نريد أن يعترف تايسون فودز لتوفير بسخاء البويضات المخصبة. وكان ارنولد شانا مفيدة أثناء تصوير هذا البروتوكول. وأيد Trenis بالمر من قبل المعاهد الوطنية للصحة تحت L. روث الجائزة الوطنية كيرشتاين دائرة البحوث (F31 المعهد الوطني للسرطان). وأيد هذا العمل جزئيا من خلال المنح CCSRI # 700537 لرابطة الدفاع اليهودية والمعاهد الوطنية للصحة / NCI منح # CA120711 - 01A1 - 01A1 وCA120711 إلى الألف إلى الياء.

<ol>
	<li>Ghiso, A. g. u. i. r. r. e., Kovalski, J. A., K, ., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+dormancy+induced+by+downregulation+of+urokinase+receptor+in+human+carcinoma+involves+integrin+and+MAPK+signaling.">Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signaling.</a> J Cell Biol. 147, 89-104 (1999).</li><li>Chambers, A. F., Shafir, R., Ling, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+system+for+studying+metastasis+using+the+embryonic+chick.">A model system for studying metastasis using the embryonic chick.</a> Cancer research. 42, 4018-4025 (1982).</li><li>Chambers, A. F., Wilson, S. M., Tuck, A. B., Denhardt, G. H., Cairncross, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+metastatic+properties+of+a+variety+of+mouse,+rat,+and+human+cells+in+assays+in+nude+mice+and+chick+embryos.">Comparison of metastatic properties of a variety of mouse, rat, and human cells in assays in nude mice and chick embryos.</a> In Vivo. 4, 215-219 (1990).</li><li>Deryugina, E. I., Zijlstra, A., Partridge, J. J., Kupriyanova, T. A., Madsen, M. A., Papagiannakopoulos, T., 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=Unexpected+effect+of+matrix+metalloproteinase+down-regulation+on+vascular+intravasation+and+metastasis+of+human+fibrosarcoma+cells+selected+in+vivo+for+high+rates+of+dissemination.">Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination.</a> Cancer Res. 65, 10959-10969 (2005).</li><li>Hahn-Dantona, E., Ramos-DeSimone, N., 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+proMMP-9+by+a+plasmin/MMP-3+cascade+in+a+tumor+cell+model.+Regulation+by+tissue+inhibitors+of+metalloproteinases.">Activation of proMMP-9 by a plasmin/MMP-3 cascade in a tumor cell model. Regulation by tissue inhibitors of metalloproteinases.</a> Ann N Y Acad Sci. 878, 372-387 (1999).</li><li>Kim, J., Yu, W., Kovalski, K., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+specific+proteases+in+cancer+cell+intravasation+as+revealed+by+a+novel+semiquantitative+PCR-based+assay.">Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay.</a> Cell. 94, 353-362 (1998).</li><li>Leong, H. S., Steinmetz, N. F., Ablack, A., Destito, G., Zijlstra, A., Stuhlmann, H., Manchester, M., Lewis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+embryonic+and+tumor+neovasculature+using+viral+nanoparticles.">Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles.</a> Nat Protoc. 5, 1406-1417 (2010).</li><li>Lewis, J. D., Destito, G., Zijlstra, A., Gonzalez, M. J., Quigley, J. P., Manchester, M., Stuhlmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+nanoparticles+as+tools+for+intravital+vascular+imaging.">Viral nanoparticles as tools for intravital vascular imaging.</a> Nat Med. 12, 354-360 (2006).</li><li>Ossowski, L., Reich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+malignant+phenotype+of+a+human+carcinoma+conditioned+by+growth+environment.">Changes in malignant phenotype of a human carcinoma conditioned by growth environment.</a> Cell. 33, 323-333 (1983).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+C(T)+method.">Analyzing real-time PCR data by the comparative C(T) method.</a> Nat Protoc. 3, 1101-1108 (2008).</li><li>Testa, J. E., Brooks, P. C., Lin, J. M., 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=Eukaryotic+expression+cloning+with+an+antimetastatic+monoclonal+antibody+identifies+a+tetraspanin+(PETA-3/CD151)+as+an+effector+of+human+tumor+cell+migration+and+metastasis.">Eukaryotic expression cloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis.</a> Cancer Res. 59, 3812-3820 (1999).</li><li>Zijlstra, A., Lewis, J., Degryse, B., Stuhlmann, H., 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=The+Inhibition+of+Tumor+Cell+Intravasation+and+Subsequent+Metastasis+via+Regulation+of+In+Vivo+Tumor+Cell+Motility+by+the+Tetraspanin+CD151.">The Inhibition of Tumor Cell Intravasation and Subsequent Metastasis via Regulation of In Vivo Tumor Cell Motility by the Tetraspanin CD151.</a> Cancer Cell. 13, 221-234 (2008).</li><li>Zijlstra, A., Mellor, R., Panzarella, G., Aimes, R. T., Hooper, J. D., Marchenko, N. D., 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=A+quantitative+analysis+of+rate-limiting+steps+in+the+metastatic+cascade+using+human-specific+real-time+polymerase+chain+reaction.">A quantitative analysis of rate-limiting steps in the metastatic cascade using human-specific real-time polymerase chain reaction.</a> Cancer Res. 62, 7083-7092 (2002).</li></ol>

الإعلان عن أي تضارب في المصالح.

وقد استخدم CAM الفرخ على مدى عقود في نظام نموذجي لدراسة الجوانب المختلفة لتطور السرطان والبيولوجيا النقيلي. وقد تم تنفيذ مبدأ تقييم القدرات المنتشر في هذا النموذج من قبل مجموعة متنوعة من الفئات بما في ذلك تحليل السكون (Ghiso أغيري وآخرون ، 1999 ؛. Ossowski والرايخ ، 1983) ، إ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف </th><th> شركة </th><th> فهرس العدد </th><th> تعليقات (اختياري) </th></tr><tr><td> 1X Dulbecco في التعديل النسر متوسطة (DMEM) </td><td> Invitrogen </td><td> 11995073 </td><td></td></tr><tr><td> Dulbecco والفوسفات مخزنة المالحة (D - PBS) (1X) والسائلة </td><td> Invitrogen </td><td> 14190250 </td><td> الرقم الهيدروجيني 7،4 </td></tr><tr><td> التربسين ، 0.05 ٪ (1X) مع 4NA EDTA والسائلة </td><td> Invitrogen </td><td> 25300054 </td><td></td></tr><tr><td> رياضي هاتشر </td><td> التوت هيل </td><td> 1550HA </td><td rowspan="2"> هذه هي بعض من حاضنات المتاحة بسهولة أكثر. ومع ذلك ، سوف تظهر معدات الفيديو المختلفة. </td></tr><tr><td> رياضي الحاضنة </td><td> التوت هيل </td><td> 1502EA </td><td></td></tr><tr><td> DREMEL أداة دوارة </td><td> DREMEL </td><td></td><td></td></tr><tr><td> عجلات قطع DREMEL لا. 36 </td><td> DREMEL </td><td> 409 </td><td></td></tr><tr><td> محمول الماصة المعونة </td><td> الصياد </td><td> 1368115E </td><td></td></tr><tr><td> يميل القطن القضبان الرفيعة </td><td> الصياد </td><td> 23-400-001) </td><td></td></tr><tr><td> بيض </td><td> مختلف </td><td></td><td> ويمكن استخدام بائع عديدة ومختلفة. يجب أن يكون البيض حوالي 35-55 غرام. والدجاجات التي تبيض لهم يجب أن تكون بين 36 و 55 أسبوعا من العمر. </td></tr><tr><td> عدادة الكريات </td><td> Hausser العلمية وVWR </td><td> 15170-090 </td><td></td></tr><tr><td> الألياف البصرية المجهر منار </td><td> Amscope </td><td> HL250 - AY </td><td> 150W </td></tr><tr><td> إبرة قياس 25 </td><td></td><td></td><td></td></tr><tr><td> Sybr الاخضر المقتطف - N - PCR الأمبير الأنسجة كيت </td><td> سيغما الدريخ </td><td> XNATRG </td><td></td></tr></tbody></table>

quantitative analysis of cancer metastasis using an avian embryo model

خلال نشر الخلايا السرطانية الانبثاث من الورم الرئيسي ، إلى غزو الأنسجة المحيطة بها ، والانتشار إلى أعضاء بعيدة. الانبثاث هي عملية معقدة يمكن أن تنطوي على العديد من أنواع الأنسجة ، وفترات زمنية متنوعة ، وغالبا ما تحدث في أعماق الأجهزة ، مما يجعل من الصعب تحقيق وكميا. بالإضافة إلى ذلك ، تأثر فعالية عملية المتنقل بخطوات متعددة في تتالي النقيلي مما يجعل من الصعب تقييم مساهمة من جانب واحد للسلوك الخلية السرطانية. نتيجة لذلك ، كثيرا ما يتم تنفيذ المقايسات الانبثاث في حيوانات التجارب لتوفير سياق واقعي بالضرورة للدراسة الانبثاث. لسوء الحظ ، فإن تعقيد هذه النماذج التي لها علم وظائف الأعضاء المعقدة. الجنين الفرخ هو نموذج فريد في الجسم الحي أن يتغلب على الكثير من القيود على دراسة ورم خبيث ، ويرجع ذلك إلى سهولة الحصول على غشاء مشيمائي (CAM) ، وأوعية دموية جيدة خارج الأنسجة الجنينية تقع تحت قشر البيض الذي يتم تقبلا لxenografting الخلايا السرطانية (الشكل 1). وعلاوة على ذلك ، لأن الجنين الفرخ هو العوز المناعي بشكل طبيعي ، وتدعم CAM engraftment بسهولة من الأنسجة الطبيعية على حد سواء والورم. الأهم من ذلك ، CAM الطيور يدعم بنجاح خصائص الخلايا السرطانية أكثر بما في ذلك النمو ، والغزو ، الأوعية الدموية ، وإعادة تشكيل المكروية. هذا يجعل نموذج مفيد بشكل استثنائي من أجل التحقيق في الممرات التي تؤدي إلى ورم خبيث من السرطان والتنبؤ استجابة للعلاجات السرطان النقيلي جديدة محتملة. الكشف عن الخلايا التي تنشرها أنواع محددة PCR ألو يجعل من الممكن إجراء تقييم كمي لورم خبيث في الأجهزة التي استعمرها قليلة مثل 25 الخلايا. باستخدام الإنسان سرطانة بشرانية خط الخلية (HEp3) نستخدم هذا النموذج لتحليل الانبثاث عفوية الخلايا السرطانية إلى أعضاء بعيدة ، بما فيها الكبد والرئة الفرخ. علاوة على ذلك ، باستخدام بروتوكول ألو - PCR نظهر حساسية واستنساخ للمقايسة كأداة لتحليل وquantitate intravasation واعتقال والتسرب ، والاستعمار بوصفها عناصر فردية من ورم خبيث.

Watch this Scientific Journal Video about Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model at JoVE.com

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

باستخدام كمية PCR ، علينا أن نظهر كيف يمكن استخدام نموذج راسخة CAM فرخ لتحليل كمي لورم خبيث من الخلايا السرطانية البشرية إلى الأجهزة البعيدة.

تحليل الكمي الإنبثاث السرطان باستخدام الأجنة النموذجي الطيور

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

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18.9K Views.  Vanderbilt University. باستخدام كمية PCR ، علينا أن نظهر كيف يمكن استخدام نموذج راسخة CAM فرخ لتحليل كمي لورم خبيث من الخلايا السرطانية البشرية إلى الأجهزة البعيدة.

Video: Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

نموذج مثلي من سرطان المثانة الفئران

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

الكمية التصور والكشف عن سرطان الجلد باستخدام التصوير الحراري الديناميكي

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

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 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

ومتعددة الوسائط التصوير من الأوعية الدموية في نموذج الفئران عاري من سرطان الثدي الإنبثاث العظام باستخدام التصوير بالرنين المغناطيسي، الحجمي التصوير المقطعي والموجات فوق الصوتية

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

نماذج من الانبثاث العظام

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

وسرطان المثانة نموذج مثلي للدراسات توصيل الجينات

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

باستخدام الكمي في الوقت الحقيقي PCR لتحديد الجهات المانحة Engraftment خلية في التنافسية نموذج الفئران زرع نخاع العظم

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

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  ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

النخاعي السوائل عن طريق الرنا الميكروي التنميط الكمي في الوقت الحقيقي PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

الكميات من البينية البريتوني ورم خبيث سرطان المبيض

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...