بدعم من المعاهد الوطنية للصحة منح P30 CA051008 وT32 CA009686 (ATR).

1. توليد الأجسام الشبه الكروية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد وحيدة الخلية تعليق لتعليق الثقافات قطرة فصل الثقافات الخلايا السرطانية ملتصقة من ~ 70٪ التقاء باستخدام PBS غسل تليها التعرض إلى 0.05٪ التربسين-EDTA الحل. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحييد الحل التربسين مع وسائل الإعلام ثقافة الخلية وعدد الخلايا باستخدام قسامة لتعليق الخلية. ملاحظة: وسائل الإعلام ثقافة الخلية معينا سوف تعتمد على خط الخلية التي يجري اختبارها. اتبع توصيات سائل الإعلام ATCC، حيث وسائل الإعلام ثقافة الخلية عادة DMEM + 10٪ FBS. ويمكن الاطلاع على مزيد من المعلومات في جدول المواد. </li><li style=";text-align:right;direction:rtl"> إجراء تخفيف للسماح لزرع البذور من 500-1،000 الخلايا في 20 قطرة ميكرولتر من وسائل الإعلام ثقافة الخلية. </li></ol></li><li style=";text-align:right;direction:rtl"> الحصول على صحن 10 سم وإضافة 5 مل من برنامج تلفزيوني العقيمة إلى أسفل. ملاحظة: هذه الخطوة تحمي قطرات شنقا من التبخر. من حيث التكلفة أقل &quot;الصف البكتريولوجية&quot; أطباق يمكن استخدامها في مكان من الأطباق الصف زراعة الخلايا، وكذلك الخلايا لا تابعالتصرف سطح الثقافة. </li><li style=";text-align:right;direction:rtl"> استخدام الماصة متعدد القنوات لنقل 20 قطرات ميكرولتر من تعليق خلية المخفف إلى السطح الداخلي للغطاء. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الماصة 40 قطرات (5 صفوف من 8 قطرات) على غطاء صحن 10 سم. </li></ol></li><li style=";text-align:right;direction:rtl"> عكس الغطاء ومكان على طبق الثقافة. احتضان الثقافات قطرة شنقا في 37 درجة مئوية لمدة 72 ساعة لتوليد الأجسام الشبه الكروية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الوجه الغطاء في اثقة، الطريقة التي تسيطر عليها حتى الان. ملاحظة: للقلب غطاء سريعة جدا أو بطيئة جدا قد يسبب قطرات للتحول. بعض خطوط الخلايا قد شكل الكروية في 48 ساعة أو أقل، في حين أن البعض الآخر قد يتطلب أكثر من 72 ساعة لإنتاج الركام المدمجة. </li></ol></li></ol> 2. تضمين من الأجسام الشبه الكروية في مصفوفة 3D <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ذوبان الجليد قسامة من النمو انخفاض عامل الطابق السفلي المواد غشاء عند 4 درجات مئوية خلال الليل قبل تضمين الكروية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اختياري: آبار طبقة مع ECM مقدما لمنع التفاعل كروي محتمل مع cultu الأنسجةإعادة السطح. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الماصة 200 ميكرولتر من ECM لكل بئر في لوحة 24-جيدا. </li><li style=";text-align:right;direction:rtl"> إمالة لوحة للتأكد من أن ECM يغطي السطح جيدا بأكمله. </li><li style=";text-align:right;direction:rtl"> إزالة بعناية ECM الزائد مع الماصة. </li><li style=";text-align:right;direction:rtl"> احتضان لوحة حتى آبار جافة: 3 ساعة في درجة حرارة الغرفة أو بين عشية وضحاها في 4 درجات مئوية. </li></ol></li></ol></li><li style=";text-align:right;direction:rtl"> جمع الكروية عن طريق إمالة غطاء صحن زراعة الأنسجة وتجمع وسائل الإعلام. نقل وسائل الإعلام مع الكروية في أنبوب microcentrifuge 1.5 مل. </li><li style=";text-align:right;direction:rtl"> سماح 10 دقيقة للالكروية ليستقر على الجزء السفلي من أنبوب microcentrifuge. وينبغي أن يكون الكروية مرئية بالعين. </li><li style=";text-align:right;direction:rtl"> مزيج 100 ميكرولتر من المواد الغشاء القاعدي مع 100 ميكرولتر من البرد (4 ° C) النوع الأول من الكولاجين في أنبوب قبل المبردة منفصل. نضع الخليط في 4 درجات مئوية لمنع أي هلام من ترسيخ قبل الأوان. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام نصائح الماصة قبل المبردة إذا نقل كميات صغيرة. </li><li style=";text-align:right;direction:rtl"> تقديم الرعاية عندماخلط المواد الغشاء القاعدي والكولاجين النوع الأول لمنع تشكيل فقاعة الهواء. ملاحظة: فقاعات الهواء قد تعيق التصوير في وقت لاحق في البروتوكول إذا أصبحت جزءا لا يتجزأ من هلام. </li></ol></li><li style=";text-align:right;direction:rtl"> نضح الكروية من 40 ميكرولتر الجزء السفلي من أنبوب microcentrifuge، ويتحد مع المواد الغشاء القاعدي / النوع الأول الكولاجين الخليط. كن حذرا لمنع تشكيل فقاعة الهواء عند خلط. ملاحظة: محلول يحتوي على 100 ميكرولتر المواد الغشاء القاعدي، 100 نوع ميكرولتر أنا الكولاجين، و 40 ميكرولتر من الكروية سيخلق ما يكفي من المواد لمدة 4 الثقافات 3D مستقلة. يمكن زيادتها هذا أعلى أو لأسفل. </li><li style=";text-align:right;direction:rtl"> الماصة 40 قطرات ميكرولتر من مزيج لزج في مراكز الآبار على طبق من ذهب 24-جيدا. يحافظ على لوحة لمنع خليط من الوقوع في جانب البئر. ملاحظة: A عمود واحد على لوحة 24-جيدا - يوصى لكل حالة لفحصها - 4 آبار. </li><li style=";text-align:right;direction:rtl"> وضع لوحة الباحثالزراعة العضوية 37 ° C الحاضنة وترك دون عائق لمدة 30 دقيقة. إن ثقافة 3D تتبلمر خلال هذه الفترة. </li><li style=";text-align:right;direction:rtl"> يغرق ببطء الثقافات 3D في 1 مل من وسائل الإعلام ثقافة الخلية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تأكد من أن وسائل الإعلام دافئة عند إضافته إلى الآبار لزيادة تعزيز هلام البلمرة. </li><li style=";text-align:right;direction:rtl"> خطوة حاسمة: إضافة بلطف وسائل الإعلام على ثقافات 3D. يمكن pipetting لوسائل الإعلام بسرعة كبيرة جدا في الآبار يؤدي إلى مفرزة من الثقافات 3D من سطح زراعة الأنسجة. </li></ol></li></ol> 3. رصد وتحليل كروي الغزو <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> غزو ​​صورة من الكروية في مصفوفة 3D المحيطة في نقطة زمنية قررت من قبل المحقق. الحصول على الصور باستخدام مجهر مقلوب مع عدسة الهدف 20X. ملاحظة: تختلف مثالية نقاط الوقت اعتمادا على خط الخلية التي يجري اختبارها. وسوف تبدأ خطوط الخلايا أكثر الغازية الخروج بهم من الكروية بعد وقت قصير من الطلاء، وبالتالي تأخذ صورة أوليةالصورة في غضون بضع ساعات بعد الطلاء. عموما، يتم أخذ الصور في 0 ساعة (بعد الطلاء)، 24 ساعة، و 48 ساعة. الغزو يخلص عادة 24 إلى 48 ساعة بعد الطلاء. </li><li style=";text-align:right;direction:rtl"> Quantitate القدرة الغازية باستخدام برمجيات تحليل الصور مثل صورة J. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحليل الغزو والمسافة خلية من على حافة كروي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام أداة رسم مستقيم بمناسبة دائرة نصف قطرها و / أو المسافة الغازية القصوى. </li><li style=";text-align:right;direction:rtl"> انقر على زر &quot;تحليل&quot; في القائمة العلوية، ثم انقر فوق &quot;القياس&quot; لعرض قياس الطول. </li></ol></li><li style=";text-align:right;direction:rtl"> تحليل الغزو كما إجمالي المساحة الغازية خارج كروي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام أداة رسم حر لتتبع حدود كروي و / أو المساحة الإجمالية الغازية. </li><li style=";text-align:right;direction:rtl"> انقر على زر &quot;تحليل&quot; في القائمة العلوية، ثم انقر فوق &quot;القياس&quot; لعرض قياس المنطقة. </li></ol></li><li style=";text-align:right;direction:rtl"> استخدام البرنامج المساعد مثل أدوات إدارة ROI لخلق قياسات كومة من الصور. </li></ol>
	</li>
</ol>

<ol>
	<li>Chambers, A. F., Groom, A. C., MacDonald, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+Dissemination+and+growth+of+cancer+cells+in+metastatic+sites.">Metastasis: Dissemination and growth of cancer cells in metastatic sites.</a> Nat Rev. Cancer. 2 (8), (2002).</li><li>Schmeichel, K. L., 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=Modeling+tissue-specific+signaling+and+organ+function+in+three+dimensions.">Modeling tissue-specific signaling and organ function in three dimensions.</a> J Cell Sci. 116 (12), 2377-2388 (2003).</li><li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue+morphogenesis+and+cancer+in+3D.">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130 (4), (2007).</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>Del Duca, ., Werbowetski, D., T, ., Del Maestro, R., F, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+preparation+from+hanging+drops:+characterization+of+a+model+of+brain+tumor+invasion.">Spheroid preparation from hanging drops: characterization of a model of brain tumor invasion.</a> J Neuro-Oncol. 67 (3), (2004).</li><li>Justus, C. R., Leffler, N., Ruiz-Echevarria, M., Yang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+migration+and+invasion+assays.">In vitro cell migration and invasion assays.</a> J Vis Exp. 88, (2014).</li><li>Ramos-DeSimone, N., Hahn-Dantona, E., Sipley, J., Nagase, H., French, D. L., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+matrix+metalloproteinase-9+(MMP-9)+via+a+converging+plasmin/stromelysin-1+cascade+enhances+tumor+cell+invasion.">Activation of matrix metalloproteinase-9 (MMP-9) via a converging plasmin/stromelysin-1 cascade enhances tumor cell invasion.</a> J Bio Chem. 274 (19), .</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nature Rev. Cancer. 3 (5), 362-374 (2003).</li><li>Kleinman, H. K., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+Basement+membrane+matrix+with+biological+activity.">Matrigel: Basement membrane matrix with biological activity.</a> Semin Cancer Biol. 15 (5), 378-386 (2005).</li><li>Shaw, L. M., Chao, C., Wewer, U. M., Mercurio, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+integrin+&#945;6&#946;1+in+metastatic+breast+carcinoma+cells+assessed+by+expression+of+a+dominant-negative+receptor.">Function of the integrin &#945;6&#946;1 in metastatic breast carcinoma cells assessed by expression of a dominant-negative receptor.</a> Cancer Res. 56 (5), 959-963 (1996).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), (2010).</li><li>Rajan, N., Habermehl, J., Cot&#233;, M. -. F., Doillon, C. J., Mantovani, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+ready-to-use,+storable+and+reconstituted+type+I+collagen+from+rat+tail+tendon+for+tissue+engineering+applications.">Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications.</a> Nat Protoc. 1 (6), (2006).</li><li>Provenzano, P. P., Eliceiri, K. W., Campbell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+reorganization+at+the+tumor-stromal+interface+facilitates+local+invasion.">Collagen reorganization at the tumor-stromal interface facilitates local invasion.</a> BMC Med. 4 (1), (2006).</li><li>Timmins, N. E., 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=Generation+of+multicellular+tumor+spheroids+by+the+hanging-drop+method.">Generation of multicellular tumor spheroids by the hanging-drop method.</a> Methods Mol Med. 140, 141-151 (2007).</li><li>Brenner, W., Gro&#223;, S., Steinbach, F., Horn, S., Hohenfellner, R., Th&#252;roff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+inhibition+of+renal+cancer+cell+invasion+mediated+by+fibronectin,+collagen+IV+and+laminin.">Differential inhibition of renal cancer cell invasion mediated by fibronectin, collagen IV and laminin.</a> Cancer Lett. 155, 199-205 (2000).</li><li>Nieman, M. T., Prudoff, R. S., Johnson, K. R., Wheelock, 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=N-cadherin+promotes+motility+in+human+breast+cancer+cells+regardless+of+their+E-cadherin+expression.">N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression.</a> J Cell Biol. 147 (3), .</li><li>Cheung, K. J., Gabrielson, E., Werb, Z., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+invasion+in+breast+cancer+requires+a+conserved+basal+epithelial+program.">Collective invasion in breast cancer requires a conserved basal epithelial program.</a> Cell. 155 (7), (2013).</li><li>Sodek, K. L., Brown, T. J., Ringuette, 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=Collagen+I+but+not+Matrigel+matrices+provide+an+MMP-dependent+barrier+to+ovarian+cancer+cell+penetration.">Collagen I but not Matrigel matrices provide an MMP-dependent barrier to ovarian cancer cell penetration.</a> BMC Cancer. 8 (1), 223-2210 (2008).</li><li>Nguyen-Ngoc, K. V., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+ductal+elongation+and+myoepithelial+migration+are+regulated+by+the+composition+of+the+extracellular+matrix.">Mammary ductal elongation and myoepithelial migration are regulated by the composition of the extracellular matrix.</a> J Microsc. 251 (3), 212-223 (2013).</li><li>Glinskii, A. B., Smith, B. A., Jiang, P., Li, X., Yang, M., Hoffman, R. M., Glinsky, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viable+circulating+metastatic+cells+produced+in+orthotopic+but+not+ectopic+prostate+cancer+models.">Viable circulating metastatic cells produced in orthotopic but not ectopic prostate cancer models.</a> Cancer Res. 63 (14), 4239-4243 (2003).</li></ol>

The authors declare that they have no competing financial interests.

قيمت هذه الدراسة أداء لجنة من خطوط الخلايا السرطانية في مقايسة غزو كروي (الجدول 1). عموما، نجد أن تتعزز تشكيل كروي في المزيد من خطوط الخلايا الظهارية المظهر، حيث وجود تقاطعات خلية خلية يشجع على تشكيل بنية تشبه كروي. خطوط الخلايا المعروفة التي خضعت لالانتقالي?...

ثبت أن الحركة الخلايا السرطانية هي التنبؤية المحتملة المتنقل، بالنظر إلى أن مثل هذا السلوك يسهل الغزو من خلال الغشاء القاعدي والدخول في الدورة الدموية 1. وقد ركزت معظم البحوث على الحركة على كيفية سلوك الخلايا في ثنائي الأبعاد (2D) الإعداد، على الرغم من أنه أصبح من المسلم به على نطاق واسع أن حركة الخلايا في ثلاثية الأبعاد (3D) المصفوفة هي أكثر تمثيلا لكيف يمكن لهذه الخلايا نفسها سوف فعلا تتصرف في الجسم الحي 2. وتستخدم نظم الاستزراع 3D على نحو متزايد لدراسة السلوكيات الخلوية التي تتراوح من مورفولوجيا الخلايا، لحركية النمو وحساسية العقاقير 3. وقد أدى الرغبة في رصد سرطان غزو الخلايا في إطار محيط 3D لتوليف التقنيات المحددة سابقا التي تنطوي على جيل من المجاميع خلية 3D (الكروية) عن طريق أسلوب ثقافة الإفلات شنقا 4، تليها تضمين هذه الأجسام الشبه الكروية إلى خارج الخلية 3D مصفوفة (ECM) تتألف من الكولاجين والغشاء القاعدي المواد 5. ويسعى هذا الأسلوب إلى تحسين هذه التقنيات المحددة سابقا من خلال توفير نهج مبسط التي يمكن استخدامها بسهولة لمقارنة الغزو في إطار مجموعة من الظروف التجريبية. الطرق التقليدية أكثر لتقييم حركية الخلية في المختبر هي فحص الجرح الصفر وفحص transwell 6. الفحص السابق يصور حركية الخلية في وضع 2D، وبالتالي فهي مستقلة عن مجموعة متنوعة من الميزات الهامة في الجسم الحي الغزو، مثل نشاط الأنزيم البروتيني 7. مقايسة transwell يمكن غزو الخلايا نموذج أفضل عندما تكون مغلفة يدرج بشكل جيد مع ركائز ECM، ولكن فقط معلمة واحدة، أي ظهور الخلايا على العكس سطح غشاء يقاس، والعديد من الفروق الدقيقة من غزو الخلايا وبالتالي لا يمكن ملاحظتها بسهولة. وعلى النقيض من هذه التقنيات، وفحص الخلايا السرطانية غزو كروي (الشكل 1 &lt;/ قوي&gt;) يسمح للرصد في الوقت الحقيقي من غزو الخلايا في إعداد هذا ليس من الناحية الفسيولوجية ذات الصلة فقط، ولكن أيضا يسمح الميزات الهامة خلية خط محدد لأن تصور، مثل هجرة الخلايا الفردية مقابل الجماعية 8. هذا الأسلوب يتيح أيضا مزايا أكثر من فحوصات النمو الثقافة 3D القياسية. توليد المجاميع الخلوية عبر طريقة انخفاض شنقا يحد في البداية حركة الخلية، بحيث يتم تحفيز الخلايا لغزو بعد رفع هذا القيد. وعلاوة على ذلك، حالما يتم رفع هذا القيد، والخروج خلية تسير في اتجاه موحد ويمكن بعد ذلك quantitated مريح. المواد ECM الأكثر شعبية المستخدمة في الخلايا السرطانية الكروية المقايسات Matrigel والنوع الأول الكولاجين، حيث كل عنصر من هذه العناصر أدوارا هامة ومميزة في التأثير على سلوك النقيلي. Matrigel هو خليط يفرز من البروتينات التي تنتجها الخلايا ساركوما Engelbreth-هولم سرب الماوس، وأثرت في basemeبروتينات الغشاء الإقليم الشمالي مثل laminin، entactin، والنوع الرابع الكولاجين 9. لهذا السبب Matrigel يشار من الآن فصاعدا باسم &quot;المواد الغشاء القاعدي&quot;. وتوفر هذه المواد الغشاء القاعدي بروابط الأساسية اللازمة لإنتغرين التصاق خلال السرطان غزو الخلايا 10 بالإضافة إلى العديد من البروتينات الأخرى التي تمارس مجموعة من الآثار على سلوك الخلية 11. في المقابل، النوع الأول الكولاجين، الذي أعد عادة من هضم الحمض الأوتار والهياكل الكولاجينية الكثيفة، 12، هو مادة مصفوفة أبسط من ذلك بكثير التي هي بمثابة العنصر الهيكلي الرئيسي للأنسجة وسدى دعم الأنسجة والأعضاء الضام في الجسم. وقد ثبت أن الخصائص الفيزيائية للالكولاجين يمكن أن تنظم عددا من ملامح الحركة الخلية؛ على سبيل المثال، في محاذاة الألياف الكولاجين في واجهة للورم انسجة تسمح الخلايا السرطانية للهجرة في وقت لاحق على طول تلك الألياف عند غزو في سدى 13. في مقايسة المقدمة هنا، سواء النوع الأول الكولاجين ويتم استخدام المواد الغشاء القاعدي وسيلة لدراسة سرطان 3D خلية سدى التفاعلات. تأثير تثبيط أو تنشيط مسارات التي تتحكم يمكن رصدها الغزو بعد أن تم جزءا لا يتجزأ من الخلايا في مصفوفة 3D. يمكن سابقة التجهيز الخلايا أثناء النمو في قطرات شنقا أو عند نقله الى ثقافة 3D، اعتمادا على ما إذا كان سوف تكون هناك حاجة إلى علاج طويل لتعديل الغزو. لعلاج أقصر، فمن المستحسن أن العقار أن تكون مختلطة مع تعليق كروي بعد جمع، وكذلك وسائل الإعلام التي ستحيط الثقافات 3D، لتسهيل التعرض الكافي للخلايا المخدرات. المقبل، خلايا انسجة طبيعية أو المرتبطة الورم يمكن ممزوجة مع مواد مصفوفة لتقييم دورها في تحوير غزو الخلايا السرطانية، أو لتحديد كيفية يشير التأثيرات سلوك الخلية نظير الصماوي وautocrine. وقد أظهرت هذه الفكرة في الدراسة حيث coculture من العقيدعلى السرطانية والخلايا البطانية في قطرات شنقا أدى إلى شبكة الأوعية الدموية داخل الكروية 14. وأخيرا، يمكن أيضا مكونات ECM يمكن تغييرها، كما يتأثر غزو الخلايا السرطانية عن طريق ركائز مختلفة 15. وبالتالي فإن الطريقة المعروضة أدناه إطارا لتقييم غزو الخلايا السرطانية في إطار مجموعة من الشروط. بشكل عام تبين أن ليس جميع خطوط الخلايا خلق الكروية في قطرات شنقا وخطوط الخلايا الظهارية المظهر عادة تشكل مجالات العادية.

<table>
	<tbody>
		<tr>
			<td>Matrigel Growth Factor Reduced</td>
			<td>Corning</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I, Rat Tail, 100 mg</td>
			<td>Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-treated Dishes</td>
			<td>Corning Incorporated</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well TC-treated Plates</td>
			<td>NEST Biotechnlology</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX-71 Inverted Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and LNCaP cells, which were mantained in RPMI + 10% FBS.</td>
		</tr>
	</tbody>
</table>

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.

تم اختبار باستخدام مقايسة غزو كروي (الشكل 1)، فريق من خطوط الخلايا السرطانية لقدرتها على تشكيل الأجسام الشبه الكروية، فضلا عن كمية من الخروج خلية عرضت بعد زرع في مصفوفة 3D تتكون من مواد الغشاء القاعدي والنوع الأول الكولاجين (الجدول 1). هذه النتائج تثب?...

Watch this Scientific Journal Video about A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting at JoVE.com

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

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 ...

a-cancer-cell-spheroid-assay-to-assess-invasion-in-a-3d-setting

Research

JoVE Journal

Medicine

32.2K Views.  Georgetown University. 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. 

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

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

في الوقت الحقيقي المعاوقة الكهربائية تقنية تستند إلى قياس غزو أحادي الطبقة الخلية البطانية من قبل خلايا السرطان

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

متعدد الفوتون التصوير من غزو خلايا الورم في الفأر نموذج مثلي الفم سرطان الخلايا الحرشفية

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

كروي الفحص لقياس TGF - β بفعل الغزو

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

النمذجة والتصوير 3 الأبعاد غزو الخلية الجماعية

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

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

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

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

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

في الفحص المختبري التخليص الظهارية النماذج أن الخطوات في وقت مبكر من الإنبثاث سرطان المبيض

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

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

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

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

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

الكمي لسرطان الثدي الغزو خلية باستخدام ثلاثي الأبعاد (3D) نموذج

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

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

والتعديل<em&gt; في المختبر</em&gt; الغزو الفحص لتحديد الدور المحتمل للالهرمونات، السيتوكينات و / أو عوامل النمو في التوسط السرطان غزو الخلايا

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

تقييم سرطان المبيض كروي مرفق وغزو الخلايا الظهارية في الوقت الحقيقي

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

التركيز تشكيل: A الفحص القائم على خلية لتحديد إمكانية أنكجنيك لجين

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

الحقن الشريان السباتي لوأضافا الدوائية وتحليل Taxanes في الفئران

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...