Bu araştırma AACR Henry Shepard Mesane Kanseri Araştırma Bursu (09-60-30-LI) tarafından finanse edilmektedir.

Hayvan denekleri Usul Kaliforniya, San Francisco Üniversitesi Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) tarafından onaylanmıştır. 1. Hücre Hazırlık <ol><li> İnsan mesane kanseri hücre hattı KU-7-luc2-GFP (Kaliper Yaşam Bilimleri, Hopkinton, MA)% 10 fetal sığır serumu ve 50 mg / ml gentamisin ile takviye edilmiş RPMI-1640 medya yetiştirilir. </li><li> Kültürler 37 ° C&#39;de muhafaza edilir, medya ile CO 2, her iki üç günde bir değişen 5%. </li><li> Hücreler tripsin sindirimi ile toplandı ve bir hemasitometre ile sayıldı. 50 ul içinde 2 x 10 6 hücrelerinin tek bir hücre süspansiyonu fosfat tamponlu tuz hazırlandı ve buz üzerinde tutuldu. </li></ol> 2. Ortotopik Mesane Tümör Modeli <ol><li> 8-10 haftalık kadın çıplak (nu / nu) farelerde (Simonsen Laboratuvarı, Gilory, CA) kullanılmıştır. </li><li> Bir hizmet için bir ısıtma pedi üzerine steril bir drape yerleştirinsa cerrahi platformu. Steril yağlama jöle, stileyi iğneler, gümüş nitrat çözeltisi ve steril su ile 24-dil bilgisi olmalıdır kateterler hazırlanır. </li><li> Genel anestezi inhale izofluran (indüksiyon için% 3, bakım için% 1-2) ve roket ucu konisi ile muhafaza ile farelerde indüklenir. Steril oftalmik merhem hayvanın göze tatbik edilir ve bir ısı pedi vücut ısısını muhafaza etmek için kullanılır. </li><li> Yatar konumda fare yerleştirin. </li><li> Yavaşça stabilizasyonu için forseps ile hayvanın vulva kavramak. </li><li> Kaldırıldı iğne stile sahip 24 dil bilgisi olmalıdır intravenöz kateter kullanarak, fare üretra kateterize edilir. Geniş yağlama kullanılmalıdır. Fare supin ile üretra vulva kıvrımlar ve vajina ön hemen arka yer almaktadır. A 45 derece açı başlangıçta pubis altında üretra kanüle ve geçmek için alınır. Bir daha sığ açı sonra mesane içine üretra geçmesine alınır. Nazik pelvik presüretra genellikle yardımcı olabilir düzeltmek için emin. Bakım Bu üretral veya mesane perforasyonu yol açabilir gibi, dirence karşı çok sert bastırıyor önlemek için alınmalıdır. Kateter içinde görülen idrar kesesi lümeninde konumunu teyit etmektedir. </li><li> Stilenin iğne kullanılarak kateter ve mesane lümeni idrar aspire. Stileyi iğne yerde kateter bırakarak, daha sonraki tüm hedefleri ve enjeksiyon için kullanılır. Bu kateter içinde ölü boşluk ortadan kaldırarak hassas hacimli enjeksiyon için izin verir ve tekrar kateterizasyonlarda gereksiz kılar. </li><li> Mesane ürotelium&#39;un glikozaminoglikan tabakası zarar için 0.5 M gümüş nitrat 10 uL enjekte ve 10 saniye durmak için izin verilir. Bu kalan idrarı ile beyaz bir çökelti meydana gelebilir. </li><li> Aspire ve bir stile iğne ile çökelti ve mesane içeriğini atın. </li><li> 100 uL steril su enjekte edilerek mesanenin yıkayın. Su aspire atın. Repe4 yıkar toplam steril su ile yıkama az 3 kez. </li><li> Kateter hazırlık üretra tıkanmak üzere meatusa etrafında 4-0 ipek kravat yerleştirin. </li><li> Daha önce enjekte stileyi iğne kullanılarak 50 uL içinde 2 x 10 KU 6-7-luc2-GFP hücreleri hazırlandı. </li><li> Aynı anda ipek kravat sıkıştırıldı üretra bırakarak, kateter ve stile iğne çıkarılır. </li><li> Ipek kravat kaldırılır ve hayvan uyanmış önce fare 2 saat için genel anestezi altında muhafaza edilecektir. </li></ol> 3. Ultrason <ol><li> Genel anestezi buharlaşmış izofluran ve burun konisi ile muhafaza ile indüklenir. </li><li> Vivo A VisualSonics VEVO 770 Yüksek Çözünürlük Mikro-Görüntüleme Sistemi (VisualSonics Inc, Toronto, Ontario, Kanada) RMV-704 40 MHz prob kullanıldı. </li><li> Ultrason platform üzerinde yatar pozisyonda fare yerleştirin. </li><li> Hayvanın arka ayakları Güvenlibant ile ultrason probu manipülasyonu sırasında aşırı hareketi önlemek için. </li><li> Fare pelvise ultrason jeli uygulayın. </li><li> Enine ya da boyuna görüntüler elde etmek için fare pelvis üzerine bir RMV-704 (40 MHz) ultrason probu indirin. </li><li> Tümör bulunursa, görüntüler tümör boyutlarının ölçümü için kaydedilebilir. </li></ol> 4. Bioluminescent Görüntüleme <ol><li> 200 uL (150 mg / kg) lusiferin substrat intraperitoneal gerçekleştirmek. </li><li> Izofluran buharlaşmış ile genel anestezi indükleyin ve burun konisi ile korumak. </li><li> Biyolüminesans görüntüleyici içinde yatar pozisyonda fareler yerleştirin. </li><li> Beş dakika lusiferin enjeksiyonundan sonra, saniyede fotonlar içinde hayvan biyolüminesans ölçmek. </li></ol> 5.. Sarna Hazırlık <ol><li> 50-100 mol ölçeğinde oligonükleotidler RNA sentezi. </li><li> Tamamlayan tek iplikli oligonükleotidler tavlama inGoogle&#39;a dubleks saRNAs. </li><li> [1,3]-dioksolanın (DLinKC2-DMA), disteroylphosphatidyl kolin, kolesterol, ve PEG-DMG - lipid-nanoparçacık (LNP)-Sarna iyonize olabilen lipid 1,2-dilinoleyl-4-(- dimetilaminoetil 2) ile hazırlanır Daha önce tarif edildiği gibi bir spontan vesikül oluşumu formülasyon prosedürü kullanarak. 1 </li></ol> 6. Formüle Sarna intravezikal İdaresi <ol><li> Ortotopik mesane tümör modeli oluşturulduktan sonra, farelerin lipid nano partiküller (LNPs) (Tablo 1) formüle intravezikal küçük aktive RNA (Sarna) ile tedavi edilir. </li><li> Genel anestezi kurulmuş ve muhafaza izofluran buharlaştırılmış, ve Steril oftalmik Yağlayıcı maddenin tatbik edilir. </li><li> Yatar konumda hayvan yerleştirin. </li><li> Daha önce açıklandığı gibi üretral kateterizasyon gerçekleştirin. </li><li> Üretra ve kateter etrafında 4-0 ipek kravat yerleştirin. </li><li> Kateter tüm idrar aspire veboş bir şırınga ve iğne kullanılarak stileyi mesane. </li><li> 50 uL (3 mg / kg) toplam dozu için intravesikal LNP-Sarna enjekte edilir. Aynı anda ipek kravat sıkıştırıldı üretra bırakarak, kateter ve stile iğne çıkarılır. </li><li> Fare 2 saat boyunca tıkanmadığı üretra ile anestetize kalacaktır. </li><li> Intravezikal LNP-Sarna ile tedavi tümör implantasyonu sonrası 4. Gün başlanmıştır. Fareler 14 tedaviler olmak üzere toplam, her 3 günde bir, seri tedavi edildi. </li></ol> 7. Temsilcisi Sonuçlar KU-7-luc2-GFP hücrelerinden kaynaklanan mesane tümörleri lusiferaz bioluminescent görüntüleme (Şekil 1A ve C) ve mesane ultrasonu (Şekil 1B ve D) ile izlenebilir. Tümörler Hücre inokülasyonundan sonra 3-5 gün içinde sık sık saptanabilir. Biz farelerin% 90&#39;ında başarılı mesane tümörü implantasyon bulundu. Intravezikal dsRNA ile muamele ensues olarak, tümör ilerlemesi Wi izlenebilirbu yöntemlerin th. Hayvan kurban sonra, tümör büyümesi, aynı zamanda GFP flöresanı kullanılarak teyit edilebilir. Genel olarak, biyolüminesans olarak Şekil 1C ve 1D görsel olarak görülen, mesane tümörleri için ortalama ultrason boyutları ile ilişkili demek. Hayvanlar kurban edildi ve tümör GFP tarafından onaylandıktan sonra Ancak, biz bu biyolüminesans ultrason ölçümleri mi daha canlı tümör varlığı ile daha yakından ilişkili bulundu. Tedavi edilen farelerde rezidüel skar dokusu genellikle ultrason ile görüntülendi ama canlı tümör ayırt edilemedi çünkü bu Sarna (as kontrolleri aksine) ile tedavi edilen farelerde arasında özellikle belirgin oldu. <img src="/files/ftp_upload/4207/4207fig1.jpg" alt="Şekil 1" /> Şekil 1. Mesane tümörlerinin tümör büyüme kinetiği. (A) Bio-ışıldama, görüntüleme konumlarına ve büyüme teyit etmek için mesane tümörleri içinde lüsiferaz faaliyeti ölçülür. (B) &#39;in vivo olarak mesane ultrasond 40 MHz prob ile olan boyutları ölçülebilir tümör görüntüleri üretir. (C) Grafikler biyolüminesans ve ultrason boyutlarda tümörün sürekli artış gösteren. 6 hayvanlardan ölçümleri - Değerler (standart sapma + /) anlamına temsil eder. <a href="http://www.jove.com/files/ftp_upload/4207/4207fig1large.jpg" target="_blank">büyük rakam görmek için buraya tıklayın</a> .

<ol>
	<li>Semple, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+cationic+lipids+for+siRNA+delivery.">Rational design of cationic lipids for siRNA delivery.</a> Nature. 28, 172-176 (2010).</li><li>Li, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+dsRNAs+induce+transcriptional+activation+in+human+cells.">Small dsRNAs induce transcriptional activation in human cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 17337-17342 (2006).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effect+of+dsRNA-induced+p21(WAF1/CIP1)+gene+activation+in+human+bladder+cancer+cells.">Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells.</a> Molecular cancer therapeutics. 7, 698-703 (2008).</li><li>Chancellor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+of+myoblast+injection+into+the+urethra+and+bladder+wall:+a+possible+method+for+the+treatment+of+stress+urinary+incontinence+and+impaired+detrusor+contractility.">Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility.</a> Neurourology and urodynamics. 19, 279-287 (2000).</li><li>Hadaschik, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+mouse+model+for+orthotopic+bladder+cancer+using+transurethral+tumour+inoculation+and+bioluminescence+imaging.">A validated mouse model for orthotopic bladder cancer using transurethral tumour inoculation and bioluminescence imaging.</a> BJU international. 100, 1377-1384 (2007).</li><li>Fu, C., Apelo, C. A., Torres, B., Thai, K. H., Hsieh, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Bladder+Wall+Injection.">Mouse Bladder Wall Injection.</a> J. Vis. Exp. (53), e2523 (2011).</li><li>Dobek, G. L., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Orthotopic+Model+of+Murine+Bladder.">An Orthotopic Model of Murine Bladder.</a> Cancer. J. Vis. Exp. (48), 2535 (2011).</li><li>Dinney, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+metastatic+variants+from+human+transitional+cell+carcinoma+passaged+by+orthotopic+implantation+in+athymic+nude+mice.">Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice.</a> The Journal of urology. 154, 1532-1538 (1995).</li></ol>

LCL California San Francisco Üniversitesi tarafından açılan ve Alnylam İlaç için lisans almış Sarna ile ilgili patent başvurularının bekleyen bir adlandırılmış mucididir.

Biz transkripsiyon aktivasyonu için p21 CIP1/WAF1 (p21) yükseltici hedefler LNP formüle Sarna ile tümörlerinin intravezikal tedavisi ardından bir fare ortotopik xenograft mesane tümörü modelinin kurulması için bir protokol mevcut. 2, 3 çıkan tümörler olabilir bioluminescent görüntüleme ve mesane ultrason ile doğrulandı ve izlenmelidir. Ortotopik modelleri idrar ve daha yakından endojen mesane kanseri yakın olduğunu ürotelium&#39;un bir ortam sağlayarak periton, subkutan vey...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> KU-7-luc-GFP hücreleri </td><td> Kaliper </td><td> 128091 </td><td></td></tr><tr><td> timik çıplak farelerde (nu / nu) </td><td> Simonsen Laboratuvarları </td><td> 6-7 haftalık </td><td> Sim: (NCR) nu / nu fisol </td></tr><tr><td> Ultrason ünitesi </td><td> Visualsonics </td><td> VEVO 770 </td><td> RMV-704 prob </td></tr><tr><td> Biyolüminesens ünitesi </td><td> Kaliper </td><td> IVIS Spektrum </td><td></td></tr><tr><td> Exel safelet kateter </td><td> Balıkçı </td><td> 14-841-21 </td><td> 24G X ¾ &quot; </td></tr><tr><td> Gümüş nitrat </td><td> Sigma </td><td> S8157 </td><td></td></tr><tr><td> Hamilton enjektörü </td><td> Hamilton company </td><td> 80301 </td><td></td></tr><tr><td> LNP-Sarna </td><td> Alnylam İlaç </td><td></td><td></td></tr></tbody></table> Tablo 1. Özel reaktifleri ve ekipmanları.

an orthotopic bladder tumor model and the evaluation of intravesical sarna treatment

Biz, ortotopik xenograft fare mesane tümörü modelinde, intravesikal teslim küçük bir aktive edici RNA (Sarna) ile mesane kanseri tedavisi için yeni bir yöntem sunmaktadır. Fare modeli inhale genel anestezi altında üretral kateterizasyon tarafından kurulmuştur. Kimyasal yanık sonra mesane glikozaminoglikan tabakası bozmaya intravezikal gümüş nitrat çözeltisi kullanılarak mesane mukozasına tanıttı ve hücrelere eklemenizi sağlar edilir. Steril su, mesane kanseri, insan KU-7-luc2-GFP hücreleri ile çok yıkamadan sonra 2 saat süreyle durmak kesesi içine kateter ile aşılanan edilir. Mesane tümörlerinin daha sonraki büyüme vivo mesane ultrasonu ve bioluminescent görüntülemede tarafından onaylandıktan ve izlenir. Tümörler daha sonra Sarna lipid nano partiküller (LNPs) formüle ile intravezikal tedavi edilir. Tümör büyüme ultrason ve biyolüminesans ile izlenir. Bu prosedür her adım, ekli video gösterilmiştir.

Watch this Scientific Journal Video about An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment at JoVE.com

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

Ultrason ve bioluminescent görüntüleme ile intravezikal teslim Sarna ve izleme tümör büyümesinin antitümör etkileri değerlendirmek için ortotopik mesane tümörü modeli oluşturmak.

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 ...

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Research

JoVE Journal

Medicine

14.6K Görüntüleme.  University of California, San Francisco. Ultrason ve bioluminescent görüntüleme ile intravezikal teslim Sarna ve izleme tümör büyümesinin antitümör etkileri değerlendirmek için ortotopik mesane tümörü modeli oluşturmak.

Video: An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

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 ...

Tıp

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 ...

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 Mouse Model of Anaplastic Thyroid Carcinoma

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

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

Anaplastik Tiroid Kanseri Ortotopik Fare Modeli

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

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  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Kemirgen Ortotopik Mesane ksenogratflarının Minimal İnvaziv kurulması

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Bir deri altı tümör modelinde tümör-süzücü lökosit subsetleri Değerlendirilmesi

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Etkinliğinin Değerlendirilmesi<em&gt; H. pylori</emOrtotopik Fare Modelinde Mesane Kanseri Tedavisinde Terapötik Aracı Olarak&gt; Protein HP-NAP

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

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

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

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

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

Bir &quot;Hasta gibi&quot; Ortotopik singenik Fare Modeli Hepatoselüler karsinomu metastazı

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