Name: a "patient-like" orthotopic syngeneic mouse model of hepatocellular carcinoma metastasis
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Description: Watch this Scientific Journal Video about A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis at JoVE.com

We would like to thank Pascal DuBois for proof-reading the article. The authors would also like to thank the personnel of the Animal Facility at Hunter College of The City University of New York. This work was supported by a NIH RCMI grant to Hunter College.

Etica Dichiarazione Tutti gli studi sugli animali sono stati approvati dalla cura e l&#39;uso Comitato istituzionale animali (IACUC) di Hunter College della City University di New York. Nota: Otto topi BALB / c sono stati usati in questa procedura sperimentale. Cinque topi BALB / c sono stati impiantati con 5 x 10 6 BNL 1ME A.7R.1 topo cellule di carcinoma epatocellulare per produrre i tumori primari. Tre topi BALB / c senza implantologia sono stati utilizzati come controlli. BNL 1ME A.7R.1 è una linea di cellule murine carcinoma epatocellulare che è stato derivato per trasformazione chimica dal murino non tumorigeniche linea cellulare di epatociti BNL CL.2. BNL 1ME A.7R.1 è stato ottenuto dalla American Type Culture Collection (ATCC). 1. Pre-chirurgia <ol><li> Assicurarsi che i topi per tutti gli interventi chirurgici di sopravvivenza sono in buona salute. Per assicurare che i topi sono sani, ispezionare segni di disidratazione tenting della pelle. Non usare i topi con qualsiasi evidenza di malattia o distress in questi esperimenti. </li><li> Pesare topi per garantire che non siano sottopeso. Assicurarsi che il peso è di almeno 16-20 grammi. </li><li> Preparare 5 × 10 6 BNL 1ME A.7R.1 cellule del mouse HCC per l&#39;impianto per il mouse: <ol><li> Aspirare i media da 10 ml 60% - piatto di coltura di cellule confluenti il ​​70% delle cellule di carcinoma epatico di topo A.7R.1 BNL 1ME. Lavare due volte con 5 ml di PBS. Aggiungere 2 ml di tripsina al piatto e incubare a 37 ° C in un incubatore umidificato con aria e 5% di CO 2 per 5 min. </li><li> Successivamente, pipette 10 ml tripsinizzate cellule in un emocitometro manuale per il conteggio delle cellule al microscopio a ingrandimento di 10X. Contare il numero totale di cellule trovate nei quattro angoli piazzali e moltiplicare il fattore di diluizione per il numero totale di cellule. Poi, dividere per il numero di quadrati contati e moltiplicare per 10 6 per la concentrazione cellulare (cellule per ml). </li><li> Utilizzando il calcolo, raccogliere 5 milioni di cellule per topo in sterili1,5 ml microprovette. Etichettare provette da 1,5 ml microcentrifuga. </li></ol></li><li> Risospendere le 5 × 10 6 celle BNL 1ME A.7R.1 topo HCC in 100 ml di tampone fosfato salino (PBS). Tenere queste cellule in ghiaccio fino impianto in chirurgia. </li><li> Svolgere attività necessarie per la preparazione di topi per la chirurgia come l&#39;induzione dell&#39;anestesia e capelli clipping lontano da attrezzature e materiali sterili per evitare la contaminazione. </li><li> Un&#39;anestesia generale nei topi per inalazione di isoflurano utilizzando un vaporizzatore isoflurano con ossigeno ad un flusso costante concentrazione di 1 L per min (circa. 2,1%). Nota: Il sistema è autosufficiente, e comprende un vaporizzatore di precisione, un apporto di ossigeno e un contenitore di carbone che è collegato a un circuito di respirazione necessario per gas / anestetici scavenging. </li><li> Assicurarsi che l&#39;anestesia sia adeguata per evitare che l&#39;animale da subire dolore durante l&#39;intervento chirurgico. Eseguire toe-pinch e osservare animali per non responsiveness per confermare l&#39;anestesia. </li><li> A quel tempo, cambiare la linea di valvole, la guarnizione alla casella e aperto al cono. </li><li> Appena il mouse è anestetizzato, applicare una piccola quantità di una pomata oftalmica alle cornee come misura preventiva da essiccazione e danni. </li><li> Successivamente, spostare l&#39;animale alla zona di preparazione, con l&#39;ogiva correttamente posizionati e connessi (per anestesia di mantenimento). Preparare il sito della incisione chirurgica per la chirurgia depilatoria in questa zona di preparazione. </li><li> Assicurarsi che l&#39;area di chirurgia è in una posizione libera di ogni altra attività. </li><li> Assicurarsi che la superficie di lavoro è robusta e adeguatamente igienizzati, unsoiled e prima della chirurgia senza matasse di cavi. Posizionare un pezzo sterile lino o tray chirurgico su di esso. </li><li> Assicurarsi che il campo operatorio è disponibile solo per persone che effettuano l&#39;intervento chirurgico. Assicurarsi che la sala operatoria è pulita e spazzata con un detergente appropriato e spruzzato con un adeguato disinfectant. </li><li> Assicurarsi che sia posizionato anche lontano da porte e finestre, come le correnti d&#39;aria può diffondere la polvere che porta alla contaminazione del sito. </li><li> Eseguire l&#39;intervento chirurgico di sopravvivenza con strumenti sterili. O immergere gli strumenti in un disinfettante secondo le raccomandazioni del fabbricante o in autoclave. Utilizzare materiali sterili di base che sono pronti per l&#39;uso. </li><li> Strumenti separati in base alla funzione per aiutare a prevenire la contaminazione delle aree sterili. </li><li> Shave circa 1 pollice intorno al sito di incisione (incisione mediana) per accedere al fegato dell&#39;animale. Rimuovere il pelo dall&#39;animale lontano dal sito chirurgico. </li><li> Dopo la rasatura, strofinare la zona con un disinfettante e sapone (betadine scrub chirurgico) e lavare il disinfettante in eccesso con la soluzione di PBS sterile. Eseguire questo più volte, con l&#39;intero processo di macchia della durata di circa 5 minuti. Rimuovere e scartato i guanti usati. </li><li> In seguito, cambiare per indumenti protettivi, come SurgicAl abito, top scrub e maschera. Avanti, disinfettare le mani con soluzione macchia adeguata prima di indossare guanti chirurgici. Nota: Questo è un passo necessario per evitare spargimento e contaminare il sito chirurgico con potenziali contaminanti. </li></ol> 2. Chirurgia <ol><li> Fornire una fonte di calore esterna per calore durante l&#39;intero periodo di anestesia. L&#39;animale può verificarsi alcuni ipotermia a causa dell&#39;anestesia provoca disturbi di termoregolazione; quindi, mantenere la temperatura corporea. </li><li> Posare il mouse sulla sua schiena per tutta la chirurgia. </li><li> Fissare gli arti anteriori, arti posteriori e la coda del mouse al tavolo operatorio con pezzi di nastri chirurgici. </li><li> Fare una linea di incisione retta immediatamente inferiore al xyphisternum. </li><li> Utilizzare uno sterile divaricatore chirurgica auto-mantenimento per esporre il fegato. </li><li> Con una siringa con un ago 20G, lentamente e con attenzione iniettare 100 ml di PBS contenente 5 × 10 6 BNL 1ME A.7R.1topo cellule HCC nel lobo destro del fegato. </li><li> Dopo aver rimosso l&#39;ago, applicare un tampone di cotone sterile per almeno 1 minuto per arrestare qualsiasi sanguinamento potenziale causa dell&#39;alta vascolarizzazione del fegato. </li><li> Chiudere il tessuto sottocutaneo con una sutura assorbibile seguita da chiusura pelle con clip chirurgiche. Successivamente, pulire la ferita ancora una volta con betadine scrub. Nota: Ciò garantirà la chiusura della ferita chirurgica. </li><li> Sottocutanea dare 100 ml di soluzione fisiologica. </li></ol> 3. Post-chirurgia <ol><li> Subito dopo l&#39;intervento chirurgico, trasferire il mouse in una gabbia pulita con una carta d&#39;identità post-operatorio per notificare custodi della condizione del mouse. Fornire cibo e acqua. Nota: Nella nostra esperienza, un mouse che ha appena subito un intervento chirurgico se la passeranno meglio se alloggiati solo con cibo umido e acqua per il periodo di 24 ore dopo il primo intervento chirurgico. </li><li> Tenere il caldo animale con una lampada di calore che genera calore non supera 38 ° C. USE un termometro per misurare la temperatura fino a quando il mouse è completamente recuperato. </li><li> Casa l&#39;animale in una gabbia, preferibilmente con biancheria da letto di carta per assicurare il comfort. </li><li> Dopo il recupero iniziale, monitorare l&#39;animale per segni di disagio, sanguinamento, dolore e angoscia. Dare buprenorfina (0,05 -0.1 mg / kg) per alleviare il dolore. Strettamente monitorare l&#39;attività, comportamento e l&#39;aspetto, così come l&#39;acqua e il consumo di cibo per circa 2 ore. Se ambulatoriale, spostare l&#39;animale torna alla zona per regolare un alloggio normale di topi e di continuare a monitorare a intervalli 6-12 hr per la post-operatorio 24 ore. Eseguire un controllo del peso se lo si desidera. </li><li> Tuttavia, se l&#39;animale sta avendo problemi recuperare, adottare le adeguate misure terapeutiche di supporto. Se i topi sono in grado di recuperare, umanamente eutanasia topi via di CO 2 asfissia e dislocazione cervicale. Seguire le linee guida istituzionali approvate per l&#39;eutanasia. </li><li> Post-operatorio, verificare la presenza di segni clinici di HCC che di solito è observable come significativa riduzione del body condition score. Nota: A questo punto, un tumore primario ha probabilmente sviluppato nel fegato e depositi metastatici hanno probabilmente sviluppata nei polmoni. </li><li> A questo punto, sacrifica tutti i topi (sperimentale e di controllo) coinvolti nella sperimentazione per eutanasia umana. Sottoporre i topi all&#39;anidride carbonica asfissia: molto lentamente rilasciare biossido di carbonio nella camera per un periodo di 5 -10 min. Ciò causerà arresto respiratorio. </li></ol>

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The authors declare that they have no competing financial interests.

In questo articolo viene fornita una descrizione approfondita del metodo che è stato recentemente riportato da Ogunwobi e colleghi di successo creazione di un modello di topo singenici ortotopico di HCC metastasi (Figura 2) 3. Il tasso di tumori per questa procedura è generalmente elevata. Abbiamo già osservato un tasso di tumore al 100% 3. Tuttavia, il tasso di utilizzo può essere variabile a seconda della competenza del ricercatore. In un recente esperimento condotto da un nu...

Hepatocellular carcinoma (HCC) is one of the most lethal cancers, with poor prognosis and low life expectancy. Nearly all cancer-related deaths are due to the metastatic spread of the disease from the originating organ to additional distant organs2-6.HCC progression is a complex process. Therefore, being able to model the tumor microenvironment that is naturally found in metastatic HCC in animal models can prove to be a successful and useful way to reveal relevant mechanisms in humans.7Unfortunately, the mechanisms of cancer metastasis are still largely unclear. Therefore, there is a need to establish animal models that will enable us to elucidate the underlying molecular mechanisms of metastases of cancers such as HCC3,5.
Mouse model systems are a very useful approach for delineating mechanisms and evaluating novel strategies for treatment of metastatic human cancers5,8. The various mouse model systems that presently exist are a testament to efforts of researchers to correctly depict the complexity of the disease5,8,9.
Ogunwobi and colleagues recently used a survival surgical approach to demonstrate establishment of a novel orthotopic syngeneic mouse model for the study of metastasis in HCC3. Their work established a &#8220;patient-like&#8221; mouse model that recapitulates features of aggressive and metastatic HCCs3. They further demonstrated that this mouse model system can be used to study the biology of circulating tumor cells, and that this holds potential for gaining novel insights into the mechanisms of cancer metastasis3.
The aim of this paper is to describe in detail the methodology used in establishing this &#8220;patient-like&#8221; orthotopic syngeneic mouse model of HCC metastasis3. The methodology of how to implant BNL 1ME A.7R.1 mouse HCC cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c3 using survival surgery will be described. Unlike other mouse xenograft tumor models where human tumor cells are implanted into immune deficient mice, this system is syngeneic and is, therefore, suitable for studying the role of the immune system in tumor metastasis3,5,8. This approach will likely gain widespread use for studying the mechanisms of metastasis in solid organ cancers.

<table><tbody><tr><td>Micro Dissecting Tweezer&amp;nbsp;</td><td>Roboz</td><td>(RS-5040)</td><td>Tip 0.20 x 0.12 mm</td></tr><tr><td>Graefe Micro Dissecting Forceps, serrated curved tip</td><td>Roboz</td><td>(RS-5111)</td><td>1 X 2 teeth, curved tip width 0.6 mm</td></tr><tr><td>Micro Dissecting Retractor-Agricola (3 by 3 prongs)</td><td>Roboz</td><td>(RS-6501)</td><td>Blunt 3 X 3 prongs, depth 4 mm, spread 25 mm</td></tr><tr><td>Micro Dissecting Retractor-Goldstein (3 by 3 prongs)</td><td>Roboz</td><td>(RS-6503)</td><td>Blunt, 3 X 3 prongs, depth 4 mm, spread 19 mm</td></tr><tr><td>Jameson Caliper (Measures tumors)</td><td>Roboz</td><td>(RS-6466)</td><td>80 mm/3 inch scale, chrome plated&amp;nbsp;</td></tr><tr><td>Micro Dissecting Scissors, large ring sissors, straight and sharp</td><td>Roboz</td><td>(RS-5852)</td><td>23 mm blades, length 4 inches, flat shanks and large rings</td></tr><tr><td>Scalpel with blades, for delicate dissecting procedures</td><td>Roboz</td><td>(RS-9861-36)</td><td></td></tr><tr><td>Scalpel handle&amp;nbsp;</td><td>Roboz</td><td>(RS-9884)</td><td>Solid</td></tr><tr><td>Littauer Stitch Sissors</td><td>Roboz</td><td>(RS-7074)</td><td>Length 4.5 inches</td></tr><tr><td>Brown needle holder, for easy suture tying</td><td>Roboz</td><td>(RS-7960)</td><td>Convex jaw, fine serrations</td></tr><tr><td>Reflex 7MM wound clips with reflex 7 clip applier</td><td>Roboz</td><td>(RS-9262)</td><td>safe, secure alternative method of wound closure</td></tr><tr><td>Instrument tray and lid</td><td>Roboz</td><td>RT-1350S</td><td></td></tr><tr><td>Mini-clipper with detachable blade</td><td>Roboz</td><td>(RS-5903)</td><td></td></tr><tr><td>Germinator 500 (the Germ Terminator) Dry Sterilizer</td><td>Roboz</td><td>Ds-400, Ds-401, DS-501</td><td>For fast decontamination of micro-dissecting instruments.&amp;nbsp;&amp;nbsp; Instruments decontaminate within 15 sec.&amp;nbsp;</td></tr><tr><td>Microinjection needles</td><td>VWR</td><td>BD305125</td><td>25G Needle</td></tr></tbody></table>

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.

Il fegato dei topi Balb / c sono stati impiantati con 5 × 10 6 BNL 1ME A.7R.1 cellule del mouse HCC. L&#39;evidenza clinica di sviluppo HCC era osservabile 63 giorni dopo l&#39;intervento. Di conseguenza, i topi sono stati umanamente eutanasia. Necroscopia è stata effettuata su topi eutanasia. I polmoni e il fegato sono stati asportati e un esame attento lordo è stata effettuata per identificare i tumori macroscopiche. In un mouse, un tumore superficiale è stata osservata sulla superficie del fegato con q...

Watch this Scientific Journal Video about A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis at JoVE.com

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

La diffusione metastatica del cancro è la principale causa di decessi correlati al cancro. Forniamo una descrizione approfondita della nostra metodologia chirurgia sopravvivenza per stabilire un modello di sistema ortotopico singenici mouse &quot;paziente-tipo&quot; per lo studio dei meccanismi di metastatizzazione dei tumori di organi solidi.

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 ...

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12.2K Views.  Hunter College of the City University of New York.  La diffusione metastatica del cancro è la principale causa di decessi correlati al cancro. Forniamo una descrizione approfondita della nostra metodologia chirurgia sopravvivenza per stabilire un modello di sistema ortotopico singenici mouse "paziente-tipo" per lo studio dei meccanismi di metastatizzazione dei tumori di organi solidi. 

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

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Avanzato modello animale di colorettale Metastasi nel fegato: tecniche di imaging e di proprietà di metastatici cloni

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

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Ricerca sul cancro

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Un modello di iniezione vena porta del fegato per studiare metastasi del cancro al seno

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Un Cell Carcinoma Singenico modello murino di renale metastatico per quantitativo e longitudinale Valutazione della preclinici Terapie

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Topo Modello ortotopico di cancro colorettale

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

Biologia

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

Un modello di topo ortotopico oncogenico indotto dall'epatocito del cancro epatocellulare in aumento nell'impostazione dell'infiammazione epatica e della fibrosi

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Modello Murine dei tumori metastatici del fegato nell'impostazione della lesione alla riperfusione dell'ischemia

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

Generazione di sottocutaneo e intraepatica umane carcinoma epatocellulare xenotrapianti in topi immunodeficienti

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Medicina

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Imaging PET/RM non invasivo in un modello murino ortotopico di carcinoma epatocellulare

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Un preclinici Modello murino di metastasi epatiche

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

A "paziente-Like" ortotopico Singenico modello murino della carcinoma epatocellulare Metastasi

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