Das Labor der AG wird vom spanischen Rat für nukleare Sicherheit finanziert. T.A. und A.G. sind Mitglieder einer von der Generalitat de Catalunya (2017-SGR-503) anerkannten Forschungsgruppe. MT hat einen Vertrag, der von der Wissenschaftlichen Stiftung Asociacion Espaéola Contra el Céncer [AECC-INVES19022TERR] finanziert wird. Der G.F.-Vertrag wird durch einen Zuschuss der Cellex Foundation finanziert.

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Stampfer, M. R., Yaswen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+models+of+human+mammary+epithelial+cell+transformation.">Culture models of human mammary epithelial cell transformation.</a> Journal of Mammary Gland Biology and Neoplasia. 5 (4), 365-378 (2000).</li><li>Schinzel, A. C., Hahn, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+transformation+and+experimental+models+of+human+cancer.">Oncogenic transformation and experimental models of human cancer.</a> Frontiers in Bioscience : A Journal and Virtual Library. 13 (13), 71 (2008).</li><li>Balani, S., Nguyen, L. V., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+process+of+human+tumorigenesis.">Modeling the process of human tumorigenesis.</a> Nature Communications. 8 (1), 15422 (2017).</li><li>Bray, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cancer+statistics+2018:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA: A Cancer Journal for Clinicians. 68 (6), 394-424 (2018).</li><li>Ince, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+different+human+breast+epithelial+cell+types+leads+to+distinct+tumor+phenotypes.">Transformation of different human breast epithelial cell types leads to distinct tumor phenotypes.</a> Cancer Cell. 12 (2), 160-170 (2007).</li><li>Repull&#233;s, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+malignant+transformation+of+preneoplastic+and+normal+breast+primary+epithelial+cells.">Radiation-induced malignant transformation of preneoplastic and normal breast primary epithelial cells.</a> Molecular Cancer Research. , 1-13 (2019).</li><li>Weigelt, B., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+microenvironmental+influences+on+the+normal+mammary+gland+and+breast+cancer.">Unraveling the microenvironmental influences on the normal mammary gland and breast cancer.</a> Seminars in Cancer Biology. 18 (5), 311-321 (2008).</li><li>Paoli, P., Giannoni, E., Chiarugi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoikis+molecular+pathways+and+its+role+in+cancer+progression.">Anoikis molecular pathways and its role in cancer progression.</a> Biochimica et Biophysica Acta. 1833 (12), 3481-3498 (2013).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+oncogenesis+of+MCF-10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures.">Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> Methods. 30 (3), 256-268 (2003).</li><li>Borowicz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+soft+agar+colony+formation+assay.">The soft agar colony formation assay.</a> Journal of Visualized Experiments. (92), (2014).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Brocher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BioVoxxel+Image+Processing+and+Analysis+Toolbox.">The BioVoxxel Image Processing and Analysis Toolbox.</a> European BioImage Analysis Symposium. 8 (2), 67112 (2015).</li><li>Torquato, S., Truskett, T. M., Debenedetti, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+random+close+packing+of+spheres+well+defined.">Is random close packing of spheres well defined.</a> Physical Review Letters. 84 (10), 2064-2067 (2000).</li><li>LaBarge, M. A., Garbe, J. C., Stampfer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+human+reduction+mammoplasty+and+mastectomy+tissues+for+cell+culture.">Processing of human reduction mammoplasty and mastectomy tissues for cell culture.</a> Journal of Visualized Experiments. (71), (2013).</li><li>Zubeldia-Plazaola, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoids+promote+transition+of+ductal+carcinoma+in+situ+to+invasive+ductal+carcinoma+by+inducing+myoepithelial+cell+apoptosis.">Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis.</a> Breast Cancer Research. 20 (1), 65 (2018).</li></ol>

Die Autoren haben nichts zu verraten.

Die in diesem Papier beschriebenen experimentellen Protokolle bieten nützliche Instrumente zur Bewertung der onkogenen Transformation von In-vitro-kulturierten Zellen. Jede Technik bewertet bestimmte Aspekte des Transformationsprozesses, und daher muss bei der Schlussfolgerungausarbeit aus einer einzigen Analyse besondere Aufmerksamkeit geschenkt werden. Wachstumskurven-Aufbau ist ein Ansatz, der Informationen erfordert, die bereits verfügbar sind, wenn Zellen für andere Zwecke kultiviert werden. Das macht diese Techn...

Für die Entwicklung von Neoplasma sind mehrere aufeinanderfolgende Ereignisse erforderlich. Im Jahr 2011 beschrieben Hanahan und Weinberg 10 Fähigkeiten, die das Wachstum, überleben und die Verbreitung transformierter Zellen ermöglichen: die sogenannten "Hallmarks of Cancer"1. Die hier beschriebene Methodik stellt drei verschiedene Werkzeuge zur Bewertung der in vitro zellulären Transformation zusammen, indem sie sich auf einige der Besonderheiten der Tumorzellen konzentriert. Diese Techniken bewerten die Zellproliferationsrate, das Verhalten von Zellen, wenn sie in 3D kultiviert werden, und ihre Fähigkeit, Kolonien mit Ankerunabhängigkeit zu bilden.
Zellmodelle sind entscheidend, um Hypothesen in vitro zu testen. Verschiedene Ansätze wurden entwickelt, um experimentelle Modelle der zellulären Transformation für die Untersuchung von Krebs zu generieren2,3,4. Da Brustkrebs weltweit die häufigste Krebsart bei Frauen ist und für etwa 15 % der Krebstoten bei Frauen5verantwortlich ist, ist die Bereitstellung geeigneter Zellmodelle von Brustepithelzellen von größter Bedeutung für die weitere Untersuchung. In diesem Artikel haben wir das Potenzial von drei Techniken zur Bewertung der zellulären Transformation anhand eines experimentellen Modells der Transformation von Breast Primary Epithelial Cells (BPECs) veranschaulicht, die ursprünglich von Ince und Kollegen im Jahr 20076 beschrieben und später in unserem Labor implementiert wurden7. Dieses experimentelle Modell basiert auf der sequenziellen Veränderung von drei gezielten Genen (SV40 Large T und small t Antigene hierin, die als Ttag, hTERTund HRASbezeichnet werden) in das Genom von nicht transformierten BPECs. Darüber hinaus begünstigt die Methode, die für die Ableitung von BPECs verwendet wird, die Aufrechterhaltung von Brustepithelzellen mit luminalen oder myoepithelialen Markern, was zu einer heterogenen Zellkultur führt, die einige der physiologischen Merkmale der Brustdrüse beibehält.
In der Brustdrüse befinden sich luminale Brustepithelzellen, die für die Milchproduktion verantwortlich sind, in der Nähe des Lumens, während Myoepithelzellen um Leuchtkörperzellen angeordnet sind und sich um Kontraktionsbewegungen kümmern, die die Milch zur Brustwarze führen. Der Verlust der richtigen Organisation zwischen diesen Zelllinien ist ein Merkmal der tumoralen Transformation8, das in vitro nach immunfluoreszierendem Nachweis von markanten Linienmarkern in 3D-Zellkulturen beurteilt werden kann. Ein weiteres wesentliches Merkmal von Tumorzellen ist ihre Fähigkeit, ohne Bindung an andere Zellen und an die extrazelluläre Matrix1zu wachsen. Wenn gesunde Zellen gezwungen werden, in Suspension zu wachsen, werden Mechanismen wie anoikis - u2012 eine Art von Zelltod induziert als Reaktion auf die Ablösung von der extrazellulären Matrix aktiviert. Die Umgehung des Zelltodes ist eines der kennzeichens Merkmale von Krebs und somit sind transformierte Zellen in der Lage, Anoikis zu inaktivieren und ankerunabhängig zu überleben. Diese Kapazität kann in vitro mit dem anchorage-unabhängigen Assay mit weichem Agar ausgewertet werden. Darüber hinaus ist ein inhärentes Merkmal von Tumorgeweben ihre anhaltende proliferative Signalfähigkeit, die leicht unter In-vitro-Bedingungen überwacht werden kann, indem der Anstieg der Zellzahl entlang der Zeit gemessen wird, nicht nur in Suspensionstests, sondern auch durch die Überwachung der Wachstumsrate von monolayer-anhaftenden Kulturen.
Trotz des besten Modells, um tumorigenic Potenzial zu testen ist die Impfung von Tumorzellen in murinen Modellen und Bewertung der Tumorentwicklung in situ, ist es wichtig, die Anzahl der Tiere in experimentellen Verfahren so weit wie möglich verwendet zu minimieren. Daher hat die Bewertung der Transformation in vitro geeignete Tests oberste Priorität. Hier stellen wir eine Reihe von Werkzeugen zur Verfügung, um das tumorgene Potenzial von teilweise und vollständig transformierten Brustepithelzellen zu bewerten, die leicht in den meisten Laboratorien implementiert werden können, die mit zellulären Transformationsmodellen arbeiten.

<table>
	<tbody>
		<tr>
			<td>1 ml Serological Pipettes</td>
			<td>Labclinics</td>
			<td>PLC91001</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorfs</td>
			<td>Thermo Fisher Scientific</td>
			<td>3451</td>
			<td>Dark eppendorfs are preferred for MTT long-term storage</td>
		</tr>
		<tr>
			<td>10 &#956;l Pipette tips w/o filter</td>
			<td>Biologix</td>
			<td>20-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>100 ml glass bottle</td>
			<td></td>
			<td></td>
			<td>With cap, autoclavable</td>
		</tr>
		<tr>
			<td>1000 &#956;l Pipette tips w/ filter</td>
			<td>Labclinics</td>
			<td>LAB1000ULFNL</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;l Pipette tips w/o filter</td>
			<td>Biologix</td>
			<td>20-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Conical tubes</td>
			<td>VWR</td>
			<td>525-0400</td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml Serological Pipettes</td>
			<td>Labclinics</td>
			<td>PLC91002</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;l Pipette tips w/ filter</td>
			<td>Labclinics</td>
			<td>FTR200-96</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml Serological Pipettes</td>
			<td>Labclinics</td>
			<td>PLC91005</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Conical Tubes</td>
			<td>VWR</td>
			<td>525-0304</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>PanReac AppliChem</td>
			<td>211007</td>
			<td>Used for 3D structure fixation prior to immunofluorescent labelling</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A1296</td>
			<td>Used for anchorage assay</td>
		</tr>
		<tr>
			<td>Anti-Claudin 4 antibody</td>
			<td>Abcam</td>
			<td>15104, RRID:AB_301650</td>
			<td>Working dilution 1:100, host: rabbit</td>
		</tr>
		<tr>
			<td>Anti-Cytokeratin 14 [RCK107] antibody</td>
			<td>Abcam</td>
			<td>9220, RRID:AB_307087</td>
			<td>Working dilution 1:100, host: mouse</td>
		</tr>
		<tr>
			<td>Anti-mouse Cyanine Cy3 antibody</td>
			<td>Jackson ImmunoResearch Inc.</td>
			<td>115-165-146, RRID:AB_2338690</td>
			<td>Working dilution 1:500, host: goat</td>
		</tr>
		<tr>
			<td>Anti-rabbit Alexa Fluor 488 antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11034, RRID:AB_2576217</td>
			<td>Working dilution 1:500, host: goat</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioVoxxel Toolbox</td>
			<td></td>
			<td>RRID:SCR_015825</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture 24-well Plate</td>
			<td>Labclinics</td>
			<td>PLC30024</td>
			<td>Used for 3D cultures in Matrigel. Flat Bottom</td>
		</tr>
		<tr>
			<td>Cell culture 6-well Plate</td>
			<td>Labclinics</td>
			<td>PLC30006</td>
			<td>Used for anchorage assay</td>
		</tr>
		<tr>
			<td>Cell incubator (37 &#186;C and 5 % CO2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers</td>
			<td>Fisherbrand</td>
			<td>11587522</td>
			<td>Mesh size: 40 &#956;m</td>
		</tr>
		<tr>
			<td>CellSense software</td>
			<td>Olympus</td>
			<td></td>
			<td>Used to image acquisition</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin from Vibrio cholerae</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td>Used to supplement cell culture medium</td>
		</tr>
		<tr>
			<td>Class II Biological Safety Cabinet</td>
			<td>Herasafe</td>
			<td>HAEREUS HS12</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal inverted Microscope</td>
			<td>Leica</td>
			<td>TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glasses</td>
			<td>Witeg Labortechnik GmbH</td>
			<td>4600122</td>
			<td>22 X 22 mm, thickness 0.13 - 0.17 mm</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td>2-(4-amidinophenyl)-1H -indole-6-carboxamidine</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>S1810</td>
			<td>Used to inactivate trypsine action</td>
		</tr>
		<tr>
			<td>Fiji software (ImageJ)</td>
			<td>National Institutes of Health</td>
			<td>RRID:SCR_002285</td>
			<td>Free download, no license needed</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisherbrand</td>
			<td>11844782</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Biowest</td>
			<td>S2000</td>
			<td>Used for immunofluorescence of 3D structures</td>
		</tr>
		<tr>
			<td>Heat-Resistant Gloves</td>
			<td></td>
			<td></td>
			<td>Used for agar manipulation after autoclave</td>
		</tr>
		<tr>
			<td>Heater bath (37 &#186;C)</td>
			<td></td>
			<td></td>
			<td>Used to temper solutions prior to cell subculture</td>
		</tr>
		<tr>
			<td>Heater bath (42 &#186;C)</td>
			<td></td>
			<td></td>
			<td>Used to keep agar warm</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td></td>
			<td></td>
			<td>Used for Matrigel dehydration</td>
		</tr>
		<tr>
			<td>Humid chamber</td>
			<td></td>
			<td></td>
			<td>Used for the incubation of antibodies during immunofluorescence</td>
		</tr>
		<tr>
			<td>Ice</td>
			<td></td>
			<td></td>
			<td>Used during Matrigel manipulation</td>
		</tr>
		<tr>
			<td>Ice-box</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Optic Microscope</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Becton Dickinson</td>
			<td>354234</td>
			<td>Store at -20 &#186;C and keep cold when in use. Referred to as basement membrane matrix</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>PanReac AppliChem</td>
			<td>131091</td>
			<td>Used for 3D structure fixation prior to immunofluorescent labelling</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td></td>
			<td></td>
			<td>p1000, p200 and p10</td>
		</tr>
		<tr>
			<td>Microsoft Office Excel</td>
			<td>Microsoft</td>
			<td>RRID:SCR_016137</td>
			<td>Used to calculate population doubling and to obtain growth rate equation</td>
		</tr>
		<tr>
			<td>MilliQ water</td>
			<td></td>
			<td></td>
			<td>Referred to as ultrapure water</td>
		</tr>
		<tr>
			<td>Nail Polish</td>
			<td></td>
			<td></td>
			<td>Used to seal samples after mounting</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM-999</td>
			<td>Used to cover antibody solution during incubation</td>
		</tr>
		<tr>
			<td>PBS pH 7.4 (w/o calcium &#38; magnesium)</td>
			<td>Gibco</td>
			<td>10010-056</td>
			<td>Sterile. Used for cell subculture</td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>Dilute in milliQ water. No sterility required. Used for immunofluorescence</td>
		</tr>
		<tr>
			<td>Pipette Aid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria T25 flasks</td>
			<td>Corning</td>
			<td>353808</td>
			<td>Used for BPEC culture</td>
		</tr>
		<tr>
			<td>Scepter Automated Cell Counter</td>
			<td>Millipore</td>
			<td>PHCC20060</td>
			<td>Alternatively, use an haemocytometer</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td>Used to cut pipette tips and parafilm</td>
		</tr>
		<tr>
			<td>Sterile filters 0.22 &#956;m</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
			<td>Used to filter MTT solution</td>
		</tr>
		<tr>
			<td>Thiazolyl Blue Tetrazolium Bromide (MTT)</td>
			<td>Sigma-Aldrich</td>
			<td>M2128</td>
			<td>Store at -20 &#186;C</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Used for immunofluorescence of 3D structures</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 10X</td>
			<td>Biowest</td>
			<td>X0930</td>
			<td>Dilute in PBS to obtain 3X solution</td>
		</tr>
		<tr>
			<td>Vectashield Antifade Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>WIT-P-NC Culture Medium</td>
			<td>Stemgent</td>
			<td>00-0051</td>
			<td>Used for primary BPEC culture</td>
		</tr>
		<tr>
			<td>WIT-T Culture Medium</td>
			<td>Stemgent</td>
			<td>00-0047</td>
			<td>Used for transformed BPEC culture</td>
		</tr>
	</tbody>
</table>

in vitro evaluation of oncogenic transformation in human mammary epithelial cells

Tumorigenese ist ein mehrstufiger Prozess, bei dem Zellen Fähigkeiten erwerben, die ihr Wachstum, überleben und verbreiten unter feindlichen Bedingungen ermöglichen. Verschiedene Tests zielen darauf ab, diese Merkmale von Krebszellen zu identifizieren und zu quantifizieren; Sie konzentrieren sich jedoch häufig auf einen einzigen Aspekt der zellulären Transformation, und tatsächlich sind mehrere Tests für ihre korrekte Charakterisierung erforderlich. Ziel dieser Arbeit ist es, Forschern eine Reihe von Instrumenten zur Verfügung zu stellen, um die zelluläre Transformation in vitro aus einer breiten Perspektive zu bewerten und so fundierte Schlussfolgerungen zu ziehen.
Eine nachhaltige proliferative Signalaktivierung ist das Hauptmerkmal von Tumorgeweben und kann leicht unter In-vitro-Bedingungen überwacht werden, indem die Anzahl der im Laufe der Zeit erreichten Bevölkerungsverdoppelungen berechnet wird. Außerdem ermöglicht das Wachstum von Zellen in 3D-Kulturen ihre Interaktion mit umgebenden Zellen, ähnlich dem, was in vivo vorkommt. Dies ermöglicht die Bewertung der zellulären Aggregation und zusammen mit der immunfluoreszenzenten Kennzeichnung von ausgeprägten zellulären Markern, Um Informationen über ein weiteres relevantes Merkmal der Tumortransformation zu erhalten: den Verlust der richtigen Organisation. Ein weiteres bemerkenswertes Merkmal transformierter Zellen ist ihre Fähigkeit, ohne Bindung an andere Zellen und an die extrazelluläre Matrix zu wachsen, die mit dem Anker-Assay ausgewertet werden kann.
Detaillierte experimentelle Verfahren zur Bewertung der Zellwachstumsrate, zur immunfluoreszenzenten Kennzeichnung von Zelllinienmarkern in 3D-Kulturen und zur Erprobung des verankerungsunabhängigen Zellwachstums in weichem Agar werden bereitgestellt. Diese Methoden sind für primäre Epithelzellen (BPEC) der Brust aufgrund ihrer Relevanz bei Brustkrebs optimiert; Nach einigen Anpassungen können jedoch Prozeduren auf andere Zelltypen angewendet werden.

Ein experimentelles Modell der zellulären Transformation mit der Einführung von drei genetischen Elementen in BPECs wurde gewählt, um repräsentative Ergebnisse der onkogenen Transformation zu generieren6,7 ( Abbildung3). Nicht transformierte BPECs (N) wurden aus krankheitsfreiem Brustgewebe abgeleitet, wie von Ince und Kollegen6 beschrieben und gemäß dem hier angegebenen Protoko...

Watch this Scientific Journal Video about In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells at JoVE.com

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Dieses Protokoll bietet experimentelle In-vitro-Werkzeuge zur Bewertung der Transformation menschlicher Brustzellen. Detaillierte Schritte zur Nachverfolgung der Zellproliferationsrate, der verankerungsunabhängigen Wachstumskapazität und der Verteilung von Zelllinien in 3D-Kulturen mit Kellermembranmatrix werden beschrieben.

In Vitro-Bewertung der onkogenen Transformation in menschlichen Mammaepithelzellen

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

in-vitro-evaluation-oncogenic-transformation-human-mammary-epithelial

Research

JoVE Journal

Cancer Research

4.4K Aufrufe.  Universitat Autònoma de Barcelona. Dieses Protokoll bietet experimentelle In-vitro-Werkzeuge zur Bewertung der Transformation menschlicher Brustzellen. Detaillierte Schritte zur Nachverfolgung der Zellproliferationsrate, der verankerungsunabhängigen Wachstumskapazität und der Verteilung von Zelllinien in 3D-Kulturen mit Kellermembranmatrix werden beschrieben.

Serie: In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Sektion Mammary Gland Ganze Mounts für Läsion Identifizierung

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Forschung

Krebsforschung

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Mit CRISPR/Cas9 gen untersuchen die Onkogene Aktivität des mutierten Calreticulin in Cytokine abhängigen hämatopoetischen Zellen bearbeiten

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

In Vitro Tumor Zelle Rechallenge für vorausschauende Bewertung der Chimeric Antigen-Rezeptor-T-Zell-Antitumor-Funktion

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Time-Lapse 2D Imaging der phagozytischen Aktivität in M1 Macrophage-4T1 Maus-Mammary Karzinom-Zellen in Kokulturen

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

Ratte Mammary Epithel Cell Transplantation in das interskapulierische weiße Fettpad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Bewertung der in vivo Antitumoraktivität von Polyanhydrid IL-1α Nanopartikeln

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Brustepithel- und Endothelzell-Sphäroide als potenzielles funktionelles In-vitro-Modell für die Brustkrebsforschung

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Reconstruct Human Retinoblastoma In Vitro

Human RB is pediatric cancer, which is lethal if no treatment is administered. As RB originates from cone precursors, which is relatively rare in rodent models, meanwhile regarding the interspecies differences between humans and rodents, a disease model derived from humans is more beneficial for uncovering the mechanisms of human RB and seeking the targets of therapy. Herein, the protocol describes the generation of two gene-edited hESC lines with a biallelic RB1 point mutation (RB1Mut/Mut) and an RB1 knockout mutation (RB1-/-), respectively. During the process of retinal development, the formation of RB is observed. The RB cell lines are also established by segregating from the RB organoids. Altogether, by differentiating the gene-edited hESC lines into the retinal organoids using a 2D and 3D combined differentiation protocol, we have successfully reconstructed the human RB in a dish and identified its cone-precursor origin. It would provide a helpful disease model for observing the retinoblastoma genesis, proliferation, and growth as well as further developing novel therapeutic agents.

We describe a method for generating human retinoblastoma (RB) by introducing biallelic RB1 mutations in human embryonic stem cells (hESC). RB cell lines could also be successfully cultured using the isolated RB in a dish.

Rekonstruktion des humanen Retinoblastoms in vitro

Human RB is pediatric cancer, which is lethal if no treatment is administered. As RB originates from cone precursors, which is relatively rare in rodent ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Erzeugung und Bildgebung von Epithelorganoiden von Mäusen und Menschen aus normalem und Tumor-Brustgewebe ohne Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play a major role in mammary gland function and are the origin of most mammary tumors. Introducing genes of interest into mouse mammary epithelial cells is a critical step in evaluating gene function in epithelial cells and generating mouse mammary tumor models. This goal can be accomplished through the intraductal injection of a viral vector carrying the genes of interest into the mouse mammary ductal tree. The injected virus subsequently infects mammary epithelial cells, bringing in the genes of interest. The viral vector can be lentiviral, retroviral, adenoviral, or adenovirus-associated viral (AAV). This study demonstrates how a gene of interest is delivered into mammary epithelial cells through mouse mammary intraductal injection of a viral vector. A lentivirus carrying GFP is used to show stable expression of a delivered gene, and a retrovirus carrying Erbb2 (HER2/Neu) is used to demonstrate oncogene-induced atypical hyperplastic lesions and mammary tumors.

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.

Im lebenden Organismus Gentransfer in Brustepithelzellen der Maus durch intraduktale Injektion in der Brust

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play ...