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 forskningen ble utført ved Keck School of Medicine, University of California, USA, og University College London, UK. KL er finansiert av National Institute of Health tilskudd 5 U19 CA148112-02. BG ble finansiert av et prosjekt stipend fra Eve Appeal gynekologisk onkologi veldedighet (UK). Noe av dette arbeidet utføres på UCLH / UCL ble delvis finansiering fra Department of Health NIHR Biomedical Research Centre støtteordning.

Figur 1 viser en oversikt over arbeidsflyten beskrevet nedenfor. 1. Isolering av Normal Ovarian Fibroblaster og utvidelse av in vitro Levetid ved Overuttrykte den katalytiske subenheten av hTERT Holoenzyme <ol><li> Eggstokkene vev kan samles med informert pasientens samtykke og godkjennelse av Institutional Review Board (for amerikanske institusjoner). Normal eggstokkene vev kan samles etter total abdominal hysterektomi eller total laparoskopisk hysterekt...

<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 tidlig stadium ovarialcancer (EOC) er dårlig forstått. Kanskje en av de viktigste hindringer i dette feltet i mange år har vært mangel på forståelse av vev konkrete opprinnelsen til sykdommen, og av betydningen av den rollen mikromiljøet i EOC utvikling. I løpet av de siste årene, har er blitt klart at EOC er en heterogen sykdom med flere forskjellige histophathological undergrupper, sannsynligvis med ulike cellulære opprinnelse for ulike undergrupper. For eksempel, dagens data tyder på at høy grad s...

<table border="1"><tbody><tr><td> Reagens </td><td> Leverandør </td><td> Katalognummer </td></tr><tr><td> PolyHEMA, egnet for cellekultur </td><td> Sigma Aldrich </td><td> P3932 </td></tr><tr><td> Molecular Biology Grade etanol </td><td> Sigma Aldrich </td><td> E7023 </td></tr><tr><td> Sterilt vann 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 Fetal bovin 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> Amfotericin 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> Celle silen (40 eller 70 uM) </td><td> VWR </td><td> 21008-949 21008-952 </td></tr><tr><td> Anti-fibroblast overflate protein antistoff, klone 1B10 </td><td> Sigma </td><td> F4771 </td></tr><tr><td> Anti-pan-cytokeratin antistoff (C11) </td><td> Santa Cruz </td><td> sc-8018 </td></tr><tr><td> Polybren (hexadimethrine bromide) </td><td> Sigma </td><td> 107689 </td></tr><tr><td> TeloTAGGG Telomerase PCR ELISA PLUS </td><td> Roche </td><td> 12013789001 </td></tr><tr><td> TeloTAGGG Telomere lengde Assay </td><td> Roche </td><td> 12209136001 </td></tr></tbody></table> Tabell 1. Reagenser og utstyr som omtales i denne studien.

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

Epiteliale eggstokkreft (EOCs) er den ledende dødsårsaken fra gynekologisk kreft i vestlige samfunn. Til tross for fremskritt innen kirurgiske behandlinger og forbedrede platina-baserte chemotherapies, har det vært lite forbedring i EOC overlevelse for mer enn fire tiår 1,2. Mens iscenesette jeg svulster har 5-års overlevelse&gt; 85%, overlevelse for stadium III / IV sykdom er &lt;40%. Dermed kunne de høye dødsrater for EOC bli vesentlig redusert hvis svulster ble oppdaget tidligere, mer treatable, scener 3-5. I dag er det molekylære genetiske og biologiske grunnlaget for tidlig stadium sykdommen utvikling dårlig forstått. Mer spesifikt, er lite kjent om rollen mikromiljøet under startfasen, men kjente risikofaktorer for EOCs (f.eks alder og paritet) tyder på at mikromiljøet spiller en nøkkelrolle i den tidlige dannelsen av EOCs. Vi har derfor utviklet tre-dimensjonale heterotypic modellerav både normale eggstokk og av tidlig fase eggstokkreft. For normal eggstokk, vi co-kultivert normal ovarian overflate epithelial (IOSE) og normal stromal fibroblast (INOF) celler, udødeliggjort av retrovrial transduksjon av den katalytiske subenheten av menneskelig telomerase holoenzyme (hTERT) å forlenge levetiden på disse cellene i kultur. Å modellere de tidligste stadier av eggstokkreft epitelcelleopprinnelse transformasjon, overuttrykte den CMYC onkogen i IOSE celler, igjen co-dyrket med INOF celler. Disse heterotypic modeller ble brukt for å undersøke effektene av aldring og senescence på transformasjon og invasjon av epitelceller. Her beskriver vi de metodiske trinnene i utviklingen av disse tre-dimensjonal modell, og disse metodene er ikke spesifikke for utvikling av normal eggstokk og eggstokkreft vev, og kan brukes til å studere andre vevstyper der stromale og epiteliale celle interaksjoner er et grunnleggende aspekt av vevet vedlikehold og diSease utvikling.

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 for å etablere<em&gt; In vitro</em&gt; Heterotypic tredimensjonale modeller som omfatter eggstokkene fibroblaster og normal ovarial overflate eller eggstokkreft epitelceller. Vi diskuterer bruken av disse modellene for å studere stromal-epiteliale interaksjoner som oppstår under eggstokkreft utvikling.

Heterotypic Tredimensjonal<em&gt; In Vitro</em&gt; Modellering av stromal-Epiteliale interaksjoner Ovarian Cancer initiering og progresjon

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

Research

JoVE Journal

Medicine

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

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

Mame Modeller for 4D Live-celle Imaging av tumor: mikromiljøet interaksjoner som Impact Ondartet Progresjon

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

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

In vitro mesothelial Clearance Assay at modeller av de tidlige trinnene på eggstokkreft Metastase

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Bioluminescent orthotopic Modell av bukspyttkjertelkreft Progresjon

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

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

En Tredimensjonal Tissue Culture modell for å studere Primær Menneskelig Bone Marrow og dets Malignancies

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

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

Tredimensjonal kulturer: Et verktøy for å studere Normal acinar Arkitektur vs malign transformasjon av brystkreft celler

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

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

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

Kvantifisering av Breast Cancer Cell Invasivitet Ved hjelp av en tredimensjonal (3D) modell

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

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

In vitro berikelse for eggstokkreft Tumor-initiere Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Tredimensjonale (3D) Tumor Spheroid invasjonen analysen

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Modellere Tidlig Steps for eggstokkreft Formidling i en organotypiske Culture of Human bukhulen

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

Modeling Chemotherapy Resistant Leukemia In Vitro

Modeling Chemotherapy Resistant Leukemia In Vitro

Modellering Kjemoterapi Resistant Leukemi<em&gt; In Vitro</em

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...