Name: in vitro evaluation of oncogenic transformation in human mammary epithelial cells
Uploaded: 2020-09-24T14:30:00
Description: Watch this Scientific Journal Video about In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells at JoVE.com

AG研究所は、スペイン原子力安全評議会によって資金提供されています。T.A.とA.G.は、 ジェネラリタット・デ・カタルーニャ (2017-SGR-503)が認めた研究グループのメンバーです。MTは、科学財団 アソシアシオン・エスパニョーラ・コントラ・エル・カンセル [AECC-INVES19022TERR]が資金を提供する契約を締結しています。G.F.契約は、セレックス財団からの助成金によって資金提供されています。

以下の実験で使用したヒト試料は、標準的な手順の同意の下で 、クリニカ・ピラール・サン・ジョルディ (バルセロナ)で行われた還元マンモパラスティから得られた。特に明記されていない限り、すべての手順はクラスII生物学的安全キャビネットで行われます。
1. ヒト乳腺上皮細胞のインビトロ培養と成長曲線プロットの蓄積
<ol><li>
乳癌原発上皮細胞(BP...

<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>

本論文に記載された実験プロトコルは、インビトロ培養細胞の発癌性転換を評価するための有用なツールを提供する。各手法は変換プロセスの特定の側面を評価するため、単一の分析から結論を導き出す際には、特別な注意が必要です。成長曲線の構築は、他の目的のために細胞を培養する際に既に利用可能な情報を要求するアプローチです。これにより、この技術は他の細胞増殖アッセイ?...

新生物の開発には、複数の連続したイベントが必要です。2011年、ハナハンとワインバーグは、形質転換細胞の成長、生存、普及を可能にする10の機能、いわゆる「がんの特徴」1を説明しました。ここで説明する方法論は、腫瘍細胞の特徴的な特徴のいくつかに焦点を当てることによって、インビトロ細胞変換を評価するための3つの異なるツールをまとめます。これらの技術は、細胞増殖速度、3Dで培養した場合の細胞の挙動、およびアンカレッジ独立性を有するコロニーを形成する能力を評価する。
細胞モデルは、インビトロで仮説をテストするために非常に重要です。癌2,3,4の研究のための細胞変換の実験モデルを生成するために、さまざまなアプローチが開発されている。乳癌は、世界中の女性の間で最も一般的な癌であり、女性5の間で癌死亡の約15%を担っているので、乳腺上皮細胞の適切な細胞モデルを提供することは、さらなる調査のために最も重要である。本稿では、2007年6月6日にInceらの研究チームが最初に説明した乳房一次上皮細胞(BFC)変換の実験モデルを用いて細胞変換を評価する3つの技術の可能性を説明した。この実験モデルは、非形質化されたBFCのゲノムに対する3つの標的遺伝子(SV40 Large Tおよび小さなt抗原をTtag、hTERT、HRASと呼ぶ)の順次変化に基づいています。 さらに、BFC誘導に使用される方法は、乳腺上皮細胞を発光または筋上皮マーカーで維持し、乳腺の生理学的形質の一部を保持する異種細胞培養をもたらす。
乳腺では、乳の生産を担う乳腺上皮細胞は内腔の近くに位置し、筋上皮細胞は発光細胞の周りに配置され、乳首にミルクを導く収縮運動の世話をします。これらの細胞系統間の適切な組織の喪失は、3D細胞培養における特徴的な系統マーカーの免疫蛍光検出後にインビトロで評価することができる腫瘍形変換8 の特徴である。腫瘍細胞のもう一つの主要な特徴は、他の細胞および細胞外マトリックス1に付着することなく増殖する能力である。健康な細胞が懸濁液で増殖することを余儀なくされると、 アノイキス \u2012のようなメカニズムは、細胞外マトリックスからの剥離に応答して誘発される細胞死の一種である\u2012が活性化される9。細胞死の回避は癌の特徴的な特徴の一つであり、したがって、形質転換細胞はア ノイキス を不活性化し、アンカーに依存しない方法で生き残ることができる。この容量は、軟寒天を用いたアンカレッジ非依存アッセイを用いてインビトロで評価することができる。さらに、腫瘍組織の固有の特徴は、その持続的な増殖シグナル伝達能力であり、懸濁アッセイだけでなく、単層接着培養物の増殖速度を監視することによって、時間に沿って細胞数の増加を測定することによって、インビトロ条件下で容易に監視することができる。
腫瘍発生の可能性をテストする最良のモデルは、マウスモデルにおける腫瘍細胞の接種およびその領域における腫瘍の発達の評価であるにもかかわらず、実験手順で使用される動物の数を可能な限り最小限に抑えることが重要である。従って、インビトロでの変換を評価するための適切な試験を有することが最優先事項である。ここでは、細胞変換モデルに作用するほとんどの研究室で容易に実施できる部分的かつ完全に形質転換された乳房上皮細胞の腫瘍発生性を評価するための一連のツールを提供する。

<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

腫瘍形成は、細胞が敵対的な条件下で増殖、生存、および普及を可能にする能力を獲得する多段階のプロセスである。異なるテストは、癌細胞のこれらの特徴を同定し、定量化しようとします。しかし、彼らはしばしば細胞の変換の単一の側面に焦点を当て、実際には、それらの適切な特性評価のために複数のテストが必要です。本研究の目的は、広い視野から細胞の形質転換を評価するための一連のツールを研究者に提供し、それによって健全な結論を導き出すことを可能にすることです。
持続的な増殖シグナル伝達活性化は、腫瘍組織の主要な特徴であり、時間の経過とともに達成される倍増する集団数を計算することによって、インビトロ条件下で容易に監視することができる。また、3D培養における細胞の増殖は、生体内で起こることに似た周囲の細胞との相互作用を可能にする。これにより、細胞凝集の評価と、特徴的な細胞マーカーの免疫蛍光標識と共に、腫瘍変態の別の関連する特徴、すなわち適切な組織の喪失に関する情報を得ることが可能になる。形質転換細胞のもう一つの顕著な特徴は、他の細胞および細胞外マトリックスに付着することなく増殖する能力であり、これは、アンカレッジアッセイで評価することができる。
細胞増殖率を評価する詳細な実験手順、3D培養における細胞系分担マーカーの免疫蛍光標識を行う、および軟寒天におけるアンカレッジ非依存性細胞増殖を試験する実験手順が提供される。これらの方法論は乳癌の関連性のために乳癌原発性上皮細胞(BPEC)のために最適化される;ただし、いくつかの調整後に、プロシージャを他のセルの種類に適用できます。

BFCに3つの遺伝子要素を導入した細胞変換の実験モデルが、発癌性変換6、7の代表的な結果を生成するために選択された(図3)。非形質化されたBFC(N)は、Inceと同僚6によって記載されているように疾患のない乳房組織に由来し、ここで示すプロトコルに従って培養した。STASIS(ストレスまたは異...

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

このプロトコルは、ヒト乳腺細胞の形質転換を評価するための実験的なインビトロツールを提供する。細胞増殖率、アンカレッジ非依存性増殖能、基基膜マトリックスを用いた3D培養における細胞系統の分布をフォローアップするための詳細なステップについて説明する。

ヒト乳腺上皮細胞における発癌性転換のインビトロ評価

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

This methodology uses the integration of different indicators to assess the degree of transformation of any cell model. This technique provides researchers with a set of time-consuming but simple-to-use tools for assessing cell transformation in vitro. This allows the method to be used in most laboratories.Demonstrating the procedure will be Teresa Anglada, a post-doctoral researcher from my laboratory, and Joan Repulles, a microscopy specialist. When the breast primary epithelial cell culture reaches 90%confluency, transfer the supernatant from the culture flask to a 15 milliliter tube containing two milliliters of fetal bovine serum and wash the cells with PBS. Treat the cells with one milliliter of trypsin for five minutes at 37 degrees Celsius.When the cells have detached, inactivate the trypsin with the reserved medium and collect the cells by centrifugation. Flick the tube to resuspend the pellet and add one to two milliliters of fresh medium for counting. After determining the cell concentration, add 300, 000 cells to a new tube and bring the final volume to five milliliters with additional fresh culture medium.Seed cells into a new T25 flask and return the cells to the cell culture incubator. Then use the formula to calculate the number of accumulated population doublings. To assess the proliferation rate of the cells, plot the accumulated number of population doublings for a specific time interval.An increased slope indicates an increased cell proliferation rate. To set up a 3D cell culture, rinse wells with a 1.9 square centimeter surface area with cold sterile PBS and coat the bottom of each well with 100 microliters of basement membrane matrix. Then place the plate in the cell culture incubator for about 20 minutes to allow the matrix to solidify.In the meantime, harvest mammary cells as just demonstrated and dilute the cells to a 400, 000 cells per milliliter of medium concentration. Mix the cells with fresh medium supplemented with 8%basement membrane matrix at a one-to-one ratio and add 500 microliters of the cells to each well of solidified matrix. Incubate the cells at 37 degrees Celsius for a few minutes, then add 500 microliters of medium supplemented with 4%basement membrane matrix to each well.Place the cells in the cell culture incubator for 14 days. The seeded cells will group and proliferate forming acini-like structures. For the immunofluorescent protein detection in the 3D cultured mammary cells, use a cutoff P200 pipette tip to remove the basement membrane matrix from each well and smear approximately 50 microliters of disaggregated matrix from each well onto individual glass slides.When the samples have completely dried, fix the cells with a one-to-one methanol-acetone solution at minus 20 degrees Celsius for 30 minutes. After fixing, block samples with blocking solution for two hours at room temperature. At the end of the incubation, add 30 microliters of the primary antibody working solution of interest and cover the slides with a strip of laboratory wrapping film for an overnight incubation at four degrees Celsius in a humid chamber.The next morning, wash the slides three times with PBS for one hour per wash and label the samples with the appropriate secondary antibody solution as just demonstrated. At the end of the incubation, wash the samples two times in PBS for two hours per wash before counter-staining with DAPI diluted in anti-fade mounting medium and covering each slide with a coverslip. Then analyze the fluorescent signal distribution for each acinus on a confocal microscope.To perform an anchorage-independent assay, first cover the bottom of the appropriate number of wells of a six-well plate with 1.5 milliliters of 0.6%agar in medium solution and let the agar solidify at room temperature. Next, prepare a five times 10 to the fourth cells per milliliter solution as demonstrated and filter the cells through a 40 micron strainer into a sterile 50 milliliter tube. Holding the tube at a 45 degree angle, add an equal volume of 0.6%agar in medium solution down the wall of the tube.And after thorough mixing, layer one milliliter of the cells in agar suspension to each well of the previously prepared six-well plate. Use an inverted microscope to confirm that the cells have been homogeneously dispersed throughout the agar. When the agar has completely solidified, carefully add one milliliter refresh medium to each well without disturbing the agar and incubate the cells for three weeks, changing the medium twice per week.At the end of the colony formation period, replace the medium with one milliliter of one milligram per milliliter MTT per well for a 24-hour incubation in the cell culture incubator. The next day, remove the MTT from each well and acquire images of each well on the inverted microscope. When all of the images have been acquired, open the images in ImageJ and click image, adjust, threshold, and apply to obtain a binary mask through thresholding of the original image.When well-delimited colonies have been obtained, select plugins and biovoxxel to launch the extended particle analyzer to identify the MTT-positive colonies. Over a specific interval of time, partially and fully transformed cells achieve a higher number of population doublings compared to non-transformed cells, indicating that the cell division rate is increased with the transformation process. Over the two-week 3D cell culture period, the cells aggregate and proliferate, increasing the acini size.The proper polarization of each acinus can be accurately assessed using protein immunofluorescent detection in conjunction with three-dimensional signal location by confocal microscopy. While all of the acini formed by non-transformed breast cells are properly organized, a loss of polarization is observed in acini formed by partially and fully transformed cells. After three weeks of embedded agar culture, cells with an anchorage independent growth capacity give rise to colonies composed of multiple cells.Only metabolically active cells are capable of cleaving the tetrazolium MTT ring, resulting in purple MTT formazan crystals after 24 hours. Measurement of the colony diameter by automatic image analysis allows the filtering of low proliferative colonies or individual cells. The number of colonies formed by non-transformed cells is negligible compared to those from partially and fully transformed cells.Colony size can also be used to discriminate between cells with different degrees of transformation. These assays can be used to screen cell lines prior to any inoculation as only those cell lines that have tested positive in vitro should be inoculated into mice.

Research

JoVE Journal

Cancer Research

Sectioning Mammary Gland Whole Mounts for Lesion Identification

病変同定のための乳腺全体マウントの分割

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

がん研究

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

CRISPR/Cas9 を使用して遺伝子変異カルレティキュリン サイトカイン依存性造血細胞での発癌性の活動を調査する編集

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

キメラ抗原受容体の T 細胞の抗腫瘍機能の予測評価のため培養腫瘍細胞の再投与に

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

M1マクロファージ-4T1マウス乳腺癌細胞における食細胞活性のタイムラプス2Dイメージング

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

間肩甲膜白脂肪パッドへのラット乳腺上皮細胞移植

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

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

ポリ無水物IL-1αナノ粒子の 生体内 抗腫瘍活性の評価

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

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

乳がん研究のための潜在的機能 インビトロ モデルとしての乳腺上皮および内皮細胞スフェロイド

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

インビトロでのヒト網膜芽細胞腫の再構築

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

継代なしの正常および腫瘍乳腺組織からのマウスおよびヒト上皮オルガノイドの生成とイメージング

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

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

インビボ 乳腺内注射によるマウス乳腺上皮細胞への遺伝子導入

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 ...