وأيد هذا العمل من قبل المعهد الوطني للأمراض العصبية والسكتة الدماغية (R01 NS048353 لSLC) ، والمعهد الوطني للسرطان (R01 CA122804 لSLC ، R01 CA134773 لكيفن روث ألف وSLC وCA13148 - 35 إلى KRZ) وإدارة الدفاع (X81XWH - 09 - 1 - 0086 لSLC). وقدمت صناديق دعم العملية الصغيرة من المركز الشامل للسرطان UAB الحيوان مرفق التصوير مشتركة من قبل لجنة التحقيق الوطنية منح الدعم الأساسية (P30 CA13148 - 35 ؛ E. الحجل ، PI). نشكر العصبية ألاباما مركز مخطط الأساسية (P30 NS57098) ، وعلم الأعصاب UAB مركز كور (P30 NS47466) لمساعدتهم. المحتوى هو فقط من مسؤولية الكتاب ولا تمثل بالضرورة وجهة النظر الرسمية للمعاهد الوطنية للصحة أو وزارة الدفاع.

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	<li>Rosenberg, M. P., Bortner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+transgenic+and+knockout+animal+models+should+be+used+(for+drug+efficacy+studies+in+cancer).">Why transgenic and knockout animal models should be used (for drug efficacy studies in cancer).</a> Cancer Met. Rev. 17, 295-299 (1999).</li><li>Killion, J. J., Radinsky, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+are+necessary+to+predict+therapy+of+transplantable+tumors+in+mice.">Orthotopic models are necessary to predict therapy of transplantable tumors in mice.</a> Cancer Met. Rev. 17, 279-284 (1999).</li><li>Carroll, S. L., Ratner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+does+the+Schwann+cell+lineage+form+tumors+in+NF1?.">How does the Schwann cell lineage form tumors in NF1?.</a> Glia. 56, 1590-1605 (2008).</li><li>Woodruff, J. M., Kourea, H. P., Louis, D. N., Scheithauer, B. W., Kleihues, P., Cavenee, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology and Genetics of Tumours of the Nervous System. , 172-174 (2000).</li><li>DeClue, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+receptor+expression+in+neurofibromatosis+type+1-related+tumors+and+NF1+animal+models.">Epidermal growth factor receptor expression in neurofibromatosis type 1-related tumors and NF1 animal models.</a> J. Clin. Invest. 105, 1233-1241 (2000).</li><li>Byer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tamoxifen+inhibits+malignant+peripheral+nerve+sheath+tumor+growth+via+an+estrogen+receptor-independent+mechanism.">Tamoxifen inhibits malignant peripheral nerve sheath tumor growth via an estrogen receptor-independent mechanism.</a> Neuro-Oncology. , (2010).</li><li>Zinn, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+bioluminescence+imaging+in+small+animals.">Noninvasive bioluminescence imaging in small animals.</a> ILAR J. 49, 103-115 (2008).</li><li>Wu, J. C., Sundaresan, G., Iyer, M., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+optical+imaging+of+firefly+luciferase+reporter+gene+expression+in+skeletal+muscles+of+living+mice.">Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice.</a> Mol. Therapy. 4, 297-306 (2001).</li><li>Luker, G. D., Leib, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luciferase+real-time+bioluminescence+imaging+for+the+study+of+viral+pathogenesis.">Luciferase real-time bioluminescence imaging for the study of viral pathogenesis.</a> Methods Mol. Biol. 292, 285-296 (2005).</li><li>Dahlberg, W. K., Little, J. B., Fletcher, J. A., Suit, H. D., Okunieff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitivity+in+vitro+of+human+soft+tissue+sarcoma+cell+lines+and+skin+fibroblasts+derived+from+the+same+patients.">Radiosensitivity in vitro of human soft tissue sarcoma cell lines and skin fibroblasts derived from the same patients.</a> Int. J. Radiat. Biol. 63, 191-198 (1993).</li><li>Badache, A., DeVries, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibrosarcoma-derived+Schwann+cells+overexpress+platelet-derived+growth+factor+(PDGF)+receptors+and+are+induced+to+proliferate+by+PDGF.">Neurofibrosarcoma-derived Schwann cells overexpress platelet-derived growth factor (PDGF) receptors and are induced to proliferate by PDGF.</a> BB. J. Cell. Physiol. 177, 334-342 (1998).</li><li>Muir, D., Neubauer, D., Lim, I. T., Yachnis, A. T., Wallace, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenic+properties+of+neurofibromin-deficient+neurofibroma+Schwann+cells.">Tumorigenic properties of neurofibromin-deficient neurofibroma Schwann cells.</a> Am. J. Pathol. 158, 501-513 (2001).</li></ol>

الإعلان عن أي تضارب في المصالح.

مثلي الطريقة التفصيلية xenografting المقدمة هنا هي تلك التي قمنا بتطويرها باستخدام الخلايا MPNST ST88 - 14 ، وهو الخط الذي يستخدم على نطاق واسع في الدراسات هذه NF1 المرتبطة غمد العصب المحيطي الأورام. ومع ذلك ، فإن هذه المنهجية هي قابلة للتكيف بسهولة مع غيرها من الدراسات قبل السريرية MPNST خطوط الخلايا. على سبيل المثال ، لقد قمنا أيضا مثلي xenografting مع الخلايا 10 STS - 26T ، وهو مشتق من الخط MPNST تحدث بشكل متقطع وT265 - 2C 11 الخلايا ، والتي هي مستمدة من MPNST NF1 المرتبطة ، مع نجاح مماثل لذلك لاحظنا مع ST88 -14 الخلايا. وقد قلت ذلك ، فإننا نلاحظ أنه لا يمكن وصف الظروف الدقيقة ونحن هنا لمثلي من xenografting ST88 - 14 خلايا سيعتمد بشكل مباشر عن عمليات ترقيع للخطوط MPNST الأخرى. بدلا من ذلك ، لا بد من المعايير الأساسية لمنهجية تحديد تجريبيا لكل خط الخلية. وتشمل هذه المعايير عدد من الخلايا المطعمة MPNST في البداية ، والوقت المسموح به للتنمية الكسب غير المشروع ، وإلى حد أقل ، في وحدة التخزين التي يتم حقنها في الخلايا السرطانية. لإنشاء هذه المعلمات لسطر جديد ، ونحن مجموعة من الفئران الكسب غير المشروع (5 الفئران في كل مجموعة) مع 03 حتي 05 أكتوبر س خلايا الورم 6 10 ، والتحقق من الخلايا two تركيزات لكل أمر من حجم (أي 10 3 خلايا ، 5 × 10 3 الخلايا ، والخلايا 10 4 ، 5 × 10 4 خلايا ، الخ). يجب أن يتم حقنه أعداد أكبر من الخلايا السرطانية (&gt; 10 6) في حجم أكبر ، ونحن قد وجدت التي يمكن حقنها يصل إلى 5 مل من دون خسارة من الخلايا العصبية من المطعمة. وقد وجدنا أيضا أنه يمكن حقن لا يزيد عن 5 × 10 6 خلايا الورم في حجم 5 مل تعليق أكثر كثافة كما أصبحت معرضة بشكل متزايد إلى القص الذي يقتل الخلايا السرطانية. نحن نتبع هذه الفئران على الأقل حتى ثلاثة حيوانات داخل كل مجموعة والأورام واضح ، وعند هذه النقطة لنا إنهاء هذه المجموعة ودراسة الأعصاب تشريحيا لتأكيد النمو الكسب غير المشروع. من الواضح ، والوقت المطلوب للوصول إلى هذه النقطة سوف تختلف ، اعتمادا على عدد من الخلايا حقن ، وبشكل عام ، ونحن نسعى للتركيز الخلايا التي ستصل إلى أقصى نمو الورم المسموح به خلال 30-60 يوما. ويمكن تحقيق المزيد من النمو السريع يجعل من الصعب تحقيق تركيزات علاجية فعالة قبل انتهاء فترة التجربة و / أو منع جمع ما يكفي من datapoints التصوير تلألؤ بيولوجي على مدار التجارب. دورة زمنية أطول من 60 يوما يجعل ضبط المعلمات التجريبية غير عملي وغير ضروري يطيل مدة التجارب المخطط لها. أخيرا ، نود أن نشير أيضا إلى أننا قد استخدمت هذه المنهجية مثلي xenografting مع ST88 - 14 المطعمة الخلايا في الفئران الثالث المعاهد الوطنية للصحة للتدليل على فعالية العلاج من 6 تاموكسيفين. يمكن العثور على وصف تفصيلي للطرق التي نستخدمها بشكل روتيني لتقييم الآثار العلاجية وكلاء المرشح على xenografts مثلي في هذا المخطوط. كما أنها أثبتت أن الخلايا يمكن أن يفي عصبي المطعمة بنجاح في الأعصاب الطرفية 12 ، مما يشير إلى أنه ، مع بعض التعديلات ، ويمكن استخدامها للإجراءات المبينة في هذا البروتوكول لإجراء التجارب قبل السريرية مع وكلاء مرشح العلاجية الموجهة ضد الأورام الليفية العصبية.

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orthotopic xenografting of human luciferase-tagged malignant peripheral nerve sheath tumor cells for in vivo testing of candidate therapeutic agents

على الرغم من الشاشات في المختبر تعتبر ضرورية لتحديد أولية من وكلاء مرشح العلاجية ، وإجراء تقييم دقيق لقدرة الدواء لمنع ويجب أن يؤدي نمو الورم في نموذج حيواني مناسبة. نوع الحيوان الذي هو النموذج الأفضل لهذا الغرض هو موضوع للمناقشة مكثفة. بعض الأدلة تشير إلى أن التجارب قبل السريرية دراسة آثار المخدرات على الأورام التي تنشأ في الفئران المعدلة وراثيا هي أكثر التنبؤية لنتائج سريرية (1) وحتى يتم اختبارها في كثير من الأحيان وكلاء مرشح العلاجية في هذه النماذج. للأسف ، ونماذج معدلة وراثيا ليست متاحة لأنواع كثيرة من السرطان. مزيد من النماذج المعدلة وراثيا في كثير من الأحيان قيود أخرى مثل القلق بشأن كيفية جيدا نماذج ورم الماوس نظيرتها الإنسان ، وانتفاذ غير مكتملة من النمط الظاهري الورم وعدم قدرة على التنبؤ عندما الأورام ستتطور. وبالتالي ، والمحققين استخدام العديد من النماذج طعم أجنبي (الخلايا السرطانية في الفئران البشرية المطعمة العوز المناعي) لتجارب قبل السريرية إذا كان ذلك مناسبا من النماذج الورم المعدلة وراثيا غير متوفرة. حتى لو النماذج المعدلة وراثيا متوفرة ، وتشاركت في كثير من الأحيان مع نماذج طعم أجنبي كدولة تسهيل التحديد السريع للنطاقات العلاجية. كذلك ، فإن هذه الشراكة تتيح للمقارنة بين فعالية عامل في الأورام المعدلة وراثيا وحقيقية الخلايا السرطانية البشرية. تاريخيا ، xenografting وكثيرا ما يقوم به عن طريق الحقن تحت الجلد خلايا الورم (xenografts خارج الرحم). هذا الأسلوب هو السريع واستنساخه ، وغير مكلفة نسبيا ، ويسمح الكميات المستمر للنمو الورم خلال فترة علاجية 2. ومع ذلك ، يمكن المساحة تحت الجلد ليست عادية بالنسبة لمعظم المكروية الأورام والنتائج التي تم الحصول عليها حتى مع xenografting خارج الرحم قد تكون مضللة نتيجة لعوامل مثل عدم وجود هيئة محددة من الأنسجة المضيف التعبير والجينات السرطانية. وهكذا كان من المستحسن أن يستعاض خارج الرحم أو ترقيع دراسات تستكمل الدراسات التي يتم فيها المطعمة الخلايا السرطانية في الأنسجة البشرية المنشأ (xenografting مثلي) 2. للأسف ، كثيرا ما أحبطت تنفيذ هذه التوصية من حقيقة أن مثلي منهجيات xenografting لم يتم تطويرها لأنواع كثيرة من السرطان. غمد العصب المحيطي الخبيثة الأورام (MPNSTs) والأورام اللحمية شديدة العدوانية التي تحدث بشكل متقطع أو بالاشتراك مع نوع الورم العصبي الليفي 1 3 الأكثر شيوعا وتنشأ في العصب الوركي 4. نحن هنا وصفا لطريقة بسيطة من الناحية الفنية التي يتم فيها xenografted orthopically يراعة luciferase الموسومة MPNST خلايا الإنسان في العصب الوركي العوز المناعي لدى الفئران. وتناقش أيضا نهجنا لتقييم نجاح العملية تطعيم الحيوانات في الفردية والعشوائية لاحقة غير المنحازة إلى مجموعات الدراسة.

Watch this Scientific Journal Video about Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents at JoVE.c

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

تم وصف طريقة لتطعيم موثوق بها الإنسان الخبيثة luciferase الموسومة غمد ورم الخلايا العصبية الطرفية في العصب الوركي العوز المناعي لدى الفئران. وتناقش أيضا استخدام التصوير تلألؤ بيولوجي لإثبات إنشاء السليم للترقيع الورم ومعايير العزل عشوائية من الحيوانات إلى مجموعات الدراسة.

من مثلي Xenografting Luciferase الموسومة الإنسان الخبيثة ورم الخلايا العصبية المحيطية لغمد في الجسم الحي اختبار وكلاء المرشح علاجي

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

orthotopic-xenografting-human-luciferase-tagged-malignant-peripheral

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Medicine

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

18.5K Views.  University of Alabama at Birmingham - UAB. تم وصف طريقة لتطعيم موثوق بها الإنسان الخبيثة luciferase الموسومة غمد ورم الخلايا العصبية الطرفية في العصب الوركي العوز المناعي لدى الفئران. وتناقش أيضا استخدام التصوير تلألؤ بيولوجي لإثبات إنشاء السليم للترقيع الورم ومعايير العزل عشوائية من الحيوانات إلى مجموعات...

Video: Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

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 ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

تقديم وكلاء العلاجية من خلال Intracerebroventricular (ICV) ، وحقن في الوريد (رابعا) في الفئران

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

نماذج مامي لتصوير الخلايا الحية 4D من ورم: التفاعلات المكروية أن التقدم تأثير الخبيثة

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

في الجسم الحي 19 F التصوير بالرنين المغناطيسي لتتبع الخليوي

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

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  ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

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

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

دراسة سرطان البنكرياس الجذعية خصائص الخلايا لتطوير استراتيجيات علاجية جديدة

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

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

جيل من Microtumors عن طريق نظام الثقافة 3D Biogel الإنسان وخلايا ورم أرومي دبقي المستمدة من المريض لKinomic التنميط والدواء استجابة اختبار

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

نضوب خلايا الفأر من Xenografts ورم الإنسان يحسن بشكل ملحوظ تحليل المصب من الخلايا المستهدفة

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

Dosators المتاح لالرئوي النفخ وكلاء العلاجية على الحيوانات الصغيرة

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

في المختبر و في فيفو كشف ميتوفاجي في الخلايا البشرية، ايليجانس جيموالفئران

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...