Questa ricerca è sostenuta da un finanziamento del programma intramurale del NIH.

I. Preparazione cellulare <ol><li> Le cellule vengono coltivate a confluenza in DMEM con 10% FBS circa un 80% confluenti 225 centimetri pallone da 3 per ogni 5 topi per essere impiantate. </li><li> Aspirare mezzi fuori delle cellule, lavare con 10 ml di PBS senza calcio o magnesio. Aspirato PBS le cellule. </li><li> Tripsinizzare le cellule con 3 ml di tripsina, attendere 10-15 min, e neutralizzare la tripsina con 15 ml di mezzo. </li><li> Pipettare tutte le cellule ei media in una ml 50 conica. Due flaconi grandi inserirsi in uno da 50 ml. </li><li> Spin celle a 1600 rpm per 7 minuti a 4 ° C. Completamente Aspirare il surnatante. Risospendere le cellule in 10 ml di PBS freddo, e risciacquare cellule delicatamente pipettando su e giù. Assicurarsi che il pellet di cellule è mescolato uniformemente con la PBS. La sospensione deve essere torbida. </li><li> In questo momento un conteggio delle cellule avviene tramite un contatore Coulter. Le cellule vengono centrifugate nuovamente a 1600 rpm per 7 minuti a 4 ° C. </li><li> Dopo la centrifugazione 2 ° delle cellule, comepirata tutti PBS e risospendere in 1 ml di PBS. </li><li> Le cellule vengono filate in 1 ml di PBS a 3000 rpm per 10 min a 4 ° C. Il PBS viene aspirata, e PBS viene aggiunto per regolare la concentrazione finale di 1x10 6 per 5 microlitri di cellule U87, altre linee cellulari possono richiedere un numero diverso e concentrazione di cellule per la crescita tumorale ottimale. </li><li> Dopo che il volume finale e concentrazione si ottiene le cellule sono tenuti in ghiaccio in una provetta Eppendorf. </li></ol> II. Xenotrapianto <ol class="ualpha"><li> Preparazione del topo <ol><li> I topi vengono anestetizzati mediante iniezione di un mix ketamina / Xylazina / Saline. La miscela è 120 mg / kg di Ketamina, 16 mg / kg di Rompun e Saline. I topi vengono pesati singolarmente e dato 0,1 ml ogni 10 g in base al loro peso. L&#39;anestesia viene praticata con ago 26G collegato al 1 IP ml siringa. Ci vorranno circa 15 min per i topi di raggiungere una fase di sonno per non risposta al dolore. È possibile controllare la risposta al dolore dellatopi di pizzicare la loro zampa. </li><li> Dopo il mouse è completamente anestetizzato, unguento oculare viene somministrato ai loro occhi per evitare che si secchi. </li><li> Il mouse è posizionato sul letto del stereotassica con i denti anteriori superiori della tacca sulla barra di bocca. Il letto viene poi inserito nel stereotassico. </li><li> La guardia naso è posta sopra il naso del mouse per impedire il movimento della testa, e le barre di auricolari sono gentilmente inserire nei canali dell&#39;orecchio dei topi. Fare attenzione a non fare né il posizionamento della barra naso o barre di auricolari troppo stretti, come il cranio possono fratturarsi e / o impedire il mouse di respirare. </li><li> Assicurarsi che la testa del mouse è il livello utilizzando le tacche sul stereotassica è utile durante questo processo. </li><li> Per la preparazione iodio topo, alcol e providine viene applicato liberamente sulla testa del mouse alternata per un totale di 3 applicazione delle applicazioni alcool e 3 di iodio. Una applicazione finale di un tampone di alcool con il 70% viene applicatala testa. </li></ol></li><li> Procedura chirurgica <ol><li> Tutti gli strumenti della procedura devono essere sterilizzati in autoclave o sia immergendo gli strumenti in etanolo al 70% prima di iniziare la procedura. </li><li> L&#39;incisione viene fatta sulla sommità del cranio dietro l&#39;occhio destro in diagonale indietro alla posteriore sinistra del cranio. L&#39;incisione dovrebbe essere circa 10 mm a 15 mm. </li><li> Usando un Q-tip, lentamente separare l&#39;incisione per esporre il cranio e spazzare via la membrana che è tra il cranio e la pelle. Utilizzare un Q-tip a cancellare il sangue e guardare per sanguinamento eccessivo. </li><li> Esporre la parte superiore del cranio per visualizzare il bregma. Utilizzando il punto di siringa Hamilton, posizionarlo sopra il bregma. La corretta posizione del foro da praticare nel cranio è 2 destra mm e 1 mm anteriore del bregma (figura 2). Un chirurgo punta di feltro o marcatore, che viene acquistato sterile, è usato per indicare la posizione del foro da forare. Si noti che il foro si trova vicino alla unosulla linea di sutura terior. </li><li> Quindi praticare un piccolo foro nel cranio nella posizione indicata usando la punta di un ago 22G in un vaso PIN o alimentato attraverso un ago 18G. </li><li> Caricare il 10 pl vetro Hamilton siringa con 5 microlitri di cellule. La siringa Hamilton è 10 microlitri con una 30G a 33G blunt ago fisso collegato ad esso. Assicurarsi di mescolare le cellule prima di caricarli, sia pipettando loro o utilizzando una siringa da 1 ml con un ago 25G attaccato. Dopo la siringa Hamilton è stato caricato, porla di nuovo in braccio per allinearla con il foro nel cranio. </li><li> Abbassare l&#39;estremità smorzata dell&#39;ago alla superficie del cranio, una volta che l&#39;ago è a livello del cranio, abbassare l&#39;ago fino a che si trova alla profondità di 3 mm sotto la superficie del cranio. </li><li> Dopo che l&#39;ago è abbassato, esso rimane per 2 minuti per evitare qualsiasi trauma grave dall&#39;ago che colpiscono il cervello. Impianto delle cellule è ad una velocità di 1 pl al minuto per un totale di 6-8 minuti, attenzione di qualsiasi backflow. Dopo l&#39;ultima delle cellule sono stati impiantati, lasciare l&#39;ago in posizione per altri 2 minuti, per permettere a tutte le cellule di sedimentare dentro </li><li> Togliere l&#39;ago dal cervello, e pulire il foro con un Q-tip. Procedere alla pulizia della ago e siringa con 50% acetone, 100% EtOH, e PBS, 3x in ciascuna soluzione e in questo ordine. </li><li> La pelle del cranio è pronto per essere suturato in questo punto. Utilizzando 5-0 PBS chiudere l&#39;incisione con circa 3 punti di sutura. </li><li> Lasciare il mouse per svegliarsi su un blocco di calore, e riapplicare unguento oculare, se necessario. Il mouse può essere restituito alla gabbia dopo che mostra il movimento da solo (cioè Sollevando la testa o spostando le spalle). </li></ol></li></ol> III. Il trattamento dell&#39;impianto IC By Radiation and Drug <ol class="ualpha"><li> Radioterapia <ol><li> A seconda della linea cellulare, il trattamento dei topi può avvenire 5 a 7 giorni dopo l&#39;impianto. Il nostro reparto utilizza una unità di radioterapia per il trattamento orthovoltage radiazionimento. </li><li> Imaging come BLI (Imaging bioluminescenza), RM o TC può essere usato per visualizzare il tumore prima della randomizzazione (figura 3A). </li><li> I topi sono anestetizzati con il cocktail di ketamina / Xylazina / soluzione salina come prima, e pomate oftalmiche viene applicata ai loro occhi. </li><li> I topi sono posizionati in una dima personalizzato radiazione fatto che è configurata come una ruota torta, in cui le teste dei topi puntare verso il centro. I corpi dei topi sono schermati dalla radiazione con il piombo. </li><li> Il trattamento farmacologico può essere somministrato prima o radiazione attraverso percorsi di consegna diversi, ad esempio SQ, di IP e IV. </li></ol></li></ol> IV. Risultati Il mouse deve svegliarsi dalla procedura entro 45-60 minuti dopo l&#39;iniezione anestesia. Se il mouse è difficilmente risveglio dopo la procedura e il tempo ha superato 1 ½ ora dopo la procedura, il mouse non può recuperare e l&#39;eutanasia può essere un&#39;alternativa efficace. Se there è il sanguinamento eccessivo o il teschio diventa incrinato in qualsiasi momento durante la procedura di eutanasia è consigliato. Se tutto va bene e, a seconda della linea cellulare, un tumore dovrebbe iniziare a formare entro 2-4 settimane dopo l&#39;intervento ha luogo. Quando si utilizzano modelli di xenotrapianto intracraniche, l&#39;efficacia terapeutica è di solito misurato come la sopravvivenza post-impianto 5. Tuttavia, poiché questo è un modello IC segni ed i sintomi si verificano prima della morte. Alcuni dei segni fisici del mouse si può presentare una postura ricurva, perdita di peso corporeo, e l&#39;inattività. Alcuni dei segni neurologici possono essere convulsioni, inclinazione testa, e la perdita di equilibrio. Figura 3B mostra un HE macchia di un tumore alla mortalità. A Kaplan-Meier è utilizzato per valutare i dati (figura 4). Il numero di giorni di sopravvivenza al 50% e vengono calcolati rispetto al controllo. Se il gruppo di combinazione vissuto più a lungo l&#39;aggiunta della radiazione singolo farmaco e braccia la combinazione è avuto successo. <img alt = &quot;Figura 1&quot; src = &quot;/ files/ftp_upload/3397/3397fig1.jpg&quot; /&gt; Figura 1. L&#39;apparecchiatura Stoelting stereotassica. <img alt="Figura 2" src="/files/ftp_upload/3397/3397fig2.jpg" /> Figura 2. Un diagramma della posizione del foro nel cranio topo. <img alt="Figura 3A" src="/files/ftp_upload/3397/3397fig3A.jpg" /> Figura 3A. BLI immagini dopo l&#39;impianto GBM. <img alt="Figura 3B" src="/files/ftp_upload/3397/3397fig3B.jpg" /> Figura 3B. H e E di tumore con una mortalità per GBM. <img alt="Figura 4" src="/files/ftp_upload/3397/3397fig4.jpg" /> Figura 4. Risultati U87 IC impianti iniettati con inibitore di PARP 7 giorni dopo l&#39;intervento chirurgico.

<ol>
	<li>Bleau, A. M., Holland, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+the+mouse+genome+to+hunt+human+alternations.">Trapping the mouse genome to hunt human alternations.</a> Proc. Nat. Acad. Sci. U.S.A. 104, 7737-7738 (2007).</li><li>McGrady, B. J., McCormick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+intracranial+invasion:+morphological+observations+on+central+nervous+system+invasion+by+melanoma+cells.">A murine model of intracranial invasion: morphological observations on central nervous system invasion by melanoma cells.</a> Clin. Exp. Metastasis. 10, 387-393 (1992).</li><li>Fei, X., Zhang, Q., Dong, J., Diao, y., Wang, Z., Li, R., Wu, Z., Wang, A., Lan, Q., Zhang, S., Huang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+clinically+relevant+orthotopic+xenograft+mouse+model+of+metastatic+lung+cancer+and+glioblastoma+through+surgical+tumor+tissues+injection+with+trocar.">Development of clinically relevant orthotopic xenograft mouse model of metastatic lung cancer and glioblastoma through surgical tumor tissues injection with trocar.</a> Journal of Experimental &#38; Clinical Cancer Research. 29, 84-84 (2010).</li><li>Fujita, M., Zhu, X., Sasaki, K., Ueda, R., Low, K., Pollack, I., Okada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+STAT3+Promotes+the+Efficacy+of+Adoptive+Transfer+Therapy+Using+Type-1+CTLs+by+Modulation+of+the+Immunological+Microenvironment+in+a+Murine+Intracranial+Glioma.">Inhibition of STAT3 Promotes the Efficacy of Adoptive Transfer Therapy Using Type-1 CTLs by Modulation of the Immunological Microenvironment in a Murine Intracranial Glioma.</a> J. Immunol. 180, 2089-2098 (2008).</li><li>Candolfi, M., Curtin, J. F., Nichols, W. S., Muhammad, A. K. M. .. . G., King, G., Pluhar, G. D., McNiel, G. E., Ohlfest, E. A., Freese, J. R., Moore, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+glioblastoma+models+in+preclinical+neuro-oncology:+neuropathlogical+characterization+and+tumor+progression.">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathlogical characterization and tumor progression.</a> J. Neurooncol. 85, 133-148 (2007).</li><li>Kaye, A., Morstyn, G., Gardner, I., Pyke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+xenograft+glioma+model+in+mouse+brain.">Development of a xenograft glioma model in mouse brain.</a> Cancer Research. 46, 1367-1373 (1986).</li><li>Zhao, Y., Xiao, A., diPierro, C., Carpenter, J., Abdel-Fattah, R., Redpath, G., Lopez, M., Hussaini, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Extensive+Invasive+Intracranial+Human+Glioblastoma+Xenograft+Model.">An Extensive Invasive Intracranial Human Glioblastoma Xenograft Model.</a> American Journal of Pathology. 176, 3032-3049 (2010).</li><li>Learn, C., Grossi, P., Schmittling, R., Xie, W., Mitchell, D., Karikari, I., Wei, Z., Dressman, H., Sampson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Analysis+of+Intracranial+Tumors+in+a+Murine+Model+of+Glioma+Demonstrate+a+Shift+in+Gene+Expression+in+Response+to+Host+Immunity.">Genetic Analysis of Intracranial Tumors in a Murine Model of Glioma Demonstrate a Shift in Gene Expression in Response to Host Immunity.</a> J. Neuroimmunol. 182, 63-72 (2007).</li><li>Martens, T., Laabs, Y., Günther, H., Kemming, D., Zhu, Z., Witte, L., Hagel, C., Westphal, M., Lamszus, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Glioblastoma+Growth+in+a+Highly+Invasive+Nude+Mouse+Model+Can+Be+Achieved+by+Targeting+Epidermal+Growth+Factor+Receptor+but+not+Vascular+Endothelial+Growth+Factor+Receptor-2.">Inhibition of Glioblastoma Growth in a Highly Invasive Nude Mouse Model Can Be Achieved by Targeting Epidermal Growth Factor Receptor but not Vascular Endothelial Growth Factor Receptor-2.</a> Clin. Cancer Res. 14, 5447-5458 (2008).</li><li>Purow, B., Schiff, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+genetics+of+glioblastoma:+are+we+reaching+critical+mass.">Advances in the genetics of glioblastoma: are we reaching critical mass.</a> Nat. Rev. Neurol. 5, 419-426 (2009).</li></ol>

Non abbiamo nulla da rivelare.

Gliomi maligni, quali glioblastoma multiforme, rappresentano i più comuni tumori cerebrali primari e hanno una prognosi infausta 4. La sopravvivenza dei pazienti affetti da GBM è rimasto praticamente invariato negli ultimi dieci anni (cioè, 9-12 mesi post-diagnosi), nonostante i progressi nella chirurgia, radioterapia e chemioterapia 5. Le caratteristiche di un modello appropriato per lo studio del trattamento glioma dovrebbe includere una posizione riproducibile tumore e la crescita delle cellu...

<table border="1"><tbody><tr><td> Lab standard stereotassico Instrutment </td><td> Stoelting mouse e adattatore Rat </td></tr><tr><td> Betadine </td><td> 100% EtOH </td></tr><tr><td> 70% EtOH </td><td> 50% Acetone </td></tr><tr><td> 6 &quot;Q-tips </td><td> Tamponi imbevuti di alcol </td></tr><tr><td> Hamilton siringa 80008 </td><td> Ethicon 5-0 PD </td></tr><tr><td> 1701 SN 31-33G / 1 &quot;/ PT3 ago </td><td> 1 ml siringhe </td></tr><tr><td> Aghi 25G </td><td> Riscaldamento pad </td></tr><tr><td> Artificial Tears Pomata </td><td> Molto raffinato marcatore permanente a punta </td></tr></tbody></table>

combination radiotherapy in an orthotopic mouse brain tumor model

Glioblastoma multiforme (GBM) sono le più comuni e aggressivi tumori cerebrali primari adulti 1. Negli ultimi anni sono stati compiuti progressi sostanziali nella comprensione dei meccanismi di invasione tumorale, e diretta inoculazione intracerebrale del tumore offre l&#39;opportunità di osservare il processo invasivo in un ambiente fisiologicamente appropriato 2. Per quanto riguarda i tumori del cervello umano sono interessati, i modelli ortotopici attualmente disponibili sono stabiliti mediante iniezione stereotassica di sospensioni cellulari o impianto di un pezzo solido di tumore mediante una procedura complicata craniotomia 3. Nella nostra tecnica si raccogliere cellule da coltura tissutale per creare una sospensione di cellule utilizzata per impiantare direttamente nel cervello. La durata dell&#39;intervento è di circa 30 minuti, e come il mouse deve essere in un piano costante chirurgico, un anestetico iniettabile viene utilizzato. Il mouse viene posizionato in un telaio stereotassico fatta da Stoetling (figura 1). A poppaer la zona chirurgica viene pulito e preparato, si pratica un&#39;incisione, e il bregma si trova per determinare la posizione del craniotomia. La posizione del craniotomia è di 2 mm verso destra e 1 mm anteriore del bregma. La profondità di 3 mm dalla superficie del cranio, e le cellule vengono iniettate ad una velocità di 2 microlitri ogni 2 minuti. La pelle viene suturata con 5-0 PDS, e il mouse è autorizzato a svegliarsi su una piastra elettrica. Dalla nostra esperienza, a seconda della linea cellulare, il trattamento può avvenire da 7-10 giorni dopo l&#39;intervento. Somministrazione di farmaci dipende dalla composizione di farmaco. Per la radioterapia i topi vengono anestetizzati, e metterlo in una maschera su misura. Piombo copre il corpo del topo e espone solo il cervello del mouse. Lo studio della tumorigenesi e la valutazione di nuove terapie per GBM richiedono accurati e riproducibili cerebrali modelli animali tumorali. Così usare questo modello ortotopico cervello per studiare l&#39;interazione del microambiente del cervello e il tumore, per testare il EFFICACIAess di diversi agenti terapeutici con e senza radiazioni.

Watch this Scientific Journal Video about Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model at JoVE.com

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Lo scopo di questo articolo è quello di descrivere l&#39;utilizzo di un modello di glioblastoma ortotopico per gli studi di chemio-radioterapia. Questo articolo passare attraverso l&#39;elaborazione delle cellule, l&#39;impianto, e la radioterapia del mouse utilizzando un modello intracranica.

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 ...

combination-radiotherapy-in-an-orthotopic-mouse-brain-tumor-model

Research

JoVE Journal

Medicine

16.3K Views.  National Cancer Institute. Lo scopo di questo articolo è quello di descrivere l'utilizzo di un modello di glioblastoma ortotopico per gli studi di chemio-radioterapia. Questo articolo passare attraverso l'elaborazione delle cellule, l'impianto, e la radioterapia del mouse utilizzando un modello intracranica.

Video: Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

An Orthotopic Model of Murine Bladder Cancer

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

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

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 ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

In Imaging bioluminescenza in vivo di Dinamica Tumore ipossia di metastasi cerebrali cancro al seno in un modello murino

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

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.

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 ...

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  ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Un ortotopico Glioblastoma modello di mouse mantenimento di vincoli Cervello parenchimale fisiche e adatto per intravitale due fotoni Microscopia

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

Tumore Trattamento Terapia del campo in combinazione con bevacizumab per il trattamento del glioblastoma ricorrente

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

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.