Name: heterotypic three-dimensional in vitro modeling of stromal-epithelial interactions during ovarian cancer initiation and progression
Uploaded: 2012-08-28T13:00:00
Description: Watch this Scientific Journal Video about Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression at JoVE.com

Denne forskning blev udført på Keck School of Medicine, University of California, USA og University College London, UK. KL er finansieret af National Institute of Health tilskud 5 U19 CA148112-02. BG blev finansieret af en projektbevilling fra Eve Appeal gynækologiske onkologiske velgørenhed (UK). Noget af dette arbejde, der udføres på UCLH / UCL blev delvist tilskud fra Department of Health er NIHR Biomedical Research Centre støtteordning.

Figur 1 illustrerer en oversigt over arbejdsgangen beskrevet nedenfor. 1. Isolering af normalt ovarialt Fibroblaster og udvidelse af in vitro Levetid ved overekspression af den katalytiske subunit af hTERT holoenzym <ol><li> Æggestokkene væv kan indsamles med patientens informerede samtykke og godkendelse af Institutional Review Board (for institutioner i USA). Normale æggestokkene væv kan indsamles efter total abdominal hysterektomi eller total lapar...

<ol>
	<li>Jelovac, D., Armstrong, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+diagnosis+and+treatment+of+ovarian+cancer.">Recent progress in the diagnosis and treatment of ovarian cancer.</a> CA Cancer J. Clin. , (2011).</li><li>Office for National Statistics. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cancer Statistics registrations: registrations of cancer diagnosed in 2008. , (2011).</li><li>K&#246;bel, M., Kalloger, S. E., Santos, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+type+and+substage+predict+survival+in+stage+I+and+II+ovarian+carcinoma:+insights+and+implications.">Tumor type and substage predict survival in stage I and II ovarian carcinoma: insights and implications.</a> Gynecol. Oncol. 116, 50-56 (2010).</li><li>K&#246;bel, M., Kalloger, S. E., Boyd, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+carcinoma+subtypes+are+different+diseases:+implications+for+biomarker+studies.">Ovarian carcinoma subtypes are different diseases: implications for biomarker studies.</a> PLoS Med. 5, e232 (2008).</li><li>Smith, L. H., Morris, C. R., Yasmeen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer:+can+we+make+the+clinical+diagnosis+earlier.">Ovarian cancer: can we make the clinical diagnosis earlier.</a> Cancer. 104, 1398-1407 (2005).</li><li>Aviv, A., Hunt, S. C., Lin, J., Cao, X., Kimura, M., Blackburn, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impartial+comparative+analysis+of+measurement+of+leukocyte+telomere+length/DNA+content+by+Southern+blots+and+qPCR.">Impartial comparative analysis of measurement of leukocyte telomere length/DNA content by Southern blots and qPCR.</a> Nucleic Acids Res. 39, e134 (2011).</li><li>Kim, N. W., Wu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+quantification+and+characterization+of+telomerase+activity+by+the+telomeric+repeat+amplification+protocol+(TRAP.">Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP.</a> Nucleic Acids Res. 25, 2595-2597 (1997).</li><li>Lawrenson, K., Grun, B., Benjamin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescent+fibroblasts+promote+neoplastic+transformation+of+partially+transformed+ovarian+epithelial+cells+in+a+three-dimensional+model+of+early+stage+ovarian+cancer.">Senescent fibroblasts promote neoplastic transformation of partially transformed ovarian epithelial cells in a three-dimensional model of early stage ovarian cancer.</a> Neoplasia. 12, 317-325 (2010).</li><li>Lawrenson, K., Benjamin, E., Turmaine, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+three-dimensional+modelling+of+human+ovarian+surface+epithelial+cells.">In vitro three-dimensional modelling of human ovarian surface epithelial cells.</a> Cell Prolif. 42, 385-393 (2009).</li><li>Lawrenson, K., Sproul, D., Grun, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+genetic+and+clinical+heterogeneity+in+epithelial+ovarian+cancers.">Modelling genetic and clinical heterogeneity in epithelial ovarian cancers.</a> Carcinogenesis. 32, 1540-1549 (2011).</li><li>Grun, B., Benjamin, E., Sinclair, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+cell+biology+models+of+ovarian+and+endometrial+cancer.">Three-dimensional in vitro cell biology models of ovarian and endometrial cancer.</a> Cell Prolif. 42, 219-228 (2009).</li><li>Zietarska, M., Maugard, C. M., Filali-Mouhim, A., Alam-Fahmy, M., Tonin, P. N., Provencher, D. M., Mes-Masson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+description+of+a+3D+in+vitro+model+for+the+study+of+epithelial+ovarian+cancer+(EOC.">Molecular description of a 3D in vitro model for the study of epithelial ovarian cancer (EOC.</a> Mol. Carcinog. 46, 872-885 (2007).</li><li>Shield, K., Ackland, M. L., Ahmed, N., Rice, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+spheroids+in+ovarian+cancer+metastases:+Biology+and+pathology.">Multicellular spheroids in ovarian cancer metastases: Biology and pathology.</a> Gynecol Oncol. 113, 143-148 (2009).</li><li>Dafou, D., Grun, B., Sinclair, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcell-mediated+chromosome+transfer+identifies+EPB41L3+as+a+functional+suppressor+of+epithelial+ovarian+cancers.">Microcell-mediated chromosome transfer identifies EPB41L3 as a functional suppressor of epithelial ovarian cancers.</a> Neoplasia. 12, 579-589 (2010).</li><li>Levanon, K., Crum, C., Drapkin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+pathogenesis+of+serous+ovarian+cancer+and+its+clinical+impact.">New insights into the pathogenesis of serous ovarian cancer and its clinical impact.</a> J. Clin. Oncol. 26, 5284-5293 (2008).</li><li>Kurman, R. J., Shih, I. e. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathogenesis+and+extraovarian+origin+of+epithelial+ovarian+cancer--shifting+the+paradigm.">Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer--shifting the paradigm.</a> Hum. Pathol. 42, 918-931 (2011).</li></ol>

Biologi tidlige fase epitelial kræft i æggestokkene (EOC) er svær at forstå. Måske en af ​​de væsentligste hindringer på dette område i mange år har været den manglende forståelse af vævsspecifikke oprindelse af sygdommen, og om betydningen af ​​den rolle, mikromiljøet i EOC udvikling. I de senere år er der blevet klart, at EOC er en heterogen sygdom med flere distinkte histophathological subtyper, formentlig med forskellige cellulære oprindelse til forskellige undertyper. For eksempel viser de ak...

<table border="1"><tbody><tr><td> Reagens </td><td> Leverandør </td><td> Katalognummer </td></tr><tr><td> PolyHEMA, egnet til cellekultur </td><td> Sigma Aldrich </td><td> P3932 </td></tr><tr><td> Molekylærbiologisk kvalitet ethanol </td><td> Sigma Aldrich </td><td> E7023 </td></tr><tr><td> Sterilt vand til cellekultur </td><td> VWR </td><td> 12001-356 </td></tr><tr><td> MCDB105 </td><td> Sigma Aldrich </td><td> M6395 </td></tr><tr><td> Medium 199 </td><td> Sigma Aldrich </td><td> M2154 </td></tr><tr><td> Hyclone Føtalt bovint serum </td><td> Thermo Scientific </td><td> SH30088.03 </td></tr><tr> &lt;td&gt; Gentamicin </td><td> Sigma Aldrich </td><td> G1397 </td></tr><tr><td> Amphotericin B </td><td> Sigma Aldrich </td><td> A2942 </td></tr><tr><td> pBABE-hygro-hTERT </td><td> Addgene </td><td> 1773 </td></tr><tr><td> PBS </td><td> VWR </td><td> 12001-766 </td></tr><tr><td> 0,25% trypsin-EDTA </td><td> Invitrogen </td><td> 25200-072 </td></tr><tr><td> Cell si (40 eller 70 um) </td><td> VWR </td><td> 21008-949 21008-952 </td></tr><tr><td> Anti-fibroblast overfladeprotein antistof, klon 1B10 </td><td> Sigma </td><td> F4771 </td></tr><tr><td> Anti-pan-cytokeratin antistof (C11) </td><td> Santa Cruz </td><td> sc-8018 </td></tr><tr><td> Polybren (hexadimethrine bromid) </td><td> Sigma </td><td> 107.689 </td></tr><tr><td> TeloTAGGG Telomerase PCR ELISA PLUS </td><td> Roche </td><td> 12013789001 </td></tr><tr><td> TeloTAGGG telomer længde Assay </td><td> Roche </td><td> 12209136001 </td></tr></tbody></table> Tabel 1. Reagenser og udstyr som omhandlet i denne undersøgelse.

heterotypic three-dimensional in vitro modeling of stromal-epithelial interactions during ovarian cancer initiation and progression

Epitelial kræft i æggestokkene (EOCs) er den hyppigste dødsårsag fra gynækologisk malignitet i de vestlige samfund. Trods fremskridt inden kirurgiske behandlinger og forbedrede platinbaserede kemoterapier, har der været en lille forbedring i EOC overlevelsesrater i mere end fire årtier 1,2. Mens fase I tumorer har 5-års overlevelsesraterne&gt; 85%, overlevelseschancerne for stadium III / IV sygdom er &lt;40%. Således kunne den høje dødelighed for EOC være signifikant nedsat, hvis tumorer blev opdaget ved tidligere, mere behandlelige, trin 3-5. På nuværende tidspunkt er den molekylære genetiske og biologiske grundlag for sygdommens tidlige stadium udvikling dårligt forstået. Mere specifikt er lidt om den rolle, mikromiljø under tumor initiering, men kendte risikofaktorer for EOCs (fx alder og paritet) tyder på, at mikromiljø spiller en central rolle i den tidlige tilblivelse EOCs. Vi har derfor udviklet tre-dimensionelle heterotypiske modelleraf både normalt ovarie og i early stage ovariecancere. For normalt ovarie, vi co-dyrket normalt ovarialt epitel overflade (IOSE) og normal stromal fibroblast (INOF) celler, immortaliseret ved retrovrial transduktion af den katalytiske underenhed af human telomerase holoenzymet (hTERT) for at forlænge levetiden af disse celler i kultur. At modellere de tidligste stadier af ovarie epitelcelle transformation, overekspression af CMYC onkogenet i IOSE celler, igen dyrket sammen med INOF celler. Disse heterotypiske modeller blev anvendt til at undersøge virkningerne af aldring og alderdom på transformation og invasion af epitelceller. Her beskriver vi de metodiske trin i udviklingen af ​​disse tre-dimensionelle model, og disse metoder er ikke specifikke for udviklingen af ​​normal æggestok og kræft i æggestokkene væv, og kan bruges til at studere andre vævstyper hvor stromale og epitel celleinteraktioner er en fundamental aspekt af vævet vedligeholdelse og diskadedyr eller sygdomme i udvikling.

Watch this Scientific Journal Video about Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression at JoVE.com

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Vi beskriver metoder til oprettelse af<em&gt; In vitro</em&gt; Heterotypiske tredimensionelle modeller omfatter æggestokkene fibroblaster og normalt ovarialt overflade eller ovariecancer epitelceller. Vi diskuterer brugen af ​​disse modeller til at studere stromal-epiteliale samspil, der opstår i løbet af kræft i æggestokkene udvikling.

Heterotypisk tredimensionale<em&gt; In Vitro</em&gt; Modellering af Stromal-Epiteliale Interactions Under Kræft i æggestokkene Indledning og Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

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