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

इस काम के भाग में, स्वास्थ्य CA131990 R01 (वित्तीय पर्यवेक्षण बोर्ड और RRM) के राष्ट्रीय संस्थानों द्वारा समर्थित किया गया. लाइव सेल इमेजिंग माइक्रोस्कोपी इमेजिंग, और Cytometry संसाधन समर्थित कोर में प्रदर्शन किया था, भाग में, एनआईएच केंद्र p30 CA22453 अनुदान द्वारा Karmanos कैंसर संस्थान, वेन स्टेट यूनिवर्सिटी और बाल स्वास्थ्य और विकास के राष्ट्रीय संस्थानों के Perinatology अनुसंधान शाखा द्वारा , वेन स्टेट यूनिवर्सिटी.

1. डीक्यू - substrates के तैयार <ol><li> DQ-substrates के खोलने शीशियों से पहले कमरे के तापमान को गर्म करने के लिए, विआयनीकृत पानी में 1 मिलीग्राम / मिलीग्राम डीक्यू सब्सट्रेट के स्टॉक समाधान तैयार, 50 μl aliquots और दुकान में 4 डिग्री ...

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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>

के रूप में प्रदर्शन किया, MAME cocultures विभिन्न सेलुलर घटक है कि एक स्तन ट्यूमर और इसके microenvironment शामिल के बीच बातचीत के वास्तविक समय में रहते सेल इमेजिंग के लिए इस्तेमाल किया जा सकता है. हमारी प्रयोगशाला में चल रह...

<table border="1"><tbody><tr><td> / अभिकर्मक और उपकरण </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियाँ </td></tr><tr><td> पुनर्गठन तहखाने झिल्ली (Cultrex) </td><td> Trevigen </td><td> 3445-005-01 </td><td> Matrigel (बी.डी. बायोसाइंसेज) करने के लिए तुलनीय </td></tr><tr><td> कोलेजन मैं </td><td> सामंजस्य लेबोरेटरीज </td><td> 5005-B </td><td></td></tr><tr><td> डीक्यू - substrates के (कोलेजन मैं, चतुर्थ) </td><td> Invitrogen </td><td> डीक्यू कर्नल मैं D12060 डीक्यू - कर्नल चतुर्थ D12052 </td><td></td></tr><tr><td> 22 मिमी प्लास्टिक coverslips </td><td> फिशर साइंटिफिक कंपनी </td><td> 12-547 </td><td> आधे में कटौती, 10 मिनट और हवा के उपयोग करने से पहले सूखे के लिए 70% इथेनॉल में छोड़ दें. </td></tr><tr><td> सेल संस्कृति माध्यम </td><td> LONZA </td><td> MEBM PRF सीसी ३,१५३ सीसी - 4136 MEGM </td><td> लाल - मुक्त phenol </td></tr><tr><td> μ-डिश ibiTreat </td><td> Ibidi </td><td> ८०,१३६ </td><td></td></tr><tr><td> Volocity सॉफ्टवेयर </td><td> Perkin - Elmer </td><td> संस्करण 5.5 </td><td> इमेजिंग सॉफ्टवेयर के अन्य ब्रांडों के लिए 3 डी पुनर्निर्माण और फिल्मों उत्पन्न करने के लिए इस्तेमाल किया जा सकता है. </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

हम उनके microenvironment में स्तन ट्यूमर कोशिकाओं और अन्य कोशिकाओं के बीच बातचीत के वास्तविक समय में रहते सेल इमेजिंग के लिए 3 डी coculture मॉडल विकसित किया है कि प्रभाव एक आक्रामक phenotype के लिए प्रगति. इन मॉडलों paracrine - प्रेरित, proteolytic, / chemokine साइटोकाइन और kinase invasiveness में फंसा रास्ते को लक्षित करने के लिए दवाओं के लिए preclinical स्क्रीन के रूप में सेवा कर सकते हैं.

MAME 4D ट्यूमर लाइव सेल इमेजिंग के लिए मॉडल: Microenvironment सहभागिता कि प्रभाव घातक प्रगति

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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