dois dias. Recomenda-se a manter as culturas de células 3D durante 3-12 dias de acordo com os parâmetros sob investigação. Para 3D PEC ácinos, recomenda-se analisar as culturas após 6 dias, e para a análise 3D PTC esferóides é recomendado após 3 dias de cultura na primeira instância. <img alt="Figura 4" src="/files/ftp_upload/54230/54230fig4.jpg" /> Figura 4:. Vista geral simples do protocolo com a passos críticos e sincronismos indicou a flodiagrama w descreve como preparar e endurecer a membrana basal reconstituída (RBM) com glicoaldeído (reação de Maillard) com passos críticos e horários indicados. Os pontos em que o protocolo pode ser parado, e géis RBM armazenados, são também indicados. RBM, membrana basal reconstituída; GLA, glicoaldeído; GEE, éster etílico de glicina; ON, durante a noite; PBS, solução salina tamponada com fosfato; RT, temperatura ambiente. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

The basement membrane (BM) is a sheet of specialized extracellular matrix (ECM) that maintains stable tissue borders by separating layers of epithelial cells from the stroma1. Covalent crosslinking between adjacent triple helices of collagen IV in the BM stabilizes their lateral association by establishing an irregular network of super-twisted helices2. These collagen IV lattices act as a scaffold for its interaction with laminin and other BM components1. The structural arrangement of the BM provides it with the mechanical strength and rigidity necessary for the normal development of glandular epithelia3. 
During aging and disease the BM progressively thickens and stiffens3,4. For example, a 3-fold increase in the elastic modulus (E) of the ocular BM occurs between the ages of 50 and 80 in the normal population, and this stiffening is further exacerbated in metabolic disorders like diabetes5. The structural and biomechanical changes in the BM that result in its increased stiffness occur when its ECM components, collagen IV and laminin, become non-enzymatically crosslinked following their exposure to advanced glycation endproducts (AGEs).
The purpose of the method described here was to establish a model for the investigation of how BM stiffness, due to AGE exposure, promotes prostate epithelial cell (PEC) and prostate tumour cell (PTC) invasiveness in the context of the switch to metastatic prostate cancer (PCa). To do this a previous method used for generating 3D glandular acini from mammary epithelial cells (MECs) in reconstituted rBM gels6 was adapted to include an additional step where the rBM gels are pre-treated with glycolaldehyde (GLA). Several techniques for assessing GLA induced crosslinking and stiffening of pre-treated rBM gels are described, including photometric analysis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), confocal microscopy and rheometric analysis. The prostate cell types selected for culture on the pre-stiffened rBM include: RWPE-1, PECs derived from a normal prostate gland7; BPH-1, PECs derived from a prostate gland affected by BPH8; and PC3, metastatic PTCs derived from a secondary tumor located in the vertebral bone of a prostate cancer (PCa) patient9. 
In addition to advancing the study of prostate gland pathology, the protocol for stiffening of rBM gels by their treatment with GLA can be adapted to investigate how BM stiffness contributes to other age-related pathologies and metabolic disorders. For example, the model can be directly applied to investigate how metastatic cancer is induced by BM stiffness in organs such as the breast, colon, ovary and pancreas by the incorporation of appropriate cell types. Furthermore, the protocol can be adapted to investigate how stiff BM promotes biomechanical mechanisms of disease progression in diabetes-related microvascular disease, retinopathy, nephropathy and neuropathy.

<table><tbody><tr><td>&lt;strong&gt;I - Material for monolayer culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BPH-1</td><td>CaP Cell Line Database</td><td>PCaCL-132</td><td>Contact: simon.hayward@mcmail.vanderbilt.edu</td></tr><tr><td>Complete keratinocyte serum-free media&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>17005-075</td><td>Do not warm at 37 &amp;ordm;C before use</td></tr><tr><td>Fetal calf serum&amp;nbsp;</td><td>First Link UK Ltd</td><td>02-00-850</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>PC3&amp;nbsp;</td><td>American Type Culture Collection</td><td>CRL-1435</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>ThermoFisher Scientific</td><td>15070-063</td><td></td></tr><tr><td>Phosphate Buffer Saline (Dulbecco A) tablets</td><td>Oxoid</td><td>BR0014G</td><td></td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Sigma-Aldrich</td><td>R5886</td><td>warm in 37 &amp;ordm;C water bath before use</td></tr><tr><td>RWPE-1&amp;nbsp;</td><td>American Type Culture Collection</td><td>CRL-11609</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich</td><td>T4049</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;II - Material for 3D culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>271004</td><td></td></tr><tr><td>Aminoguanidine hydrochloride</td><td>Sigma-Aldrich</td><td>396494</td><td>Irritating to eyes, respiratory system and skin</td></tr><tr><td>Chamber slides, 8-well&amp;nbsp;</td><td>Thermo Scientific Nunc Lab-Tek</td><td>TKT-210-816M</td><td></td></tr><tr><td>Culture Matrix reconstituted basement membrane (rBM) reduced growth factor extract&amp;nbsp;</td><td>AMS Biotechnology</td><td>3445-005-01</td><td>Store Basement membrane (BM) at -80 &amp;ordm;C in aliquots</td></tr><tr><td>Cyanogen bromide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C91492</td><td>Toxic by contact skin and inhalation</td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>695076</td><td></td></tr><tr><td>Glycine ethyl ester hydrochloride (GEE)</td><td>Sigma-Aldrich</td><td>50060</td><td>Irritating to eyes</td></tr><tr><td>Glycolaldehyde dimer (GLA)</td><td>Sigma-Aldrich</td><td>G6805</td><td></td></tr><tr><td>Sodium cyanoborohydride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>71435</td><td>Highly flammable; Toxic by contact skin and inhalation</td></tr><tr><td>Syringe filter 0.22 microns</td><td>Appleton Woods</td><td>BC680</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;III - Material to quantify Maillard reaction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>ThemoFisher Scientific</td><td>D3571</td><td>light sensitive and store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Cloning cylinders</td><td>Sigma-Aldrich</td><td>C1059</td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td><td>ThemoFisher Scientific</td><td>A-11001</td><td>light sensitive</td></tr><tr><td>Goat anti-rabbit IgG (H+L) Alexa Fluor 488 conjugate</td><td>ThemoFisher Scientific</td><td>A-11034</td><td>light sensitive</td></tr><tr><td>Goat serum</td><td>Abcam</td><td>ab7481</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Vectashield mounting media&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>Mouse anti-pentosidine clone PEN-12 mAb</td><td>TransGenic Inc</td><td>KH012</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>F8775</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Rabbit anti-human collagen IV polyclonal antibody&amp;nbsp;&amp;nbsp;</td><td>Acris Antibodies</td><td>R1041</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Rabbit anti-laminin A/C pAb</td><td>Santa Cruz Biotechnology Inc</td><td>sc-7292</td><td>Store at -20 &amp;ordm;C in aliquots</td></tr><tr><td>Polyethylene glycol tert-octylphenyl ether (Triton-X100)</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>Polyethylene glycol sorbitan monolaurate (Tween-20)</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Dialysis cassette Slide-A-Lyzer</td><td>ThemoFisher Scientific</td><td>66333</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;IV - Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ARG2 controlled strain rotational rheometer</td><td>T.A. Instruments</td><td></td><td></td></tr><tr><td>Axiovert S100 (20X magnification) microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>CO2 controlled humidified incubation chamber for Zeiss Axio S100 microscope</td><td>Solent Scientific</td><td></td><td></td></tr><tr><td>Confocal Axiovert 200M (40X, 63X magnification) microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Olympus LH50A microscope fitted with a digital camera using phase-contrast&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td></td><td></td></tr><tr><td>PHERAstar Plus plate reader spectrophotometer</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;V - Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Image J 1.47v</td><td>National Institute of Health, USA</td><td></td><td></td></tr><tr><td>MetaXpress</td><td>Molecular Divices</td><td></td><td></td></tr></tbody></table>

how to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases

Descrevemos aqui um protocolo que pode ser utilizado para estudar o microambiente biofísico relacionadas ao aumento da espessura e rigidez da membrana basal (BM) durante patologias relacionadas com a idade e distúrbios metabólicos (por exemplo, cancro, diabetes, doença microvascular, retinopatia, nefropatia e neuropatia) . A premissa de que o modelo é de ligação cruzada não enzimática de BM matriz reconstituído (RBM), por tratamento com glicolaldeído (GLA) para promover a glicação avançada produto final geração (FGA), através da reacção de Maillard. Exemplos de técnicas laboratoriais que podem ser utilizados para confirmar a geração de AGE, a reticulação não enzimática e aumento da rigidez em GLA tratada RBM são descritas. Estes incluem a preparação de MAE nativa (tratados com solução salina tamponada com fosfato, PBS) e rígida RBM (tratadas com GLA) para a determinação de: o seu conteúdo de AGE por análise fotométrica e de microscopia de imunofluorescência, a sua ligação cruzada não enzimática de poliacrilamida de sulfato de dodecilo de sódioelectroforese em gel (SDS-PAGE), bem como microscopia confocal, e a sua rigidez aumentada utilizando reometria. O processo descrito aqui pode ser utilizado para aumentar a rigidez (módulos elásticos, E) de MAE-se para 3,2 vezes, consistente com as medições efectuadas em saudável contra o tecido da próstata humano doente. Para recriar o microambiente biofísico associado ao envelhecimento e próstata doente glândula três tipos de células da próstata foram introduzidas para RBM nativa e dura RBM: RWPE-1, células epiteliais da próstata (PECs) derivada de uma próstata normal; HBP-1, PEC derivadas de uma próstata afectados por hiperplasia prostática benigna (BPH); e PC3, células metastáticas derivado de um tumor ósseo secundário proveniente de cancro da próstata. Vários parâmetros podem ser medidos, incluindo o tamanho, forma e características invasoras do acini glandular 3D formado por RWPE-1 e BPH-1 no nativa contra RBM duro, e duração média de células, a velocidade de migração e persistência de circulação de células de spher 3Doids formados por células PC3 sob as mesmas condições. Vias celular e a localização subcelular de proteínas de sinalização pode igualmente ser avaliada.

3D próstata Acini cultivadas em RBM Stiff Após 6 dias de cultura, os PEC derivada de tecido de próstata normal (RWPE-1) (Figura 1A) e no tecido HBP (BPH-1) (Figura 1B) forma ácinos no nativo (tratados com PBS) RBM que estão organizados em esferóides uniformes de epitelial células. Estes ácinos também têm as características de PECs altamente organizadas com espaço apical-para-basal polaridade e um luminal visível 13,20. O ácinos formado por PEC derivadas a partir de tecido de próstata normal (RWPE-1) (Figura 1A) e no tecido HBP (BPH-1) (Figura 1B) em endureceu RBM (rico em AGE) (tratado com GLA) têm uma arquitectura interrompido (deslocando de esferoidal de forma poligonal e células salientes / migração do ácinos no RBM rico-AGE) (Figura 1A). Estes<html> ácinos são também caracterizados por PEC altamente desorganizada que perderam a sua polaridade para-apical-basal com uma pequena ou inexistente espaço luminal 13. <img alt="figura 1" src="/files/ftp_upload/54230/54230fig1.jpg" /> Figura 1:. Células da próstata epiteliais cultivadas como 3D glandular Acini em Native e Stiff membrana basal reconstituída (RBM) (A) imagens de campo claro de células RWPE-1 cultivadas durante 12 h até 6 dias em géis RBM tratados com PBS (nativo) ou glicoaldeído 50 mM durante 14 h (-AGE rica; rígida); Barra de escala = 50 mm. (B) as células BPH-1, crescidas como descrito no painel A; Barras de escala = 50 um; dados são representativos de 3 experiências independentes. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <html> Tabela 1 &quot;src =&quot; / files / ftp_upload / 54230 / 54230table1.jpg &quot;/&gt; Tabela 1:. Características da próstata epitelial RWPE-1 ácinos cultivadas em Native, Semi-Stiff e Stiff basal reconstituída Membrane (RBM) RWPE-1 ácinos foram cultivadas em RBM pré-tratados com PBS, durante 14 horas (nativo), glicoaldeído (GLA ) durante 6 horas (semi-rígidos) ou GLA, durante 14 horas (dura). Por forma acinar, o percentual (%) ± desvio padrão (SD) da rodada, acini semi-poligonal e poligonais foram calculados a partir de 5 experiências independentes (50 ácinos quantificados por condição). tamanho acinar relativo foi calculado (nativo RBM = 100%) a partir de 3 experiências independentes. Para invasividade,% ± SD ácinos com uma ou mais células protuberantes foram calculadas a partir de 3 experiências independentes. mudança da dobra é calculado dividindo o valor médio obtido em condições semi-rígidas ou rígidas por o valor correspondente para condições nativas. Valores de p calculados utilizando o teste t de Student (α = 0,05). onteúdo &quot;fo: manter-together.within-page =&quot; 1 &quot;&gt; AGE dependente aumento da rigidez RBM promove a migração de células tumorais PC3 próstata Células PC3 cultivadas em RBM nativa migrar mantendo contato célula-célula contínua, enquanto que as células PC3 cultivadas em rich-AGE (dura) RBM movimento independentemente um do outro (Figura 2A). Depois de 72 horas em células PC3 cultura formar focos (esferóides) sobre nativa (tratado PBS) RBM, enquanto que as células PC3 sobre dura RBM (rico em AGE) não fazer a partir de esferóides e migrar de forma independente (Figura 2B). Células PC3 sobre dura RBM (rico em AGE) são mais alongado do que as células PC3 cultivadas em RBM nativa (Figura 2C). Células PC3 sobre RBM dura migrar mais rapidamente do que células PC3 cultivadas em RBM nativa (Figura 2D). Células PC3 sobre RBM dura exibir uma diminuição na persistência em comparação com células PC3 cultivadas em RBM nativa (Figura 2E). <img alt = &quot;Figura 2&quot; src = &quot;/ files / ftp_upload / 54230 / 54230fig2.jpg&quot; /&gt; Figura 2:. A migração do tumor de células da próstata em Native e Stiff membrana basal reconstituída (RBM) (A) imagens de campo claro de células PC3 cultivados em géis RBM tratados com PBS (nativo) ou glicoaldeído 50 mM durante 14 h (rico em AGE, duro) . As células foram fotografadas utilizando um microscópio de campo claro (objetiva de 10X) e uma taxa de aquisição de uma imagem por hora durante 12 horas seguido de células de rastreamento para gerar trajetórias. As imagens mostradas correspondem aos pontos de tempo após 0, 3, 6, 9 e 12 horas. Trajetórias de células individuais são mostrados para o ponto de tempo de 12 horas. Barra de escala = 100 pm. Células (B) PC3 cultivadas em RBM nativa ou rígida, durante 72 h, e visualizada como se descreveu no painel (A). Barra de escala = 100 pm. Detalhe mostra a área selecionada a 2X ampliação. (C) Média ± SD comprimento célula (um); diferença significativa entre RBM nativa e RBM rígida (p = 1,2 x 10 -23). ( <strong&gt; D) Média ± SD velocidade (pM / hr) calculado a partir de trajectórias de células; diferença significativa entre RBM nativa e RBM rígida (p = 0,004). (E) Média ± SD persistência do movimento celular (razão D / T, em que D = distância do início ao fim da trajectória de célula, T = comprimento total da trajectória de célula); diferença significativa entre RBM nativa e RBM rígida (p = 0,0007). Para os painéis CE&gt; 10 células foram analisados, os dados são representativos de 3 experiências independentes. <a href="https://www.jove.com/files/ftp_upload/54230/54230fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases at JoVE.com

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Aqui nós explicar um protocolo para modelar o microambiente biofísico onde reticulação e o aumento da rigidez da membrana basal (BM) induzida por produtos finais de glicação avançada (AGEs) tem relevância patológica.

Como Estudar Basement Membrane Rigidez como um gatilho Biofísica no cancro da próstata e outras patologias relacionadas com a idade ou Doenças Metabólicas

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

how-to-study-basement-membrane-stiffness-as-biophysical-trigger

Research

JoVE Journal

Cancer Research

9.6K visualizações.  Universidad de la República (UDELAR).  Aqui nós explicar um protocolo para modelar o microambiente biofísico onde reticulação e o aumento da rigidez da membrana basal (BM) induzida por produtos finais de glicação avançada (AGEs) tem relevância patológica. 

Vídeo: How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Aspiração micropipeta de Células-substrato anexados para Estimar Rigidez celular

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Bioengenharia

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Medir as propriedades mecânicas das células vivas Usando Microscopia de Força Atômica

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Medição Longitudinal de Matriz Extracelular Rigidez em 3D tumorais modelos com o uso de rastreamento de partículas Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Isolamento de células do cólon tumor primário Humanos a partir de tecidos cirúrgicos e cultivando-as Diretamente macias elásticas Substratos para Traction Citometria

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Estudando os efeitos da rigidez Matrix em função celular usando acrilamida baseado Hidrogéis

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Biologia

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Micro-Mecânica Caracterização do tecido pulmonar Usando Microscopia de Força Atômica

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Um ensaio de poliacrilamida-baseado formato multi bem para estudar o efeito da rigidez da matriz extracelular sobre a infecção bacteriana das células aderentes

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Imunologia e Infecção

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Microscopia confocal e força atômica (Conpokal) em células vivas

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Extracting the Young's Modulus of Native Murine Pulmonary Basement Membranes from Atomic Force Microscopy Derived Force Maps

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of nanometers. Because mammalian cells sense and react to the mechanics of their immediate microenvironment, the characterization of biomechanical properties of tissues with high spatial resolution is crucial for understanding various developmental, homeostatic, and pathological processes. The basement membrane (BM), a roughly 100 - 400 nm thin extracellular matrix (ECM) substructure, plays a significant role in tumor progression and metastasis formation. Although determining Young&#39;s modulus of such a thin ECM substructure is challenging, biomechanical data of the BM provides fundamental new insights into how the BM affects cell behavior and, in addition, offers valuable diagnostic potential. Here, we present a visualized protocol for assessing BM mechanics in murine lung tissue, which is one of the major organs prone to metastasis. We describe an efficient workflow for determining the Young&#39;s modulus of the BM, which is located between the endothelial and epithelial cell layers in lung tissue. The step-by-step instructions comprise murine lung tissue freezing, cryosectioning, and AFM force-map recording on tissue sections. Additionally, we provide a semi-automatic data analysis procedure using the CANTER Processing Toolbox, an in-house developed user-friendly AFM data analysis software. This tool enables automatic loading of recorded force maps, conversion of force versus piezo-extension curves to force versus indentation curves, computation of Young&#39;s moduli, and generation of Young&#39;s modulus maps. Finally, it shows how to determine and isolate Young&#39;s modulus values derived from the pulmonary BM through the use of a spatial filtering tool.

This protocol visualizes how to prepare cryosections of murine lung tissue, perform atomic force microscopy force map experiments, and analyze the data to determine Young's modulus of the native murine pulmonary basement membrane.

Extraindo o módulo de Young de membranas basais pulmonares murinas nativas de mapas de força derivados de microscopia de força atômica

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Medindo a rigidez<em&gt; Ex Vivo</em&gt; Mouse aortas Usando Microscopia de Força Atômica

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Como Estudar Basement Membrane Rigidez como um gatilho Biofísica no cancro da próstata e outras patologias relacionadas com a idade ou Doenças Metabólicas

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