Name: mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression
Uploaded: 2012-02-17T12:00:00
Description: Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

Эта работа была поддержана, в частности, от Национального института здоровья R01 CA131990 (BFS и RRM). Онлайн-клеток изображений проводили в основной микроскопии и обработки изображений цитометрии ресурсов поддерживается, в частности, по гранту Центра NIH P30 CA22453 к Karmanos института рака, Wayne State University и отделения перинатологии исследований Национального института детского здоровья и развития , Wayne State University.

1. Подготовить DQ-подложки <ol><li> Позвольте лиофилизированный DQ-подложки нагревали до комнатной температуры перед открытием флаконов, готовят маточный раствор 1 мг / мл DQ-подложки в деионизованной воды, разделить на 50 мкл аликвоты и хранят при температуре 4 ° C. </li></ol> Это мож...

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	<li>Sameni, M., Dosescu, J., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+proteolysis:+stromal+and+inflammatory+cells+increase+tumor+proteolysis.">Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis.</a> Mol. Imaging. 2, 159-175 (2003).</li><li>Jedeszko, C., Sameni, M., Olive, M. B., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+protease+activity+in+living+cells:+from+two+dimensions+to+four+dimensions.">Visualizing protease activity in living cells: from two dimensions to four dimensions.</a> Curr. Protoc. Cell Biol. 39, (2008).</li><li>Jedeszko, C., Victor, B. C., Podgorski, I., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+hepatocyte+growth+factor+promotes+invasion+of+human+mammary+ductal+carcinoma+in+situ.">Fibroblast hepatocyte growth factor promotes invasion of human mammary ductal carcinoma in situ.</a> Cancer Res. 69, 9148-9155 (2009).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+live-cell+imaging+demonstrates+that+beta1-integrin+promotes+type+IV+collagen+degradation+by+breast+and+prostate+cancer+cells.">Functional live-cell imaging demonstrates that beta1-integrin promotes type IV collagen degradation by breast and prostate cancer cells.</a> Mol. Imaging. 7, 199-213 (2008).</li><li>Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p21-activated+kinase+1+coordinates+aberrant+cell+survival+and+pericellular+proteolysis+in+a+three-dimensional+culture+model+for+premalignant+progression+of+human+breast+cancer.">p21-activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer.</a> Neoplasia. 10, 314-328 (2008).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantifying+the+dynamics+of+tumor+associated+proteolysis.">Imaging and quantifying the dynamics of tumor associated proteolysis.</a> Clin. Exp. Metastasis. 26, 299-309 (2009).</li><li>Cavallo-Medved, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+demonstrates+that+proteases+in+caveolae+of+endothelial+cells+degrade+extracellular+matrix.">Live-cell imaging demonstrates that proteases in caveolae of endothelial cells degrade extracellular matrix.</a> Exp. Cell. Res. 315, 1234-1246 (2009).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li></ol>

Как показано, MAME cocultures может быть использован для живых клеток изображений в режиме реального времени взаимодействия между различными сотовой избирателей, что составляет опухоли молочной железы и его микросреды. В текущие исследования в нашей лаборатории, мы использовали MAME cocultures оп?...

<table border="1"><tbody><tr><td> Реагент / Оборудование </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> Восстановленный базальной мембраны (Cultrex) </td><td> Trevigen </td><td> 3445-005-01 </td><td> Сопоставимые с Матригель (BD Biosciences) </td></tr><tr><td> Коллаген я </td><td> Сплоченность лаборатории </td><td> 5005-B </td><td></td></tr><tr><td> DQ-подложки (Коллаген I, IV) </td><td> Invitrogen </td><td> DQ-кол I-D12060 DQ-кол IV D12052 </td><td></td></tr><tr><td> 22-мм пластиковыми покровные </td><td> Компания Fisher Scientific </td><td> 12-547 </td><td> Разрежьте пополам, оставит в 70% этаноле в течение 10 мин и воздушно-сухой перед использованием. </td></tr><tr><td> Среду клеточной культуры </td><td> Lonza </td><td> MEBM CC-PRF-3153 MEGM CC-4136 </td><td> Фенол красный бесплатно </td></tr><tr><td> ibiTreat μ-Блюдо </td><td> Ibidi </td><td> 80136 </td><td></td></tr><tr><td> Volocity программного обеспечения </td><td> Перкин-Элмер </td><td> Версия 5.5 </td><td> Другие бренды изображений программное обеспечение может быть использован для создания 3D-реконструкции и фильмов. </td></tr></tbody></table>

mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

Мы разработали 3D-моделей coculture для живых клеток изображений в режиме реального времени взаимодействия между опухолевых клетках молочной железы и других клеток в их микросреды, влияющих на прогрессирование инвазивного фенотипа. Эти модели могут служить в качестве экранов для доклинических наркотиков целевой паракринной вызванных протеолитических, хемокинов / цитокинов и киназы пути причастных к инвазии.

MAME Модели 4D Онлайн-клеточного изображения опухоли: микроокружение взаимодействия, что воздействие злокачественной прогрессии

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

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