الكتاب الامتنان الدكتور فاسيلي Gurchenkov (معهد كوري) لصورة اكتساب ومعالجة في الشكل 3 و مرفق التصوير PICT-IBISA (معهد كوري). وأيد هذا العمل من قبل ANR-09-JCJC0023-01، ARC SFI20111203863 والموافقة المسبقة عن علم 3D - مجمع في المختبر نماذج الخلوية.

<ol>
	<li>Schiro, J. A., Chan, B. M., 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=Integrin+alpha+2+beta+1+(VLA-2)+mediates+reorganization+and+contraction+of+collagen+matrices+by+human+cells.">Integrin alpha 2 beta 1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells.</a> Cell. 67 (2), 403-410 (1991).</li><li>Klein, C. E., Dressel, D., 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=Integrin+alpha+2+beta+1+is+upregulated+in+fibroblasts+and+highly+aggressive+melanoma+cells+in+three-dimensional+collagen+lattices+and+mediates+the+reorganization+of+collagen+I+fibrils.">Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.</a> J. Cell Biol. 115 (5), 1427-1436 (1991).</li><li>Friedl, P., Maaser, K., Klein, C. E., Niggemann, B., Krohne, G., Z&#228;nker, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+highly+aggressive+MV3+melanoma+cells+in+3-dimensional+collagen+lattices+results+in+local+matrix+reorganization+and+shedding+of+alpha2+and+beta1+integrins+and+CD44.">Migration of highly aggressive MV3 melanoma cells in 3-dimensional collagen lattices results in local matrix reorganization and shedding of alpha2 and beta1 integrins and CD44.</a> Cancer Res. 57 (10), 2061-2070 (1997).</li><li>Wirtz, D., Konstantopoulos, K., Searson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physics+of+cancer:+the+role+of+physical+interactions+and+mechanical+forces+in+metastasis.">The physics of cancer: the role of physical interactions and mechanical forces in metastasis.</a> Nat. Rev. Cancer. 11 (7), 512-522 (2011).</li><li>Zaman, M. H., Trapani, L. M., 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=Migration+of+tumor+cells+in+3D+matrices+is+governed+by+matrix+stiffness+along+with+cell-matrix+adhesion+and+proteolysis.">Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis.</a> Proc. Natl. Acad. Sci. U.S.A. 103 (29), 10889-10894 (2006).</li><li>Doyle, A. D., Wang, F. W., Matsumoto, K., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-dimensional+topography+underlies+three-dimensional+fibrillar+cell+migration.">One-dimensional topography underlies three-dimensional fibrillar cell migration.</a> J. Cell Biol. 184 (4), 481-490 (2009).</li><li>Fraley, S. I., Feng, Y., 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=A+distinctive+role+for+focal+adhesion+proteins+in+three-dimensional+cell+motility.">A distinctive role for focal adhesion proteins in three-dimensional cell motility.</a> Nat. Cell Biol. 12 (6), 598-604 (2010).</li><li>Harunaga, J. S., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-matrix+adhesions+in+3D.">Cell-matrix adhesions in 3D.</a> Matrix Biol. 30 (7-8), 363-368 (2011).</li><li>Raub, C. B., Suresh, V., 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=Noninvasive+assessment+of+collagen+gel+microstructure+and+mechanics+using+multiphoton+microscopy.">Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy.</a> Biophys. J. 92 (6), 2212-2222 (2007).</li><li>Geraldo, S., Simon, A., Elkhatib, N., Louvard, D., Fetler, L., Vignjevic, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+cancer+cells+have+distinct+adhesions+in+3D+collagen+matrices+and+in+vivo?.">Do cancer cells have distinct adhesions in 3D collagen matrices and in vivo?.</a> Eur. J. Cell Biol. 91 (11-12), 930-937 (2012).</li><li>Geraldo, S., Gordon-Weeks, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+dynamics+in+growth-cone+steering.">Cytoskeletal dynamics in growth-cone steering.</a> J. Cell Sci. 122 (Pt 20), 3595-3604 (2009).</li><li>Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+generation+of+homogeneous+multicellular+tumor+spheroids+applicable+to+a+wide+variety+of+cell+types.">Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types.</a> Biotechnol. Bioeng. 83 (2), 173-180 (2003).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hanging+drop+cell+culture+protocol+for+generation+of+3D+spheroids.">A simple hanging drop cell culture protocol for generation of 3D spheroids.</a> J. Vis. Exp. (51), (2011).</li><li>Haji-Karim, M., Carlsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+viability+in+cellular+spheroids+of+human+origin.">Proliferation and viability in cellular spheroids of human origin.</a> Cancer Res. 38 (5), 1457-1464 (1978).</li><li>Eyre, D. R., Paz, M. A., Gallop, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+in+collagen+and+elastin.">Cross-linking in collagen and elastin.</a> Ann. Rev. Biochem. 53, 717-748 (1984).</li><li>Baici, A., Cohen, G., Fehr, K., B&#246;ni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+handy+assay+for+collagenase+using+reconstituted+fluorescein-labeled+collagen+fibrils.">A handy assay for collagenase using reconstituted fluorescein-labeled collagen fibrils.</a> Anal. Biochem. 108 (2), 230-232 (1980).</li><li>Stein, A. M., Vader, D. A., Jawerth, L. M., Weitz, D. A., Sander, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+algorithm+for+extracting+the+network+geometry+of+three-dimensional+collagen+gels.">An algorithm for extracting the network geometry of three-dimensional collagen gels.</a> J. Microsc. 232 (3), 463-475 (2008).</li><li>Sabeh, F., Ota, I., 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=Tumor+cell+traffic+through+the+extracellular+matrix+is+controlled+by+the+membrane-anchored+collagenase+MT1-MMP.">Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP.</a> J. Cell Biol. 167 (4), 769-781 (2004).</li><li>Artym, V. V., Matsumoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+in+three-dimensional+collagen+matrix.">Imaging cells in three-dimensional collagen matrix.</a> Curr. Prot. Cell Biol. Chapter 10, Unit 10.18.1-Unit 10.18.20 (2010).</li><li>Jawerth, L. M., M&#252;nster, S., Vader, D. A., Fabry, B., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+blind+spot+in+confocal+reflection+microscopy:+the+dependence+of+fiber+brightness+on+fiber+orientation+in+imaging+biopolymer+networks.">A blind spot in confocal reflection microscopy: the dependence of fiber brightness on fiber orientation in imaging biopolymer networks.</a> Biophys. J. 98 (3), L1-L3 (2010).</li><li>Cicchi, R., Vogler, N., Kapsokalyvas, D., Dietzek, B., Popp, J., Pavone, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+molecular+structure+to+tissue+architecture:+collagen+organization+probed+by+SHG+microscopy.">From molecular structure to tissue architecture: collagen organization probed by SHG microscopy.</a> J. Biophotonics. 6 (2), 129-142 (2013).</li></ol>

يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

بروتوكول الموصوفة هنا لتسمية fluorescently الكولاجين أنا باستخدام TAMRA يوفر وسيلة ممتازة للسماح التصور السهل للمنظمة شبكة الكولاجين، وذلك باستخدام المجهر متحد البؤر القياسية مجهزة ليزر نانومتر 561. ميزة هذه التقنية مقارنة مع الانعكاس متحد البؤر المجهري هو القدرة على صورة أ...

مجال الهجرة الخلية وقد تحدوا في العلامة التجارية الجديدة في العالم الثالث الأبعاد. فمن بديهية لدراسة الهجرة الخلية في بيئة التي تمثل الأكثر تطابقا واحد الفسيولوجية، وبالتالي، ثلاثي الأبعاد (3D). ومع ذلك، بسبب القيود التقنية، أجريت دراسات الهجرة الأكثر خلية تحليل حركة الخلية عبر ثنائية البعد (2D) ركائز صلبة، إما غير المعالجة أو المغلفة مع البروتينات المصفوفة خارج الخلية المناسب (ECM). الدراسات الأولى مخصصة لهجرة الخلية في ثلاث السياج الكولاجين الأبعاد أعود أكثر من 20 عاما 1-3. ومع ذلك، فقط على مدى السنوات ال 5 الماضية أصبح من الواضح أن الخلايا المهاجرة يمكن أن تختلف إلى حد كبير في التشكل، ووضع الهجرة اعتمادا على أبعاد من الركيزة. في 2D، وخلايا الاتصال فقط الركيزة مع سطحها البطني باستخدام الالتصاقات البؤرية، مما أدى إلى تشكيل نتوءات مسطحة عريضة (أقدام صفاحية) معجزءا لا يتجزأ من الإصبع تشبه نتوءات (أرجل كاذبة خيطية) في قدرتها الرائدة. هذه الهياكل، جنبا إلى جنب مع الألياف الإجهاد التي تربط الجبهة الخلية إلى الحافة، ويعتقد أن تكون حاسمة بالنسبة لحركة الخلية في 2D. في المصفوفات 3D، والخلايا عادة ما تكون أكثر ممدود، مع سطح الخلية بأكملها الاتصال ECM، مما تسبب تغييرات كبيرة في تشكيل وصلة وظيفية لكثير من هذه الهياكل. وعلى العكس، ملامح الخلوية الأخرى تكتسب أهمية في الهجرة 3D، مثل تشوه والهياكل النووية تشارك في إعادة عرض ECM 4. على الرغم من هذه التغيرات المورفولوجية المعروفة، فضلا عن الاختلافات في طرق الهجرة 5-7، والتي يمكن أن تختلف تبعا لECM وأنواع الخلايا، التحليل البنيوي والوظيفي للخلايا جزءا لا يتجزأ من داخل المصفوفات 3D لا يزال غير عادية. العمل مع سميكة وكثيفة المصفوفات 3D يحمل الصعوبات الفنية، مثل التصوير المجهري عالية الدقة، وعدم التوافق مع معظم ستابروتوكولات ndard الأمثل للثقافات 2D، مثل وضع العلامات مناعي من البروتينات الذاتية. أيضا، لأن استخدام المصفوفات 3D هو نهج جديد نسبيا، والباحثين مازالوا يحققون أفضل الظروف لتشبه محددة في حالات الجسم الحي، مثل الهندسة المعمارية انسجة طبيعية من الأنسجة المختلفة أو أجهزة المنظمة ECM حول الورم. تناقضات في النتائج من قبل مجموعات مختلفة فيما يتعلق، على سبيل المثال، قد ولدت وسائط الخلايا السرطانية من الهجرة أو وجود الالتصاقات البؤرية، بعض الجدل 8. وهناك الكثير من الجهد فقد تم تخصيص مؤخرا للتوصل إلى توافق في الآراء من حيث ECM الكيميائية طبيعة وحجم المسام، وسمك الألياف، وتصلب المصفوفة. وتستخدم حاليا العديد من أنواع مختلفة من موبينيل 3D، تتفاوت من خلية إلى مصفوفات المستمدة matrigel المتاحة تجاريا، والكولاجين البقري pepsinized أنا، أو nonpepsinized ذيل فأر الكولاجين I. كل من هذه المصفوفات له خصائص فيزيائية وكيميائية محددة، ويحتاج المرء إلى العالقاتالشركة المصرية للاتصالات مصفوفة من خيار لعملية الفسيولوجية التي تجري دراستها. وبالإضافة إلى ذلك، يمكن أن حجم المسام وسماكة الألياف تعتمد على ظروف البلمرة، مثل الرقم الهيدروجيني ودرجة الحرارة 9،10. يمكن الربط إلى والمسافة من ركائز صلبة مثل الزجاج، وأيضا تغيير خصائص المرونة في مصفوفة 10،11. توضح هذه المقالة الطرق لإعداد وتصوير 3D الثقافات الخلايا السرطانية، إما كخلايا مفردة أو الأجسام الشبه الكروية. وقد سبق وصف طرق لجعل الأجسام الشبه الكروية الخلايا السرطانية، وأكثرها شعبية يجري عملية الشنق قطرة أسلوب 12،13 وطريقة لوحة الاغاروز المغلفة 14. باعتبارها الركيزة ECM المناسبة للهجرة الخلايا السرطانية، ويستخدم nonpepsinized الفئران الكولاجين ذيل أنا بلمرة في درجة حرارة الغرفة في 2 ملغ / مل. Nonpepsinized الكولاجين حمض المستخرج أنا من ذيل الفئران يحتفظ كل من N-C ومحطة telopeptides، أجزاء nonhelical للجزيء الكولاجين المسؤولة عن الأم الكولاجين intermolecيشابك جزيئية والاستقرار fibrilar 15. معا، وهذه الظروف تسمح بتشكيل شبكات الكولاجين التي تشبه أوثق تلك التي لوحظت في الجسم الحي 10. للسماح التصور من ألياف الكولاجين، سواء في الثقافات الثابتة والمعيشة، ويتم توفير بروتوكول مفصلة لتسمية fluorescently الكولاجين في المختبر باستخدام 10 5 - (و-6)-carboxytetramethylrhodamine (TAMRA)، succinimidyl استر. وقد تم تكييف هذا البروتوكول من Baici وآخرون. 16،17، حيث يتم استخدام فلوريسئين ثيوسيانات لتسمية جزيئات الكولاجين القابل للذوبان. كما فلوريسئين، TAMRA هو صبغة الفلورسنت الأمينية المتفاعل أن يتفاعل مع nonprotonated الأليفاتية المجموعات الأمينية للبروتينات، مثل مجموعة الأمينية الحرة في N-محطة، والأهم، والمجموعة الأمينية من الجانب lysines. رد الفعل هذا لا يحدث إلا في درجة الحموضة الأساسية، عندما يكون للمجموعة الأمينية ليسين في شكل nonprotonated. بالإضافة إلى TAMRA كونها أكثر استقرارا من خلال فلوريسئينالوقت، أطياف الانبعاثات ليسقط على نطاق برتقالي / أحمر (م ت / م = 555/518 نانومتر)، والتي يمكن دمجها بشكل مفيد لتصوير الخلايا الحية من البروتينات GFP الموسومة. باستخدام الكولاجين للذوبان جزيئات المسمى مع الأصباغ الأمينية التفاعل لا يؤثر على عملية البلمرة ولا كثافة، حجم المسام وحالة يشابك من المصفوفة الكولاجين 10،16،18،19. ويشمل هذا البروتوكول أيضا وسيلة لوضع العلامات مناعي 3D من البروتينات الذاتية، التي تم تحسين أخرى لتسمية الهيكل الخلوي أو البروتينات المرتبطة الهيكل الخلوي. التركيز النهائي من هذا البروتوكول هو على طرق للحصول على الصور عالية الدقة من الثقافات 3D باستخدام المجهر متحد البؤر مع انخفاض مساهمة من coverslips الزجاج جامدة على التوتر مصفوفة الكولاجين.

<table border="0" cellpadding="0" cellspacing="0" width="603">
	<colgroup>
		<col span="2" />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">TAMRA, SE</td>
			<td style="width:195px;">Invitrogen</td>
			<td style="width:136px;">C-1171</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat tail Collagen High Concentration</td>
			<td>BD Biosciences</td>
			<td>354249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat tail Collagen</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P2141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taxol (Paxitel)</td>
			<td>Sigma</td>
			<td>T7402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-alpha Tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488 Phalloidin</td>
			<td>Invitrogen</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 633 Goat anti-Mouse antibody</td>
			<td>Invitrogen</td>
			<td>A21052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting media</td>
			<td>Fisher Scientific</td>
			<td>106 226 89 </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name of Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis Cassette</td>
			<td>Pierce</td>
			<td>66380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottom dishes</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MetaMorph Microscopy Automation &#38; Image Analysis Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris 7.2.3</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

revealing the cytoskeletal organization of invasive cancer cells in 3d

وقد جرت العادة على دراسة الهجرة الخلية في ركائز 2D. ومع ذلك، فقد أصبح من الواضح بشكل متزايد أن هناك حاجة لدراسة الهجرة خلية في بيئات 3D أكثر ملاءمة، التي تشبه بشكل أفضل الأبعاد من العمليات الفسيولوجية في السؤال. الخلايا المهاجرة يمكن أن تختلف إلى حد كبير في التشكل، ووضع الهجرة اعتمادا على ما إذا كانت تتحرك على ركائز 2D أو 3D. ونظرا للصعوبات التقنية وعدم التوافق مع بروتوكولات معظم القياسية، التحليل البنيوي والوظيفي للخلايا جزءا لا يتجزأ من داخل المصفوفات 3D لا يزال من غير المألوف. توضح هذه المقالة الطرق لإعداد وتصوير 3D الثقافات الخلايا السرطانية، إما كخلايا مفردة أو الأجسام الشبه الكروية. باعتبارها الركيزة ECM المناسبة للهجرة الخلايا السرطانية، ونحن نستخدم nonpepsinized الفئران الكولاجين ذيل أنا بلمرة في درجة حرارة الغرفة وfluorescently المسمى لتسهيل التصور باستخدام المجاهر مبائر القياسية. ويشمل هذا العمل أيضا protocرأ لوضع العلامات مناعي 3D من الهيكل الخلوي الخلية الذاتية. استخدام هذه البروتوكولات ونحن نأمل أن تسهم في وصف أفضل من التركيب الجزيئي، التعريب، ومهام الهياكل الخلوية في 3D.

وسم الفئران الذيل الكولاجين أنا مع TAMRA يسمح للإعداد سهلة والتصور شبكات الكولاجين 3D. البلمرة بطيئة في النتائج درجة حرارة الغرفة في تشكيل شبكات الكولاجين مع منظمة مماثلة لتلك التي وجدت في الجسم الحي 10. هنا نقدم بروتوكول ?...

Watch this Scientific Journal Video about Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D at JoVE.com

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

تقدم هذه المقالة طريقة لتسمية fluorescently الكولاجين التي يمكن استخدامها لمزيد من الإصلاح على حد سواء والتصوير الحي من الثقافات الخلية 3D. كما نقوم بتوفير بروتوكول الأمثل لتصور البروتينات هيكل الخلية الذاتية من الخلايا المستزرعة في بيئات 3D.

وكشف عن المنظمة هيكل الخلية من خلايا سرطان الغازية في 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

revealing-the-cytoskeletal-organization-of-invasive-cancer-cells-in-3d

Research

JoVE Journal

Medicine

15.6K Views.  Institut Curie.  تقدم هذه المقالة طريقة لتسمية fluorescently الكولاجين التي يمكن استخدامها لمزيد من الإصلاح على حد سواء والتصوير الحي من الثقافات الخلية 3D. كما نقوم بتوفير بروتوكول الأمثل لتصور البروتينات هيكل الخلية الذاتية من الخلايا المستزرعة في بيئات 3D. 

Video: Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

المؤشرات الحيوية في نموذج حيواني لكشف الاقتران العصبية، الدموية، والسلوكية للPTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

التنميط الجزيئي للالمكروية ورم الغازية في نموذج 3 الأبعاد للخلايا سرطان القولون والمستقيم و<em&gt; خارج الحي</em&gt; الخلايا الليفية

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

تقييم غير الغازية من فعالية العلاجات الجديدة لعلوم الأمراض المعوية عن طريق التصوير بالمنظار المسلسل من الفئران الحية

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

ثلاثي الأبعاد (3D) ورم الجسم الشبه الكروي الغزو الفحص

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

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

فرخ القلب الغزو الفحص لاختبار الغازية من خلايا سرطان وآخر للمركبات يحتمل مكافحة الغازية

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

دراسة سرطان البنكرياس الجذعية خصائص الخلايا لتطوير استراتيجيات علاجية جديدة

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

التغليف حرارة Preadipocytes للزراعة إلى الدهنية مستودعات الأنسجة

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

العزلة ونشر تعميم الخلايا السرطانية من ماوس السرطان نموذج

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

خلية السرطان كروي الفحص لتقييم الغزو في إعداد 3D

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

المناظير مستعرض الأورطي انقباض في الفئران

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...