Name: Murine Bioluminescent Hepatic Tumour Model (Video) | JoVE Journal
Uploaded: 2010-07-17T17:00:00.000+00:00
Duration: 5 min 23 s
Description: Learn a surgical technique for inducing orthotopic bioluminescent liver tumors in mice. This method enables non-invasive monitoring of tumor growth using live whole body luminescence imaging for cancer research.

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5 minutes

This work was funded through the Cork South Infirmary Victoria University Hospital Breast fund, the Irish Cancer Society (CRI07TAN) and the Cork Cancer Research Centre. Lewis lung cell line stably expressing luciferase was a kind gift from Dr. Karin Jooss, Cell Genesys, Inc., San Francisco, USA.

Introduction
The liver is often the first site of metastatic disease and may be the only site of spread in as many as 30 40% of patients with advanced disease. However options for treating metastatic liver tumours are few with a minority of patients suitable for resection and only 23% major responders to chemotherapeutics. Preclinical research in this field has been limited by the absence of a suitable mouse model to study isolated liver disease. We describe a split-spleen approach that can be used to reliably establish uniform, diffuse liver metastases. Resection of this half of the spleen avoids the consequences of splenic tumour growth and the other half of the spleen is returned with an intact portal circulation to maintain splenic immune protective properties. The model we describe is quite simple to perform and can be done quickly through a single incision. The bioluminescent properties of the tumour cell line allows accurate quantification of treatment response without sacrifice of animals.
Protocol
I. Cell Preparation
<ol>
 <li>Lewis lung carcinoma cells are grown Dulbecco's Modified Essential Medium supplemented with 10% fetal calf serum at 37°C, 5 % CO2 and harvested by trypsinisation when sub-confluent.</li>
 <li>Cells were counted using a Nucleocounter (Chemo-metec, Denmark) and a single cell suspension of 1 X 106 cells in 200 μl was prepared in serum free media.</li>
</ol>
II. Mouse Preparation
Note: All procedures were approved by the ethics committee of University College Cork. In our laboratory we use injectable anaesthetics to anesthetize the mouse; alternatively, one can use inhaled anaesthetics to achieve the same effect. The mouse strain we used is the hairless MF1 nu/nu strain obtained from Harlan Laboratories (Oxfordshire, England). Female MF1 nu/nu mice weighing 16-22 g and of 6-8 weeks of age were included in this experiment. Other strains such as the C57 and Balb/c may also be used with appropriate tumour cell lines. 
<ol>
 <li>Anaesthesia was delivered by intraperitoneal injection of 100ul of 1.5 mg ketamine and 300 ug xylazine. </li>
 <li>The depth of anaesthesia is assessed using toe pinch. There should be no withdrawal reflex with toe pinch.</li>
 <li>The anesthetized mouse is properly positioned and taped over a heating pad.</li>
 <li>The abdomen is prepped with a 10% betadine solution.</li>
 <li>The abdomen and surgical site are draped in a sterile fashion.</li>
</ol>
III. Abdominal Incision
<ol>
 <li>A small subcostal incision is made in the skin.</li>
 <li>The incision is extended to 2-3 cm.</li>
 <li>The abdominal wall musculature were divided and peritoneal cavity entered.</li>
</ol>
IV. Exposure and Division of Spleen
<ol>
 <li>The spleen is mobilised and gently exteriorized on a pre-cut sterile gauze.</li>
 <li>Two 4-0 vicryl ligatures are tied around the mid body of the spleen.</li>
 <li>Spleen is divided between the two ligatures creating two hemi-spleens.</li>
</ol>
V. Injection of Cells into the Hemi-spleen
<ol>
 <li>A 27 G is used to inject a 100 μL volume of cells into one hemi-spleen.</li>
 <li>Tumour cells are allowed to drain to the liver for ten minutes providing sufficient time for the majority of the injected cells to reach the liver.</li>
</ol>
VI. Excision of Hemi-spleen, Abdominal closure and Recovery
<ol>
 <li>The injected hemi-spleen is excised.</li>
 <li>The other hemi-spleen is returned to the abdominal cavity.</li>
 <li>The muscle layer is closed with 4-0 PDS suture using a simple running stitch.</li>
 <li>The abdominal skin is closed with 4-0 Prolene using interrupted stitches.</li>
 <li>Fluid bolus is administered and post-operative analgesia, Carpofen (5 mg/kg) is administered by IP injection for 24 h post surgery.</li>
 <li>The mouse is allowed to recover from anaesthesia</li>
</ol>
VII. IVIS Whole Body Bioluminescent Imaging
<ol>
 <li>Mouse is anaesthetised using injectable anaesthetic. </li>
 <li>Luciferin substrate is given by intra-peritoneal injection ten minutes before imaging</li>
 <li>Mice are placed on the stage and imaged for 3 min using IVIS Whole body imaging system</li>
 <li>Luminescence from the liver can be measured using a region of interest tool</li>
</ol>
VIII. Exposure of mouse liver
<ol>
<li>Mouse is anaesthetised using injectable anaesthetic.</li>
<li>A rooftop abdominal incision allows visualisation of diffuse liver metastasis in vivo.</li>
<li>The mouse is euthanized by cervical dislocation and the liver may be excised for further analysis.</li>
</ol>
<img src="/files/ftp_upload/1977/1977fig1.jpg" alt="Normal vs tumor-bearing liver tissue comparison, surgical sample, pathology, research visualization." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Freshly excised murine untreated and tumour-infiltrated livers.
<img src="/files/ftp_upload/1977/1977fig2.jpg" alt="Bioluminescence imaging results; tumor growth over 14 days on mice; luminescence graph analysis." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Kinetics of hepatic tumour growth as evidenced by IVIS whole body imaging.

<ol><li>Kasuya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+of+subcutaneous+spleen+reservoir+for+multiple+portal+venous+injections+to+treat+liver+malignancies%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Mouse models of subcutaneous spleen reservoir for multiple portal venous injections to treat liver malignancies.</a> Cancer Res. 65, 3823-3827 (2005).</li>
<li>Liu, Q. Z., Tuo, C. W., Wang, B., Wu, B. Q., Zhang, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liver+metastasis+models+of+human+colorectal+carcinoma+established+in+nude+mice+by+orthotopic+transplantation+and+their+biologic+characteristic%5BTitle%5D)+AND+%22World+J+Gastroenterol%22%5BJournal%5D)">Liver metastasis models of human colorectal carcinoma established in nude mice by orthotopic transplantation and their biologic characteristic.</a> World J Gastroenterol. 4, 409-411 (1998).</li>
<li>Li, Z. Y., Ni, S., Yang, X., Kiviat, N., Lieber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenograft+models+for+liver+metastasis%3A+Relationship+between+tumor+morphology+and+adenovirus+vector+transduction%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Xenograft models for liver metastasis: Relationship between tumor morphology and adenovirus vector transduction.</a> Mol Ther. 9, 650-657 (2004).</li>
<li>Cashman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+gene+therapy+as+a+neoadjuvant+to+surgical+excision+to+control+metastatic+cancers%5BTitle%5D)+AND+%22Cancer+Lett%22%5BJournal%5D)">Immune gene therapy as a neoadjuvant to surgical excision to control metastatic cancers.</a> Cancer Lett. , (2008).</li>
</ol>

No conflicts of interest declared.

The development of reliable in vivo models for the study of liver metastasis is becoming increasing important with the advent of new therapeutic strategies. For the accurate study of new strategies, animal models should be relatively easy to perform, form a significant number of metastasis, involve all the steps of the metastatic cascade and have a reporter system that allows detection and counting of a limited number of metastatic cells. The split-spleen approach as described involves a single operation and is ...

<table><tbody><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Ketamine</td><td>V&amp;eacute;toquinol</td><td></td><td></td></tr><tr><td>Xylazine</td><td>V&amp;eacute;toquinol</td><td></td><td></td></tr><tr><td>Carprofen</td><td>V&amp;eacute;toquinol</td><td></td><td></td></tr><tr><td>Videne</td><td>Ecolab</td><td></td><td></td></tr><tr><td>Vicryl 4-0 suture</td><td>Ethicon Inc.</td><td></td><td></td></tr><tr><td>PDS 4-0 suture</td><td>Ethicon Inc.</td><td></td><td></td></tr><tr><td>Prolene 4-0 suture</td><td>Ethicon Inc.</td><td></td><td></td></tr><tr><td>Firefly Luciferin</td><td>Biosynth International, Inc</td><td></td><td></td></tr></tbody></table>

murine bioluminescent hepatic tumour model

Murine Bioluminescent Hepatic Tumour Model

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

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

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

Hello everyone. My name is Simon Regenron. I'm one of the surgical registrars in the car cancer research center here in Ireland.Today I'll be showing you a surgical technique of inducing liver metastases. In a neuron model, the liver is a common site of metastatic spread and a major cause of cancer related mortality. Only a small percentage of patients are suitable for surgical resection with advanced liver disease.Therefore, novel therapeutics are needed for the treatment of this condition. Many animal models of hepatic metastases do not result in disease resembling the clinical setting. The model I'm showing you today is a clinically relevant model and it is relatively easy to learn and with very good success rates.This model involves dividing the spleen into two hemi spleens. One Hemi spleen is used for the inoculation of tumor cells. These tumor cells travel through the porter circulation into the liver.The other Hemi spleen is left intact maintaining its immunological properties. The cell line I'm using is stably expressing luciferase, allowing for the bioluminescent analysis of tumor growth in vivo. Therefore, this model will allow for the non-invasive monitoring of therapeutics of liver disease.Okay, let's go ahead and start this for the procedure today. The instruments that we need are dissecting scissors, needle holder, two small faucets, size 10 scalpel five oh proline suture for skin closure, four oh PIA, suture for closure of the peritoneal layer three oh Vicryl ties for dividing the spleen saline 29 gauge insulin needle for anesthetic injection, 26 gauge insulin needle for tumor suspension, injection tumor cell suspension in serum free media, injectable anesthetic, skin marker, precut sterile gauze swabs Ine. The murn model that I will use today is the Athymic MF one nude mouse.Other models such as the C 57 and BC may be used with appropriate tumor cell lines. A heating pad is used injectable general anesthetic is used in a laboratory. The depth of anesthesia is assessed using the toe pinch.There should not be a toe withdrawal reflex on toe pinch. The knee size mouse is properly positioned and taped, and abdomen is prepped with Betadine and draped in a sterile fashion. A subcostal incision is made, an abdominal cavity is entered, the spleen is identified and exteriorized.The spleen is isolated from the rest of the mouse using a precut sterile gauze. Two vertical ties are passed between the two vascular pedicles and gently tied. The spleen is divided into two equal hemi spleens.One hemi spleen is used for the inoculation of Tumor cells. Tumor cells are slowly injected into the Hemi spleen to avoid leakage. Tumor cells travel through the portal circulation and arrive at the liver.After 10 Minutes of incubation, the injected hemi spleen is excised and the other hemi spleen is returned into the abdomen. Absorbable sutures and a running stitch are used to close the peritoneal cavity and non-absorbable five oh proline suture is used for skin closure. Recovery is carried out routinely.The development of liver disease can be assessed over time using 2D ivus imaging. The mouse is anesthetized as before, and Lucifer substrate is given intraperitoneal. The mouse is placed on the stage in the 37 degree chamber.Imaging is carried out for three minutes and a superimposed image of mouse with detected luminescence is produced Three weeks after treatment, abdomen is opened and diffuse liver disease is found. In conclusion, this technique permits the induction of liver localized metastatic tumor cell growth by luminescent imaging enables visualization and quantification of tumor growth and application in assessment of therapeutic responses.

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Learn a surgical technique for inducing orthotopic bioluminescent liver tumors in mice. This method enables non-invasive monitoring of tumor growth using live whole body luminescence imaging for cancer research.

Murine Bioluminescent Hepatic Tumour Model

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