Bu çalışma, Ulusal Sağlık ve Tıbbi Araştırma Konseyi, Avustralya (1008865), Avustralya Araştırma Konseyi (LE110100125), Ulusal Kanser Enstitüsü (CA138687-01), Erica Sloan Ulusal Meme Kanseri bir Erken Kariyer Bursu tarafından desteklenen tarafından desteklenmiştir Vakfı, Avustralya. Corina Kim-Fuchs İsviçre Kanser Ligi ve İlaç Bilimleri Enstitüsü Monash bir HDR burs bir burs ile desteklenmektedir. Eliane Angst Bern Kanser Ligi bir hibe ile desteklenmektedir.

Gösterdi olan Protokol yazarın kurumun hayvan bakımı ve kullanımı komitesi rehberlik ve onay altında yapılır. Tüm deneyler ilgili tüm kurallar, düzenlemeler ve düzenleyici kurumlar uygun olarak yürütülür. 1. Pankreas Kanseri Hücre Hatları iletici <ol><li> Daha önce tarif edildiği gibi transdüksiyonu pankreas kanseri hücrelerinin lusiferaz ifade etmek. 12,13 Panc-1 ve Capan-1 pankreas kanseri hücre hatlarının ateşböceği lusiferaz ile transduse burada da kullanılmıştır. </li></ol> Not: Renilla lusiferaz ve bakteriyel lusiferaz da kullanılabilir. 2. Pankreas Kanseri Hücre Hazırlık <ol><li> Kültür% 70 konfluent kadar pankreas kanseri hücrelerinin transduse. </li><li> Pankreas hücreleri kaldırın ve canlılığı% 90 daha büyük olduğundan emin olun. </li><li> Matrigel soğutulmuş, 3:2 karışımı içinde 2 x 10 7 hücre / ml &#39;de yeniden süspanse: Fosfat tamponlu tuzine (PBS). </li><li> Önce pankreas içine enjeksiyon için buz üzerinde Matrigel-hücre süspansiyonu tutun. </li></ol> Notlar: Matrigel hızlı katılaşma sağlamak için, hücre pelet hacmi hesaba PBS hacmini azaltmak. Enjeksiyondan önce katılaşma önlemek için her zaman buz gibi araçlar ve şırınga kullanarak Matrigel tutun. Önerilen hücre sayısı bir kılavuzdur ve her bir hücre hattı için ampirik olarak belirlenmelidir. 3. Fare Hazırlık <ol><li> % 2-3 izofluran teneffüs kullanarak fare anestezisi. Hafif bir ayak tutam için pedal refleks eksikliğinden anestezi derinliği belirler. </li><li> Kuruma önlemek için gözleri bir miktar gres sürülmesi. </li><li> 37 ° C ısıtma yastığı sırtında fare yerleştirin ve yavaşça karın sol tarafında yükseltmek için fareyi açın. </li><li> % 10 povidon iyot çözeltisi ile karın hazırlayın. </li></ol> Notlar: Injectable anestezi inhale anestezi yerine kullanılabilir. Ameliyat öncesi açlık gerekli değildir. 4. Laparotomi <ol><li> Steril cerrahi aletler kullanarak yaklaşık 1 cm orta hat yanal sol deride 1.5 cm kesi yapmak. </li><li> Temel karın kas bir 1.5 cm kesi olun. </li><li> Forseps kullanarak dalak bulun ve yavaşça karın boşluğundan dalak çıkarın. Altta yatan pankreas ortaya çıkarmak için steril bir pamuklu çubuk boyunca dalak sabitleyin. </li><li> Dalak bitişik pankreas kuyruk bulun. </li><li> Bir 29 g, 0.3 ml insülin şırınga kullanarak, pankreas içine Matrigel hücre süspansiyonu, 20 ul enjekte edilir. </li><li> Matrigel katılaşmış kadar enjeksiyonu takiben, 30-60 saniye pankreas şırınga tutun. Bu önemli bir adım hücre kaçağı en aza indirir. </li><li> Herhangi bir sızıntı meydana sağlamak için enjeksiyon yerinde kontrol edin. </li><li> Karın boşluğuna dalak ve pankreas dönün. </li></ol> Not: ince olabilir pankreas sırt tarafında delinmesiyle önlemek için dikkat edin. 5. Karın Duvarı Kapatma <ol><li> Sürekli bir dikiş kullanarak bir yuvarlak iğne ile emilebilir örgülü 4-0 sütür ile fare karın kas kapatın. </li><li> Sürekli bir dikiş kullanarak bir kesici iğne ile bir emilmeyen monofilament 6-0 sütür ile dış deri kapatın. </li><li> Inhale anestezik ve deri altına enjekte 0.05-0.1 mg / kg buprenorfin gelen fareyi çıkarın. </li><li> Fare yiyecek ve su ücretsiz erişim ile 37 ° C ısıtma yastığı yerleştirilir kafesine kurtarmak için izin verin. Farenin bu hunching veya azalan hareketlilik gibi ağrı belirtileri gösteriyorsa, buprenorfin, bir 36 saat süre boyunca her 12 saat verilebilir. </li><li> Yara iyileşmesi (7-10 gün) sonra, fare uyutmak ve dış dikişler çıkarın. </li></ol> 6. Pankreas Canc bir Bioluminescent İzlemeer ilerleme <ol><li> Izofluran teneffüs kullanarak fare anestezisi. </li><li> Kuyruk ven yoluyla 150 mg / kg D-luciferase enjekte edilir. </li><li> Bioluminescent görüntüleme sisteminde fare yerleştirin ve daha önce açıklandığı gibi beyaz ışık ve biyolüminesans görüntüleri yakalayabilir. 14,15 </li><li> Inhale anestezik gelen fareyi çıkarın ve onun evde kafes içinde kurtarmak için izin verir. </li></ol> Not: Bioluminescent görüntüleme non-invaziv ve tümör büyüme kinetiği araştırmak için periyodik olarak yapılabilir. Pankreas kuyruk görüntü tümör için onun sol tarafında fare koymak önemlidir, kameraya doğru tümör noktaları çok. Frekans önce FOV 12.5 binning 4 Oturma Görüntüleme 4.3.1 yazılımı çalıştıran bir Lumina II görüntüleme sistemi (Perkin-Elmer, eski Kaliper Yaşam Bilimleri), kullanarak deneysel nokta için haftada üç katına kadar artırılabilir ile Biz, haftada bir kez görüntülendi F-stop 1, pozlama 1 - 60 saniye (en yüksek exp tarafından belirlenirpiksel doygunluk olmadan osure).

<ol>
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(56), e3175 (2011).</li><li>Lim, 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=Monitoring+tumor+metastases+and+osteolytic+lesions+with+bioluminescence+and+micro+CT+imaging.">Monitoring tumor metastases and osteolytic lesions with bioluminescence and micro CT imaging.</a> J. Vis. Exp. (52), e2775 (2011).</li><li>Burton, J. B., 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=Adenovirus-mediated+gene+expression+imaging+to+directly+detect+sentinel+lymph+node+metastasis+of+prostate+cancer.">Adenovirus-mediated gene expression imaging to directly detect sentinel lymph node metastasis of prostate cancer.</a> Nat Med. 14, 882-888 (2008).</li><li>Vezeridis, M. P., Doremus, C. M., Tibbetts, L. M., Tzanakakis, G., Jackson, B. 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U.S.A. 89, 5645-5649 (1992).</li><li>Reyes, G., 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=Orthotopic+xenografts+of+human+pancreatic+carcinomas+acquire+genetic+aberrations+during+dissemination+in+nude+mice.">Orthotopic xenografts of human pancreatic carcinomas acquire genetic aberrations during dissemination in nude mice.</a> Cancer Res. 56, 5713-5719 (1996).</li><li>Kim, M. P., 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=Generation+of+orthotopic+and+heterotopic+human+pancreatic+cancer+xenografts+in+immunodeficient+mice.">Generation of orthotopic and heterotopic human pancreatic cancer xenografts in immunodeficient mice.</a> Nat Protoc. 4, 1670-1680 (2009).</li><li>Furukawa, T., Kubota, T., Watanabe, M., Kitajima, M., Hoffman, R. M. <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+"patient-like"+treatment+model+of+human+pancreatic+cancer+constructed+using+orthotopic+transplantation+of+histologically+intact+human+tumor+tissue+in+nude+mice.">A novel "patient-like" treatment model of human pancreatic cancer constructed using orthotopic transplantation of histologically intact human tumor tissue in nude mice.</a> Cancer Res. 53, 3070-3072 (1993).</li><li>Lewis, C. E., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+role+of+macrophages+in+different+tumor+microenvironments.">Distinct role of macrophages in different tumor microenvironments.</a> Cancer Res. 66, 605-612 (2006).</li><li>Brembeck, F. 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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Burada pankreas tümör gelişimi ve ilerlemesi boyuna değerlendirilmesi için ortotopik modeli tarif. Primer tümör büyüme kinetiği tekrarlanabilir (Şekil 3) ve yeni anti-pankreas kanseri tedavi tümör yanıtı analizleri için örneğin lusiferaz-etiketli hücreleri biyoparlaklık görüntüleme kullanarak non-invaziv takip olabilir. , Insan hastalığı ile uyumlu olarak, model pankreas mikro tümör hücre etkileşimleri inceleme sağlayan yerel pankreas invazyonu (Şekil 4A)...

Pankreas kanseri% 4-6 bir 5-yıllık sağkalım oranı ile, kansere bağlı ölüm dördüncü önde gelen nedenidir. Hastaların 1,2 sadece 15% cerrahi için uygun olduğu hastalığı zamanında teşhis ve tümörler tekrarlayabilir edilir &gt; bu hastaların% 80&#39;inde. 3,4 Gemsitabin pankreas adenokanserlerinin tedavisinde kullanılan, ancak, kemoterapi yaygındır ve genellikle ilaç genel sağkalım üzerinde çok az etkisi vardır. pankreas kanseri tedavisi için 5 Yeni farmakolojik stratejiler kritik ihtiyaç vardır. Onların gelişimi terapötik müdahale duyarlı olabilir hastalığın ilerlemesi en önemli adımlardan önemli ölçüde geliştirilmiş anlayış bağlıdır. Pankreas kanseri ortotopik modelleri pankreas kanseri biyoloji eğitim için ideal alet yapımında, insan hastalığın temel yönlerini taklit. Pankreas kanseri hücre davranış in vitro hücre tabanlı deneyleri aksine bir olarak 6-9pankreas kanseri in vivo modellerde d deri altı, ortotopik modelleri pankreas mikro ile tümör hücre etkileşimlerinin incelenmesi sağlar. Hastalığın ilerlemesi kinetik ortotopik modelleri son derece tekrarlanabilir ve onları da yeni tedavi öncesi klinik test için uygun hale getirir kısa bir süre (hafta), üzerinde meydana gelir. Bu hastalık başlangıcı daha uzun ve daha değişken bir zaman dilimi (ay 1 yıl). 10 daha agresif hücre hatları ile kullanılan gerçekleşir transgenik modelleri aksine olan, pankreas kanseri ortotopik modellerinde görülen benzer spontan metastaz desenler var hasta. gibi ateşböceği lusiferaz gibi bioluminescent muhabiri genlerin 8 İfade tümör büyümesinin boyuna izlenmesi, metastatik yayılması, nüks ve tedavi yanıt kolaylaştırır. 6,11 Burada Matr kullanır pankreas kanseri ortotopik modeli tariftümör progresyonu non-invazif izleme için lokalize hücre teslimat ve in vivo biyoparlaklık görüntüleme için igel. Pankreas kanseri Bu ortotopik modeli hastalığın ilerlemesi ve syngenic veya xenograft modellerinde tedavi girişimlerine yanıt non-invaziv analiz sağlar.

<table border="1">
 <tbody>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Clean Bench coat</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td></td>
 <td></td>
 <td>Set to 37 &deg;C </td>
 </tr>
 <tr>
 <td>Ivis Lumina ll Bioluminescent imager</td>
 <td>Caliper </td>
 <td></td>
 <td>Alternative bioluminescent imaging systems include In vivo F PRO (Carestream) and Photon Imager (Biospace Lab)</td>
 </tr>
 <tr>
 <td>Dissecting scissors</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Iris forceps (serrated)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holder</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>27 G 0.3 ml insulin syringe</td>
 <td>Terumo</td>
 <td>T35525M2913</td>
 <td></td>
 </tr>
 </tbody>
</table>

bioluminescent orthotopic model of pancreatic cancer progression

<p class="jove_content"&gt; Pankreas kanseri% 4-6 son derece kötü beş yıllık sağ kalım oranına sahip. Yeni tedavi seçenekleri kritik gerekli ve pankreas kanseri biyoloji geliştirilmiş anlayış bağlıdır vardır. Daha iyi pankreas mikro ile kanser hücrelerinin etkileşimi anlamak için, kanser ilerleme non-invazif izleme izin pankreas kanseri ortotopik modeli göstermektedir. Lusiferaz etiketli pankreas kanser hücrelerinin Matrigel yeniden süspanse ve laparotomi sırasında pankreas kuyruk içine teslim edilir. Matrigel enjeksiyon sırasında kanser hücrelerinin sızmasını önlemek için, vücut sıcaklığında katılaşır. Uzak organlara birincil tümör büyümesi ve metastazı kullanılarak, lusiferaz substrat luciferase enjeksiyonu takiben izlenir<em&gt; In vivo</emKanser hücrelerinden biyolüminesans emisyon&gt; görüntüleme.<em&gt; In vivo</em&gt; Görüntüleme da rezeksiyon sonrası primer tümörün nüksü izlemek için kullanılabilir. Bu ortotopik modeli singeneik ve ksenograft modelleri hem için uygundur ve birincil pankreas tümörü ve metastaz üzerinde yeni anti-kanser tedavi etkisini araştırmak için ön-klinik çalışmalarda kullanılabilir.</p&gt;

Bu yöntem, anestezi indüksiyonu, laparotomi, Matrigel ve karın kapatılması (Şekil 1A) kanser hücrelerinin enjeksiyonu da dahil olmak üzere cerrahi işlemler, kullanarak pankreas kanseri ortotopik model tanımlamaktadır. Enjekte edilen hücreler, pankreas yüzeyinde bir balon (Şekil 1B) oluşturur. Pankreas kanseri ilerleme non-invaziv kanser hücre çoğalması ve yayılması (Şekil 2) izlemek için in vivo biyolüminesans görüntüleme kul...

Watch this Scientific Journal Video about Bioluminescent Orthotopic Model of Pancreatic Cancer Progression at JoVE.com

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pankreas kanseri biyoloji Geliştirilmiş anlayış eleştirel pankreas kanseri tedavisi için daha iyi tedavi seçeneklerinin gelişimi için gereklidir. Bu ihtiyacı karşılamak için, bizim kullandığımız kanser ilerlemesi non-invazif izleme izin pankreas kanseri ortotopik modeli göstermek<em&gt; In vivo</em&gt; Biyoparlaklık görüntüleme.

Pankreas Kanseri İlerleme Bioluminescent Ortotopik Model

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

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26.7K Görüntüleme.  Monash University. Pankreas kanseri biyoloji Geliştirilmiş anlayış eleştirel pankreas kanseri tedavisi için daha iyi tedavi seçeneklerinin gelişimi için gereklidir. Bu ihtiyacı karşılamak için, bizim kullandığımız kanser ilerlemesi non-invazif izleme izin pankreas kanseri ortotopik modeli göstermek In vivo Biyoparlaklık görüntüleme.

Video: Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Murin Bioluminescent Karaciğer Tümör Modeli

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

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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.

Murin Mesane Kanseri bir Ortotopik Model

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

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.

Oral Skuamöz Hücre Karsinomu Ortotopik Fare Modeli Tümör Hücre Invasion Multi-foton Görüntüleme

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 Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Vivo Çift Yüzey Bioluminescent Imaging&#39;de

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

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.

Bir Ortotopik Fare Beyin Tümörü Modelinde Kombinasyon Radyoterapi

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 Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

Bir Ortotopik Mesane Tümörü Model ve intravezikal Sarna Tedavisinin Değerlendirilmesi

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Hepatik Arter İmar Kısmi Ortotopik Karaciğer Nakli Bir Rat Modeli Cerrahi İşlemleri

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

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.

Gene Teslim Çalışmaları Ortotopik Mesane Kanseri Modeli

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

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.

İnsan Prostat Kanser metastaz Ortotopik fare modeli

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  ...

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.