We would like to thank Pascal DuBois for proof-reading the article. The authors would also like to thank the personnel of the Animal Facility at Hunter College of The City University of New York. This work was supported by a NIH RCMI grant to Hunter College.

بيان الأخلاق وقد وافق جميع الدراسات على الحيوانات من قبل اللجنة المؤسسية رعاية الحيوان واستخدام (IACUC) من كلية هنتر في جامعة مدينة نيويورك. ملاحظة: استخدمت ثمانية BALB / ج الفئران في هذا الإجراء التجريبي. تم زرع خمسة BALB / ج الفئران مع 5 X 10 6 BNL 1ME A.7R.1 الماوس خلايا سرطان الكبد لإنتاج الأورام الأولية. واستخدمت ثلاثة الفئران BALB / ج بدون زرع والضوابط. BNL 1ME A.7R.1 هو الكبد خط خلية سرطان الفئران التي كانت التي يجنيها التحول الكيميائي من غير مكون للأورام الخلية الكبدية خط الفئران BNL CL.2. تم الحصول على BNL 1ME A.7R.1 من نوع الثقافة مجموعة أمريكا (آي تي ​​سي سي). 1. ما قبل الجراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تأكد من أن الفئران لجميع العمليات الجراحية البقاء في صحة جيدة. للتأكد من أن الفئران هي صحية، وفحص علامات الجفاف التي كتبها خيام من الجلد. لا تستخدم الفئران مع أي دليل على مرض أو دistress في هذه التجارب. </li><li style=";text-align:right;direction:rtl"> وزن الفئران للتأكد من أنهم ليسوا ناقصي الوزن. تأكد من أن الوزن هو 16-20 غراما على الأقل. </li><li style=";text-align:right;direction:rtl"> إعداد 5 × 10 6 BNL 1ME A.7R.1 خلايا فأر HCC لزرع في الماوس: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وسائل الإعلام نضح من 10 مل 60٪ - 70٪ طبق خلية ثقافة متكدسة من BNL 1ME خلايا HCC A.7R.1 الماوس. يغسل مرتين مع 5 مل PBS. إضافة 2 مل من التربسين إلى الطبق واحتضان عند 37 درجة مئوية في حاضنة ترطيب مع الهواء و 5٪ CO 2 لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> وفي وقت لاحق، ماصة 10 ميكرولتر trypsinized الخلايا في عدادة الكريات يدوية لفرز الخلايا تحت المجهر في التكبير من 10X. عد العدد الكلي للخلايا الموجودة في أربعة مربعات زاوية كبيرة ومضاعفة عامل التخفيف من العدد الكلي للخلايا. ثم، القسمة على عدد من الساحات عد وضرب من قبل 10 6 للتركيز الخلية (الخلايا لكل مل). </li><li style=";text-align:right;direction:rtl"> باستخدام حساب، جمع 5 ملايين الخلايا في الماوس في عقيمة1.5 مل أنابيب microcentrifuge. تسمية 1.5 مل أنابيب microcentrifuge. </li></ol></li><li style=";text-align:right;direction:rtl"> Resuspend و5 × 10 6 خلايا BNL 1ME A.7R.1 الماوس HCC في 100 ميكرولتر من الفوسفات مخزنة المالحة (PBS). الحفاظ على هذه الخلايا على الجليد حتى زرع في الجراحة. </li><li style=";text-align:right;direction:rtl"> أداء الأنشطة المطلوبة لإعداد الفئران لإجراء عملية جراحية مثل تحريض التخدير والشعر قصاصات بعيدا عن المعدات والمواد المعقمة لتجنب التلوث. </li><li style=";text-align:right;direction:rtl"> حمل التخدير العام في الفئران عن طريق استنشاق الأيزوفلورين استخدام المرذاذ الأيزوفلورين جنبا إلى جنب مع الأكسجين بتركيز معدل تدفق ثابت من 1 L لكل دقيقة (تقريبا 2.1٪). ملاحظة: هذا النظام هو الاعتماد على الذات، ويشمل المرذاذ الدقة، وإمدادات الأكسجين وعلبة الفحم الذي يرتبط دارة التنفس حاجة للغاز / مخدر الكسح. </li><li style=";text-align:right;direction:rtl"> ضمان التخدير كافية لمنع الحيوانات من الألم أثناء خضوعه لعملية جراحية. أداء إصبع القدم قرصة ومراقبة الحيوان لغير respon-siveness لتأكيد التخدير. </li><li style=";text-align:right;direction:rtl"> في ذلك الوقت، والتحول خط صمام، وختم لمنطقة الجزاء، ومفتوحة للمخروط الأنف. </li><li style=";text-align:right;direction:rtl"> بمجرد أن يتم تخدير الماوس، وتطبيق كمية صغيرة من مرهم للعين لالقرنيات كإجراء وقائي من الجفاف والتلف. </li><li style=";text-align:right;direction:rtl"> وفي وقت لاحق، نقل الحيوان إلى منطقة التحضير، مع مخروط الأنف وضعها بشكل صحيح ومتصل (للتخدير الصيانة). إعداد الموقع للشق الجراحي لعملية جراحية قبل إزالة الشعر في هذا المجال التحضير. </li><li style=";text-align:right;direction:rtl"> تأكد من أن مجال الجراحة في مكان خال من كل نشاط آخر. </li><li style=";text-align:right;direction:rtl"> تأكد من أن سطح العمل هو قوي وتعقيم مناسب، unsoiled وقبل الجراحة خالية من الفوضى. وضع قطعة معقمة من الكتان أو علبة الجراحية أكثر من ذلك. </li><li style=";text-align:right;direction:rtl"> تأكد من أن حقل من المنطوق متاح للأشخاص فقط تنفيذ عملية جراحية. تأكد من أن غرفة العمليات الجراحية يتم تنظيفها واجتاحت مع نظافة المناسبة ورش مع disinfe المناسبctant. </li><li style=";text-align:right;direction:rtl"> تأكد من وضعه أيضا بعيدا عن الأبواب والنوافذ كما تيارات الهواء يمكن أن ينتشر الغبار مما يؤدي إلى تلوث الموقع. </li><li style=";text-align:right;direction:rtl"> إجراء الجراحة البقاء على قيد الحياة مع أدوات معقمة. إما نقع الصكوك في مطهر وفقا لتوصية الشركة الصانعة أو الأوتوكلاف. استخدم مستلزمات معقمة الأساسية التي هي جاهزة للاستخدام. </li><li style=";text-align:right;direction:rtl"> أدوات منفصلة وفقا لوظيفة للمساعدة في منع التلوث من المناطق العقيمة. </li><li style=";text-align:right;direction:rtl"> يحلق حوالي 1 بوصة حول موقع شق (شق خط الوسط) للوصول إلى كبد الحيوان. إزالة الشعر من الحيوانات بعيدا عن موقع الجراحة. </li><li style=";text-align:right;direction:rtl"> بعد الحلاقة، فرك المنطقة مع مطهر والصابون (betadine فرك الجراحية) وغسل مطهر الزائد بعيدا مع حل PBS العقيمة. تنفيذ هذا عدة مرات، مع عملية فرك كامل دائم حوالي 5 دقائق. إزالة والتخلص من القفازات المستخدمة. </li><li style=";text-align:right;direction:rtl"> بعد ذلك، تغيير الملابس الواقية مثل surgicآل ثوب، أعلى فرك، وقناع الوجه. المقبل، تطهير الأيدي بمحلول فرك الصحيح قبل وضع القفازات الجراحية. ملاحظة: هذه هي خطوة ضرورية لمنع سفك وتلويث موقع الجراحية مع الملوثات المحتملة. </li></ol> 2. جراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> توفير مصدر الحرارة الخارجية للدفء طوال فترة التخدير. قد تواجه بعض الحيوانات انخفاض حرارة الجسم بسبب التخدير يسبب اضطرابات الحراري. وبالتالي، والحفاظ على درجة حرارة الجسم. </li><li style=";text-align:right;direction:rtl"> وضع الماوس على ظهرها طوال الجراحة. </li><li style=";text-align:right;direction:rtl"> إصلاح أطرافه الصدارة، أطرافه الخلفية وذيل الماوس إلى طاولة الجراحة مع قطعة من الأشرطة الجراحية. </li><li style=";text-align:right;direction:rtl"> جعل شق خط مستقيم أقل شأنا على الفور إلى xyphisternum. </li><li style=";text-align:right;direction:rtl"> استخدام الذاتي الاحتفاظ ضام جراحي معقم لفضح الكبد. </li><li style=";text-align:right;direction:rtl"> مع حقنة ذات إبرة 20G، ببطء وبعناية ضخ 100 ميكرولتر من برنامج تلفزيوني يحتوي على 5 × 10 6 BNL 1ME A.7R.1خلايا فأر HCC في الفص الأيمن من الكبد. </li><li style=";text-align:right;direction:rtl"> بعد إزالة الإبرة، وتطبيق مسحة القطن المعقم لمدة 1 دقيقة على الأقل لوقف أي نزيف محتمل بسبب ارتفاع الأوعية الدموية للكبد. </li><li style=";text-align:right;direction:rtl"> إغلاق الأنسجة تحت الجلد مع خياطة للامتصاص تليها إغلاق الجلد باستخدام مقاطع الجراحية. وفي وقت لاحق، وتنظيف الجرح مرة أخرى مع betadine فرك. ملاحظة: وهذا يضمن إغلاق الجروح. </li><li style=";text-align:right;direction:rtl"> تحت الجلد تعطي 100 ميكرولتر من محلول ملحي. </li></ol> 3. ما بعد الجراحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مباشرة بعد عملية جراحية، ونقل الماوس إلى قفص نظيفة مع بطاقة الهوية بعد الجراحة لإعلام القائمين على شرط الماوس. توفير الغذاء والماء. ملاحظة: في تجربتنا، والفأر الذي خضع لعملية جراحية فقط سوف يحقق نتائج أفضل إذا يضم وحده مع الغذاء الرطب والماء لفترة أولية 24 ساعة بعد الجراحة. </li><li style=";text-align:right;direction:rtl"> الحفاظ على الحارة الحيوان مع مصباح الحرارة التي تولد الحرارة لا يتجاوز 38 درجة مئوية. Uحد ذاتها ترمومتر لقياس درجة الحرارة حتى يتم استرداد الماوس بشكل كامل. </li><li style=";text-align:right;direction:rtl"> بيت الحيوانات في قفص ويفضل مع الفراش ورقة لضمان الراحة. </li><li style=";text-align:right;direction:rtl"> بعد الانتعاش الأولي، ومراقبة الحيوانات لعلامات الانزعاج، والنزيف والألم والضيق. إعطاء البوبرينورفين (0.05 -0.1 ملغم / كغم) لتخفيف الألم. تراقب عن كثب النشاط والسلوك والمظهر، وكذلك استهلاك الماء والغذاء لحوالي 2 ساعة. إذا العيادات الخارجية، نقل الحيوان إلى منطقة للسكن العادي العادي من الفئران ومواصلة رصد في فترات 12/06 ساعة لمدة 24 ساعة بعد الجراحة. إجراء فحص الوزن إذا رغبت في ذلك. </li><li style=";text-align:right;direction:rtl"> ولكن إذا كان الحيوان هو وجود قضايا يتعافى، واتخاذ إجراءات الرعاية الداعمة المناسبة. إذا الفئران غير قادرة على التعافي، الموت ببطء إنسانية الفئران عبر CO 2 الخنق وخلع عنق الرحم. اتباع المبادئ التوجيهية المؤسسية المعتمدة للقتل الرحيم. </li><li style=";text-align:right;direction:rtl"> بعد الجراحة، لفحص الأدلة السريرية من HCC التي عادة ما تكون observabلو خفض كبير كما في حالة الجسم النتيجة. ملاحظة: عند هذه النقطة، والورم الرئيسي المحتمل وضعت في الكبد، وقد الودائع المتنقل المرجح ضعت في الرئتين. </li><li style=";text-align:right;direction:rtl"> عند هذه النقطة، تضحية بكل الفئران (التجريبية والضابطة) تشارك في التجربة التي كتبها القتل الرحيم إنسانية. تعرض الفئران لثاني أكسيد الكربون الاختناق: ببطء شديد الافراج عن ثاني أكسيد الكربون في الغرفة خلال الفترة من 5 -10 دقائق. وهذا سوف يسبب توقف التنفس. </li></ol>

<ol>
	<li>Sheng, W., 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=Capture,+release+and+culture+of+circulating+tumor+cells+from+pancreatic+cancer+patients+using+an+enhanced+mixing+chip.">Capture, release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip.</a> Lab on a Chip. 14, 89-98 (2014).</li><li>Ogunwobi, O. O., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+and+prognostic+importance+of+epithelial-mesenchymal+transition+in+liver+cancers:+insights+from+experimental+models.">Therapeutic and prognostic importance of epithelial-mesenchymal transition in liver cancers: insights from experimental models.</a> Critical Reviews in Oncology/Hematology. 83, 319-328 (2012).</li><li>Ogunwobi, O. O., Puszyk, W., Dong, H. J., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+upregulation+of+HGF+and+c-Met+drives+metastasis+in+hepatocellular+carcinoma.">Epigenetic upregulation of HGF and c-Met drives metastasis in hepatocellular carcinoma.</a> PloS One. 8, e63765 (2013).</li><li>Ogunwobi, O. O., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+growth+factor+upregulation+promotes+carcinogenesis+and+epithelial-mesenchymal+transition+in+hepatocellular+carcinoma+via+Akt+and+COX-2+pathways.">Hepatocyte growth factor upregulation promotes carcinogenesis and epithelial-mesenchymal transition in hepatocellular carcinoma via Akt and COX-2 pathways.</a> Clinical and Experimental Metastasis. 28, 721-731 (2011).</li><li>Fujiwara, 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=A+novel+animal+model+for+in+vivo+study+of+liver+cancer+metastasis.">A novel animal model for in vivo study of liver cancer metastasis.</a> World Journal of Gastroenterology : WJG. 18, 3875-3882 (2012).</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> Nature Reviews Cancer. 11, 512-522 (2011).</li><li>Heindryckx, F., Colle, I., Van Vlierberghe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+mouse+models+for+hepatocellular+carcinoma+research.">Experimental mouse models for hepatocellular carcinoma research.</a> International Journal of Experimental Pathology. 90, 367-386 (2009).</li><li>Zhang, L., 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=An+in+vivo+mouse+model+of+metastatic+human+thyroid+cancer.">An in vivo mouse model of metastatic human thyroid cancer.</a> Thyroid Official Journal of the American Thyroid Association. 24, 695-704 (2014).</li><li>Menezes, M. E., 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=Genetically+engineered+mice+as+experimental+tools+to+dissect+the+critical+events+in+breast+cancer.">Genetically engineered mice as experimental tools to dissect the critical events in breast cancer.</a> Advances in Cancer Research. 121, 331-382 (2014).</li><li>Ogunwobi, O. O., Wang, T., Zhang, L., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclooxygenase-2+and+Akt+mediate+multiple+growth-factor-induced+epithelial-mesenchymal+transition+in+human+hepatocellular+carcinoma.">Cyclooxygenase-2 and Akt mediate multiple growth-factor-induced epithelial-mesenchymal transition in human hepatocellular carcinoma.</a> Journal of Gastroenterology and Hepatology. 27, 566-578 (2012).</li><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=The+hallmarks+of+cancer.">The hallmarks of cancer.</a> Cell. 100, 57-70 (2000).</li><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=Dissemination+and+growth+of+cancer+cells+in+metastatic+sites.">Dissemination and growth of cancer cells in metastatic sites.</a> Nature Reviews Cancer. 2, 563-572 (2002).</li><li>Kim, M. 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=Tumor+self-seeding+by+circulating+cancer+cells.">Tumor self-seeding by circulating cancer cells.</a> Cell. 139, 1315-1326 (2009).</li><li>Stolpe, A., Pantel, K., Sleijfer, S., Terstappen, L. W., den Toonder, 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=Circulating+tumor+cell+isolation+and+diagnostics:+toward+routine+clinical+use.">Circulating tumor cell isolation and diagnostics: toward routine clinical use.</a> Cancer Research. 71, 5955-5960 (2011).</li><li>Saad, F., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+role+of+circulating+tumor+cells+in+the+diagnosis+and+management+of+bone+metastases+in+advanced+prostate+cancer.">The current role of circulating tumor cells in the diagnosis and management of bone metastases in advanced prostate cancer.</a> Future Oncology. 8, 321-331 (2012).</li></ol>

The authors declare that they have no competing financial interests.

في هذه المقالة يتم إعطاء وصف متعمقة للطريقة التي تم الإبلاغ عنها مؤخرا من قبل Ogunwobi وزملاؤه من إنشاء الناجح لنموذج الفأر مسانج مثلي من سرطان الكبد ورم خبيث (الشكل 2) 3. معدل تأخذ من أورام لهذا الإجراء مرتفع عموما. وقد لاحظنا سابقا بمعدل اتخاذ الورم من 100٪ <s...

Hepatocellular carcinoma (HCC) is one of the most lethal cancers, with poor prognosis and low life expectancy. Nearly all cancer-related deaths are due to the metastatic spread of the disease from the originating organ to additional distant organs2-6.HCC progression is a complex process. Therefore, being able to model the tumor microenvironment that is naturally found in metastatic HCC in animal models can prove to be a successful and useful way to reveal relevant mechanisms in humans.7Unfortunately, the mechanisms of cancer metastasis are still largely unclear. Therefore, there is a need to establish animal models that will enable us to elucidate the underlying molecular mechanisms of metastases of cancers such as HCC3,5.
Mouse model systems are a very useful approach for delineating mechanisms and evaluating novel strategies for treatment of metastatic human cancers5,8. The various mouse model systems that presently exist are a testament to efforts of researchers to correctly depict the complexity of the disease5,8,9.
Ogunwobi and colleagues recently used a survival surgical approach to demonstrate establishment of a novel orthotopic syngeneic mouse model for the study of metastasis in HCC3. Their work established a &#8220;patient-like&#8221; mouse model that recapitulates features of aggressive and metastatic HCCs3. They further demonstrated that this mouse model system can be used to study the biology of circulating tumor cells, and that this holds potential for gaining novel insights into the mechanisms of cancer metastasis3.
The aim of this paper is to describe in detail the methodology used in establishing this &#8220;patient-like&#8221; orthotopic syngeneic mouse model of HCC metastasis3. The methodology of how to implant BNL 1ME A.7R.1 mouse HCC cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c3 using survival surgery will be described. Unlike other mouse xenograft tumor models where human tumor cells are implanted into immune deficient mice, this system is syngeneic and is, therefore, suitable for studying the role of the immune system in tumor metastasis3,5,8. This approach will likely gain widespread use for studying the mechanisms of metastasis in solid organ cancers.

<table border="0">
	<tbody>
		<tr>
			<td>Micro Dissecting Tweezer&#160;</td>
			<td>Roboz</td>
			<td>(RS-5040)</td>
			<td>Tip .20 x .12mm</td>
		</tr>
		<tr>
			<td>Graefe Micro Dissecting Forceps, serrated curved tip</td>
			<td>Roboz</td>
			<td>(RS-5111)</td>
			<td>1 X 2 teeth, curved tip width 0.6mm</td>
		</tr>
		<tr>
			<td>Micro Dissecting Retractor-Agricola (3 by 3 prongs)</td>
			<td>Roboz</td>
			<td>(RS-6501)</td>
			<td>Blunt 3 X 3 prongs, depth 4mm, spread 25mm</td>
		</tr>
		<tr>
			<td>Micro Dissecting Retractor-Goldstein (3 by 3 prongs)</td>
			<td>Roboz</td>
			<td>(RS-6503)</td>
			<td>Blunt, 3 X 3 prongs, depth 4mm, spread 19mm</td>
		</tr>
		<tr>
			<td>Jameson Caliper (Measures tumors)</td>
			<td>Roboz</td>
			<td>(RS-6466)</td>
			<td>80mm/3 inch scale, chrome plated&#160;</td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors, large ring sissors, straight and sharp</td>
			<td>Roboz</td>
			<td>(RS-5852)</td>
			<td>23mm blades, length 4 inches, flat shanks and large rings</td>
		</tr>
		<tr>
			<td>Scalpel with blades, for delicate dissecting procedures</td>
			<td>Roboz</td>
			<td>(RS-9861-36)</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle&#160;</td>
			<td>Roboz</td>
			<td>(RS-9884)</td>
			<td>Solid</td>
		</tr>
		<tr>
			<td>Littauer Stitch Sissors</td>
			<td>Roboz</td>
			<td>(RS-7074)</td>
			<td>Length 4.5 inches</td>
		</tr>
		<tr>
			<td>Brown needle holder, for easy suture tying</td>
			<td>Roboz</td>
			<td>(RS-7960)</td>
			<td>Convex jaw, fine serrations</td>
		</tr>
		<tr>
			<td>Reflex 7MM wound clips with reflex 7 clip applier</td>
			<td>Roboz</td>
			<td>(RS-9262)</td>
			<td>safe, secure alternative method of wound closure</td>
		</tr>
		<tr>
			<td>Instrument tray and lid</td>
			<td>Roboz</td>
			<td>RT-1350S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-clipper with detachable blade</td>
			<td>Roboz</td>
			<td>(RS-5903)</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500 (the Germ Terminator) Dry Sterilizer</td>
			<td>Roboz</td>
			<td>Ds-400, Ds-401, DS-501</td>
			<td>For fast decontamination of micro-dissecting instruments.&#160;&#160; Instruments decontaminate within 15 seconds.&#160;</td>
		</tr>
		<tr>
			<td>Microinjection needles</td>
			<td>VWR</td>
			<td>BD305125</td>
			<td>25G Needle</td>
		</tr>
	</tbody>
</table>

a "patient-like" orthotopic syngeneic mouse model of hepatocellular carcinoma metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

تم زرع كبد BALB / ج الفئران مع 5 × 10 6 BNL 1ME A.7R.1 خلايا فأر HCC. والأدلة السريرية التنمية HCC يمكن ملاحظتها 63 أيام بعد الجراحة. ونتيجة لذلك، الموت الرحيم كانت الفئران إنسانية. تم إجراء تشريح على الفئران الموت الرحيم. كانت مقطوعة الرئتين والكبد وتم إجراء الفحص الإجمالي دقي?...

Watch this Scientific Journal Video about A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis at JoVE.com

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

انتشار النقيلي من السرطان هو السبب الرئيسي للوفيات المرتبطة بالسرطان. ونحن نقدم وصفا متعمقة لمنهجية عملية جراحية بقائنا لإنشاء نظام نموذجي الماوس مسانج مثلي &quot;المريض مثل&quot; لدراسة آليات الانبثاث في أورام الجهاز الصلبة.

A &quot;المريض مثل&quot; مثلي مسانج نموذج الفأر من سرطان الكبد الانبثاث

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

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JoVE Journal

Medicine

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

12.1K Views.  Hunter College of the City University of New York.  انتشار النقيلي من السرطان هو السبب الرئيسي للوفيات المرتبطة بالسرطان. ونحن نقدم وصفا متعمقة لمنهجية عملية جراحية بقائنا لإنشاء نظام نموذجي الماوس مسانج مثلي "المريض مثل" لدراسة آليات الانبثاث في أورام الجهاز الصلبة. 

Video: A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

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 ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

في ضوء بارد التصوير حيوية الجسم الحي من نقص الأكسجة ورم من الدماغ الانبثاث سرطان الثدي في نموذج الفأر

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

العلاج الإشعاعي مجموعة في نموذج الفأر Orthotopic استئصال ورم من الدماغ

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

ماوس نموذج مثلي من سرطان الغدة الدرقية كشمي

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

جيل من تحت الجلد والخلايا الكبدية داخل الكبد سرطان Xenografts الإنسان في الفئران العوز المناعي

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

على الفئران نموذج مثلي من سرطان البروستاتا البشرية الانبثاث

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

بدء النقيلي سرطان الثدي عن طريق استهداف ظهارة الأقنية مع اتش، لجنة المساواة العرقية: رواية المعدلة وراثيا نموذج الفأر من سرطان الثدي

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

ال<em&gt; في البيضة</em&gt; الفرخ مشيمائي غشاء (CAM) الفحص بمثابة طعم أجنبي نموذج فعال لسرطان الكبد

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

التباين الديناميكي المحسن التصوير بالرنين المغناطيسي لنموذج مثلي سرطان البنكرياس ماوس

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

مثلي زرع والأجهزة الطرفية المناعي رصد خلية في نموذج II-45 مسانج الجرذ ورم الظهارة المتوسطة

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...