Questa ricerca è finanziata dalla vescica AACR Henry Shepard Cancer Research Grant (09-60-30-LI).

Procedure che coinvolgono soggetti animali sono stati approvati dal Animal Care istituzionale e Comitato uso (IACUC) presso l&#39;University of California, San Francisco. 1. Preparazione cellulare <ol><li> La linea di carcinoma della vescica umana cellule KU-7-luc2-GFP (Caliper Life Sciences, Hopkinton, MA) viene coltivato in RPMI-1640 integrato con 10% siero fetale bovino e 50 ug / ml di gentamicina. </li><li> Le colture vengono mantenute a 37 ° C, 5% CO 2 con mezzi cambia ogni due o tre giorni. </li><li> Le cellule sono state raccolte mediante digestione con tripsina e contate con un emocitometro. Una sospensione singola cella di 2 x 10 6 cellule in 50 pl è stato preparato in PBS e mantenuto in ghiaccio. </li></ol> 2. Ortotopico modello di tumore della vescica <ol><li> Nudo femminile (nu / nu) topi 8-10 settimane di età (Simonsen Laboratory, Gilory, CA) sono stati utilizzati. </li><li> Posizionare un telino sterile su una piastra elettrica per servire unadi una piattaforma chirurgica. Sterile gel lubrificante, 24-linguistiche cateteri con aghi mandrino, soluzione di nitrato d&#39;argento, e l&#39;acqua sterile sono preparati. </li><li> Anestesia generale viene indotta nei topi con isoflurano per via inalatoria (3% per l&#39;induzione, 1-2% per la manutenzione) e mantenuto via dell&#39;ogiva. Pomata oftalmica sterile viene applicato agli occhi dell&#39;animale, e un cuscinetto di calore viene utilizzato per mantenere la temperatura corporea. </li><li> Posizionare il mouse in posizione supina. </li><li> Afferrare vulva dell&#39;animale con le pinze per la stabilizzazione. </li><li> Utilizzando un 24-gua catetere endovenoso con l&#39;ago con stiletto rimosso, l&#39;uretra mouse è cateterismo. Ampio lubrificazione devono essere utilizzati. Con la posizione supina del mouse, l&#39;uretra si trova immediatamente posteriore alle pieghe della vulva e anteriore della vagina. Un angolo di 45 gradi è inizialmente adottare al fine di incannulare l&#39;uretra e passare sotto l&#39;osso pubico. Un angolo più superficiale viene poi portato a passare attraverso l&#39;uretra nella vescica. Gentle pres pelviciAssicurarsi di raddrizzare l&#39;uretra può essere d&#39;aiuto. Si deve prestare attenzione per evitare di spingere troppo contro la resistenza, in quanto questo può portare a perforazione della vescica o uretrale. Urine visto all&#39;interno del catetere conferma la sua posizione all&#39;interno del lume vescicale. </li><li> Aspirare urina dal lume del catetere vescicale e con l&#39;ago mandrino. L&#39;ago stiletto viene utilizzato per tutte le aspirazioni e successive iniezioni, lasciando il catetere in posizione. Ciò consente di iniezione di volumi precisi eliminando il spazio morto all&#39;interno del catetere, e rende superflua cateterizzazioni ripetute. </li><li> Per mettere in pericolo il livello glicosaminoglicano dell&#39;urotelio vescica, 10 pl di 0,5 M di nitrato d&#39;argento viene iniettato e lasciato abitare per 10 secondi. Ciò può formare un precipitato bianco con urina residua. </li><li> Aspirare ed eliminare i contenuti precipitato e la vescica utilizzando un ago con stiletto. </li><li> Lavare la vescica mediante iniezione di 100 microlitri di acqua sterile. Aspirare ed eliminare l&#39;acqua. REPEa lavaggio con acqua sterile 3 volte per un totale di 4 lavaggi. </li><li> Posizionare una cravatta di seta attorno al 4-0 meato uretrale per occludere l&#39;uretra in preparazione per la rimozione del catetere. </li><li> Iniettare precedentemente preparato 2 x 10 6 KU-7-luc2-GFP cellule in 50 pl usando l&#39;ago mandrino. </li><li> Contemporaneamente rimuovere il catetere e l&#39;ago mandrino, lasciando l&#39;uretra occluso dalla cravatta di seta. </li><li> Il mouse sarà mantenuto in anestesia generale per 2 ore prima che la cravatta di seta viene rimosso e l&#39;animale è risvegliata. </li></ol> 3. Ultrasuono <ol><li> Anestesia generale viene indotta con isofluorano vaporizzato e mantenuto tramite cono di naso. </li><li> A VisualSonics Vevo 770 In Vivo ad alta risoluzione Micro-Imaging System (VisualSonics Inc, Toronto, Ontario, Canada) con il RMV-704 40 MHz sonda è stata utilizzata. </li><li> Posizionare il mouse in posizione supina sulla piattaforma ultrasuoni. </li><li> Fissare zampe posteriori dell&#39;animalecon nastro per evitare movimenti in eccesso durante la manipolazione della sonda ad ultrasuoni. </li><li> Applicare il gel per ultrasuoni al bacino del mouse. </li><li> Abbassare uno RMV-704 (40 MHz) sonda ad ultrasuoni sul bacino del mouse per ottenere immagini trasversali o longitudinali. </li><li> Se il tumore si trova, le immagini possono essere salvate per la misurazione delle dimensioni del tumore. </li></ol> 4. Bioluminescent Imaging <ol><li> Eseguire iniezione intraperitoneale di 200 pl (150 mg / kg) substrato luciferina. </li><li> Indurre anestesia generale con isofluorano vaporizzato e mantenere via cono di naso. </li><li> Mettere i topi in posizione supina nel imager bioluminescenza. </li><li> Cinque minuti dopo l&#39;iniezione luciferina, misurare la bioluminescenza dall&#39;animale in fotoni al secondo. </li></ol> 5. Preparazione sarna <ol><li> Sintetizzare RNA oligonucleotidi ad una scala di 50-100 mol. </li><li> Ricottura complementari a singolo filamento oligonucleotidi into duplex saRNAs. </li><li> Lipid-nanoparticelle (LNP)-sarna vengono preparati con il lipide ionizzabile 1,2-dilinoleyl-4-(2 - dimetilamminoetil) - [1,3]-diossolano (DLinKC2-DMA), disteroylphosphatidyl colina, colesterolo, e PEG-DMG utilizzando una procedura di formazione spontanea vescicola formulazione come precedentemente descritto. 1 </li></ol> 6. La somministrazione intravescicale di Sarna Formulato <ol><li> Dopo aver stabilito il ortotopico modello di tumore della vescica, i topi sono trattati con attivazione intravescicale piccolo RNA (sarna) formulati in nanoparticelle lipidiche (LNPs) (Tabella 1). </li><li> Anestesia generale è stabilito e mantenuto con isofluorano vaporizzato, e lubrificante oftalmica sterile viene applicato. </li><li> Mettete l&#39;animale in posizione supina. </li><li> Eseguire uretrale cateterismo come descritto in precedenza. </li><li> Posizionare una cravatta di seta 4-0 intorno all&#39;uretra e il catetere. </li><li> Aspirare tutto urina dal catetere edella vescica utilizzando una siringa vuota e l&#39;ago mandrino. </li><li> Iniettare LNP-sarna via endovescicale per una dose totale di 50 ml (3 mg / kg). Contemporaneamente rimuovere il catetere e l&#39;ago mandrino, lasciando l&#39;uretra occluso dalla cravatta di seta. </li><li> Il mouse rimarrà anestetizzati con l&#39;uretra occluso per 2 ore. </li><li> Il trattamento con intravescicale LNP-sarna fu iniziata il 4 ° giorno dopo l&#39;impianto del tumore. I topi sono stati trattati in serie, ogni 3 giorni, per un totale di 14 trattamenti. </li></ol> 7. Risultati rappresentativi Tumori della vescica derivanti da KU-7-luc2-GFP cellule può essere monitorato da luciferasi bioluminescenza immagini (Figura 1A e C) e ultrasuoni vescica (Figura 1B e D). I tumori sono spesso rilevabili entro 3-5 giorni dopo l&#39;inoculazione delle cellule. Abbiamo trovato successo l&#39;impianto del tumore della vescica in oltre il 90% dei topi. Come il trattamento con intravescicale dsRNA deriva, la progressione del tumore può essere monitorato with queste modalità. Dopo che l&#39;animale viene sacrificato, la crescita tumorale può anche essere confermata mediante GFP fluorescenza. In generale, la media bioluminescenza correlata con le dimensioni medie ultrasuoni per tumori della vescica, come si vede visivamente in figure 1C e 1D. Tuttavia, dopo gli animali sono stati sacrificati e tumore è stata confermata da GFP, abbiamo trovato che bioluminescenza più strettamente correlata con la presenza di tumore conveniente rispetto fatto misurazioni ultrasuoni. Ciò è particolarmente evidente tra topi trattati con sarna (rispetto ai controlli) perché il tessuto cicatriziale residuo in topi trattati era spesso visualizzata mediante ultrasuoni, ma potrebbe non essere differenziata da tumore vivo. <img src="/files/ftp_upload/4207/4207fig1.jpg" alt="Figura 1" /> Figura 1. Cinetica di crescita tumorale del tumori della vescica. (A) di imaging bioluminescenza misura l&#39;attività della luciferasi all&#39;interno del tumore della vescica per confermare la loro posizione e la crescita. (B) In ultrasoun vescica vivod utilizzando una sonda 40MHz produce immagini tumorali cui dimensioni possono essere misurata con precisione. (C) grafici che illustrano aumento progressivo del tumore in dimensioni e bioluminescenza ultrasuoni. I valori rappresentano la media (+ / - deviazione standard) le misure da 6 animali. <a href="http://www.jove.com/files/ftp_upload/4207/4207fig1large.jpg" target="_blank">Clicca qui per ingrandire la figura</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 è un inventore di nome in attesa di domande di brevetto relative a sarna che siano stati presentati dalla University of California San Francisco e concesso in licenza a Alnylam Pharmaceuticals.

Vi presentiamo un protocollo per l&#39;istituzione di un modello murino ortotopica tumore della vescica xenotrapianto seguito da un trattamento intravescicale dei tumori con sarna LNP formulato che bersaglia il promotore di CIP1/WAF1 p21 (p21) per l&#39;attivazione della trascrizione. 2, 3 I tumori risultanti possono essere confermata e monitorata con immagini e bioluminescenti ultrasuoni vescica. Modelli ortotopica può essere superiore a intraperitoneale, sottocutanea, endovenosa o modelli fornen...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> KU-7-luc-GFP le cellule </td><td> Pinza </td><td> 128091 </td><td></td></tr><tr><td> timici topi nudi (nu / nu) </td><td> Simonsen Laboratories </td><td> 6-7 settimane </td><td> Sim: (NCR) nu / nu fisol </td></tr><tr><td> Ultrasuoni unità </td><td> Visualsonics </td><td> Vevo 770 </td><td> RMV-704 sonda </td></tr><tr><td> Bioluminescenza unità </td><td> Pinza </td><td> IVIS Spectrum </td><td></td></tr><tr><td> Exel safelet catetere </td><td> Pescatore </td><td> 14-841-21 </td><td> 24G X ¾ &quot; </td></tr><tr><td> Di nitrato d&#39;argento </td><td> Sigma </td><td> S8157 </td><td></td></tr><tr><td> Hamilton siringa </td><td> Hamilton company </td><td> 80301 </td><td></td></tr><tr><td> LNP-sarna </td><td> Alnylam Pharmaceuticals </td><td></td><td></td></tr></tbody></table> Tabella 1. Reagenti e attrezzature specifiche.

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

Presentiamo un nuovo metodo per il trattamento del cancro della vescica con attivante RNA via endovescicale consegnato piccolo (sarna) in un modello murino ortotopico eterotrapianto tumore della vescica. Il modello murino è stabilito dal cateterismo uretrale sotto anestesia generale per via inalatoria. Combustioni chimiche viene quindi introdotto alla mucosa della vescica utilizzando intravescicale soluzione di nitrato d&#39;argento di distruggere lo strato di glicosaminoglicani vescica e permette alle cellule di allegare. Dopo alcuni lavaggi con acqua sterile, cancro della vescica umana KU-7-luc2-GFP cellule sono instillati attraverso il catetere in vescica di sosta per 2 ore. Successiva crescita dei tumori vescicali è confermata e monitorata da ecografia vescicale in vivo e di imaging bioluminescente. I tumori vengono poi trattati per via endovescicale con sarna formulata in nanoparticelle lipidiche (LNPs). La crescita del tumore è monitorata con gli ultrasuoni e la bioluminescenza. Tutte le fasi di questa procedura sono illustrati nel video allegato.

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

Stabilire un modello di tumore della vescica ortotopica per valutare gli effetti antitumorali di sarna via endovescicale consegnate e monitoraggio della crescita tumorale con l&#39;ecografia e imaging bioluminescente.

Un modello ortotopico tumore della vescica e la valutazione del trattamento intravescicale sarna

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 Views.  University of California, San Francisco. Stabilire un modello di tumore della vescica ortotopica per valutare gli effetti antitumorali di sarna via endovescicale consegnate e monitoraggio della crescita tumorale con l'ecografia e imaging bioluminescente.

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.

Un modello ortotopico di cancro alla vescica Murine

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

Ricerca

Medicina

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.

Multi-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

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.

La radioterapia combinata in un modello di tumore ortotopico cervello di topo

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.

Un modello di mouse ortotopico di anaplastico della tiroide Carcinoma

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.

Un ortotopico cancro della vescica Modello di Gene Studi di consegna

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.

Un ortotopico Modello murino di Human Prostate Cancer Metastasi

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.

Mini-Invasiva Istituzione di murini ortotopico vescica Xenotrapianti

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.

Valutazione del tumore infiltrante leucociti sottoinsiemi in un tumore sottocutaneo modello

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.

Valutazione dell&#39;efficacia della<em&gt; H. pylori</em&gt; Proteine ​​HP-NAP come strumento terapeutico per il trattamento del cancro della vescica in un ortotopico Modello murino

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.

A &quot;paziente-Like&quot; ortotopico Singenico modello murino della carcinoma epatocellulare Metastasi

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 ...