Name: a preclinical murine model of hepatic metastases
Uploaded: 2014-09-27T13:00:00.000+00:00
Duration: 6 min 51 s
Description: Watch this Scientific Journal Video about A Preclinical Murine Model of Hepatic Metastases at JoVE.com

KF and KO are co first authors along with KS

This work was supported in part by the AHPBA Fellowship (K.S.), NIH K23 CA148964-01 (L.Z.), Johns Hopkins School of Medicine Clinical Scientist Award (L.Z.), Viragh Foundation and the Skip Viragh Pancreatic Cancer Center at Johns Hopkins (E.M.J. and L.Z.), the National Pancreas Foundation (L.Z.), the Lefkofsky Family Foundation (L.Z.), the NCI SPORE in Gastrointestinal Cancers P50 CA062924 (E.M.J., L.Z.), the Lustgarten Foundation (L.Z.), and the Sol Goldman Pancreatic Cancer Center grants (K.S., B.H.E., L.Z.).

All animal experiments conformed to the guidelines of the Animal Care and Use Committee of the Johns Hopkins University, and animals were maintained in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Care (AAALAC).&#160;The surgical procedure is performed under sterile conditions in an operating room that undergoes a minimum of fifteen air exchanges per hour. All instruments are sterilized by autoclaving prior to the procedure and again with 70% ethanol in between each mouse during the procedure. All mice not expected to survive the surgical procedure are euthanized under CO2 for 3 min&#160;followed by cervical dislocation while still under anesthesia.
1. Cell Preparation
<ol>
	<li>Resuspend cultured Panc02 cells in phosphate buffered saline at 2 x107 cells/ml, and keep on ice.</li>
</ol>
2. Mouse Preparation
<ol>
	<li>Administer Ketamine (100 mg/kg) mixed with Xylazine (10 mg/kg) intraperitoneally to 8-12 week old C57BL/6 female mice in divided doses.</li>
	<li>Check to see that the toe pinch withdrawal reflex is abolished to ensure that the mouse is fully anesthetized.</li>
	<li>Administer additional doses of Ketamine (100 mg/kg) mixed with Xylazine (10 mg/kg) as needed to fully anesthetize the mice.</li>
	<li>Apply Puralube Vet Ointment to the eyes of the mice to prevent drying while the mice are under anesthesia.</li>
	<li>Shave the surgical area of the mice to avoid contamination of the wound.</li>
	<li>Prep the left subcostal area of the incision with 70% ethanol and then iodine.</li>
	<li>Place a surgical drape around the abdomen to maintain sterility.</li>
</ol>
3. Laparotomy
<ol>
	<li>Make a left subcostal incision in line with the left ear through the skin and through the peritoneum using a scalpel.</li>
	<li>Express the spleen through the incision by applying simultaneous digital pressure along the cranial and caudal aspects of the incision.</li>
	<li>Locate the splenic blood vessels at the inferior end of the spleen.</li>
	<li>Divide the spleen by placing two Horizon medium size ligating clips in the center of the spleen being careful to avoid damaging the splenic blood vessels at the splenic poles.</li>
	<li>Place the upper pole of the spleen back into the peritoneum to avoid contamination.</li>
</ol>
4. Tumor Injection
<ol>
	<li>Draw up 150 &#956;l of phosphate buffered saline into a 26 G&#160;x 5/8&#8221; syringe.</li>
	<li>Draw up 100 &#956;l&#160;of Panc02 (2 x106) cells into the same syringe maintaining the syringe in an upright position at all times during the injection.</li>
	<li>Dip the needle in a 70% ethanol solution.&#160;The needle is dipped in 70% ethanol to ensure sterility.</li>
	<li>Inject the cells slowly into the exposed hemispleen being sure the syringe is kept upright at all times. The bevel of the syringe should be observed at all times through the splenic capsule to avoid injecting the cells under the spleen. A whitening of the spleen and blood vessels should be observed upon injection.&#160;</li>
	<li>Elevate the spleen and apply one medium Horizon clip under the spleen to occlude the splenic blood vessels.</li>
	<li>Apply one small Horizon clip to the most distal aspect of the pancreas and splenic vessels.</li>
	<li>Ligate the pancreas and splenic vessels from the hemispleen by cutting above the Horizon clips.</li>
	<li>Place the mouse on its&#8217; side, and irrigate the incision with distilled water.</li>
</ol>
5. Abdominal Closure and Recovery from Anesthesia
<ol>
	<li>Close the peritoneum with a 4-0 running stitch.</li>
	<li>Apply two to three skin clips to close the skin incision.</li>
	<li>Administer 0.1 mg/kg buprenorphine or 5-10&#160;mg/kg Carprofen subcutaneously to alleviate post surgical pain.</li>
	<li>Place the mouse on a heating pad for recovery until mobile and the mouse demonstrates regular breathing patterns. Animals are only returned to the company of other animals after fully recovering from the surgical procedure.</li>
</ol>
6. Necropsy Examination and Harvesting of the Liver
<ol>
	<li>Between 30 to 60 days following the surgery, mice will begin to show clinical symptoms of metastasis and will require euthanasia. Signs of metastatic disease requiring humane euthanasia include enlarged abdomens and the development of ascites. Euthanize mice by inhalation of CO2.</li>
	<li>Following inhalation, perform cervical dislocation.</li>
	<li>After the animal has been determined to be non responsive, make an incision through the peritoneum to expose the abdomen using scissors.</li>
	<li>Using forceps and scissors, gently cut the liver out of the abdomen.</li>
	<li>Place the liver in OCT compound, and flash freeze using liquid nitrogen. Store the liver at -80&#176;C. Alternatively, the liver can be fixed in 16% neutral buffered formalin at RT&#160;for 24 hr and paraffin&#160;embedded.</li>
</ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMT+and+dissemination+precede+pancreatic+tumor+formation.">EMT and dissemination precede pancreatic tumor formation.</a> Cell. 148, 349-361 (2012).</li><li>Frese, K. K., Tuveson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+mouse+cancer+models.">Maximizing mouse cancer models.</a> Nature reviews. Cancer. 7, 645-658 (2007).</li><li>Corbett, T. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+depletion+of+regulatory+T+cells+allows+the+recruitment+of+mesothelin-specific+CD8+T+cells+to+the+antitumor+immune+response+against+a+mesothelin-expressing+mouse+pancreatic+adenocarcinoma.">Effective depletion of regulatory T cells allows the recruitment of mesothelin-specific CD8 T cells to the antitumor immune response against a mesothelin-expressing mouse pancreatic adenocarcinoma.</a> Clinical and translational science. 1, 228-239 (2008).</li><li>Jain, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+effect+of+a+granulocyte-macrophage+colony-stimulating+factor-transduced+tumor+vaccine+and+systemic+interleukin-2+in+the+treatment+of+murine+colorectal+cancer+hepatic+metastases.">Synergistic effect of a granulocyte-macrophage colony-stimulating factor-transduced tumor vaccine and systemic interleukin-2 in the treatment of murine colorectal cancer hepatic metastases.</a> Annals of surgical oncology. 10, 810-820 (2003).</li><li>Yoshimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+targeting+of+antitumor+immune+responses+with+engineered+live-attenuated+Listeria+monocytogenes.">Selective targeting of antitumor immune responses with engineered live-attenuated Listeria monocytogenes.</a> Cancer research. 66, 1096-1104 (2006).</li><li>Yoshimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+attenuated+Listeria+monocytogenes+effectively+treats+hepatic+colorectal+cancer+metastases+and+is+strongly+enhanced+by+depletion+of+regulatory+T+cells.">Live attenuated Listeria monocytogenes effectively treats hepatic colorectal cancer metastases and is strongly enhanced by depletion of regulatory T cells.</a> Cancer research. 67, 10058-10066 (2007).</li><li>Olino, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-associated+antigen+expressing+Listeria+monocytogenes+induces+effective+primary+and+memory+T-cell+responses+against+hepatic+colorectal+cancer+metastases.">Tumor-associated antigen expressing Listeria monocytogenes induces effective primary and memory T-cell responses against hepatic colorectal cancer metastases.</a> Annals of surgical oncology. 19, 597-607 (2012).</li><li>Hingorani, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trp53R172H+and+KrasG12D+cooperate+to+promote+chromosomal+instability+and+widely+metastatic+pancreatic+ductal+adenocarcinoma+in+mice.">Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.</a> Cancer cell. 7, 469-483 (2005).</li><li>Marion, A., Aoudi, W., Basarab, A., Delachartre, P., Vray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+evaluation+in+high-frequency,+40+MHz+imaging:+a+comparative+study+of+four+vector+velocity+estimation+methods.">Blood flow evaluation in high-frequency, 40 MHz imaging: a comparative study of four vector velocity estimation methods.</a> Ultrasonics. 50, 683-690 (2010).</li><li>Zheng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+23+phosphorylation-dependent+cell-surface+localization+of+annexin+A2+is+required+for+invasion+and+metastases+of+pancreatic+cancer.">Tyrosine 23 phosphorylation-dependent cell-surface localization of annexin A2 is required for invasion and metastases of pancreatic cancer.</a> PloS one. 6, e19390 (2011).</li></ol>

The authors have no relevant conflicts of interest to disclose.

This preclinical murine model of pancreatic cancer metastasis to the liver via a hemispleen injection technique is the first model described that mirrors many of the clinical and immunological conditions of patients with metastatic disease to the liver. This model offers several advantages over other murine models. First, unlike transgenic models of pancreatic cancer in which only 30% of the mice develop metastases to the liver and the timing of metastases formation is quite variable, this model offers the advantage of consistently obtaining metastasis to the liver in 100% of mice at a uniform time, enabling researchers to study metastatic disease in this setting. Additionally, this model retains a naive hemispleen to allow for peripheral blood immune analysis in a reproducible model. Furthermore, in the absence of anti-neoplastic therapies, mice die from tumor progression at a consistent time point depending on the initial tumor load, and survival is consistent from one experiment to the next. Finally, this model has a variety of applications including the study of liver metastasis13, tumor immunology10, and experimental therapy testing9.
The critical steps of this technique are the cell suspension preparation, the tumor cell injection, the placement of the Horizon clips, and the placement of the skin clips. To ensure a successful surgery and avoid potential skewing of the results, the following modifications of the critical steps may be necessary. First, during cell suspension preparation, it is imperative that a single suspension of cells be obtained and injected into the hemispleen. Minor clumping of tumor cells will occlude the splenic vessels and result in premature death during surgery. This can be avoided by resuspending the cells in a declumping agent and by preparing each syringe for injection immediately prior to the injection. Additionally, during the preparation of the syringe, it is imperative that the cells not mix with the phosphate buffered saline flush. This can be achieved by first drawing up the phosphate buffered saline and then very slowly drawing up the cells to avoid mixing, maintaining the syringe in an upright position at all times. Second, during the tumor cell injection step, the bevel of the syringe should be observed at all times through the splenic capsule to avoid injecting the cells under the spleen. A whitening of the spleen and blood vessels should be observed upon injection, which indicates that the tumor cells have been successfully injected into the spleen. In younger mice with smaller spleens, it may be necessary to inject a smaller volume of flush than risk the cells leaking from the spleen. If any leaking is suspected, lay the mice on their side and wash the spleen with distilled water before suturing the mice to help remove any cells that may have leaked during the injection step. Failure to remove these cells will result in tumor growth at the injection site, which may cause death prior to achieving adequate metastases. Third, the placement of the Horizon clips under the hemispleen is important for ensuring that there is no bleeding following removal of the hemispleen. Additional Horizon clips may be used to occlude vessels as necessary. The final critical step of this technique is the placement of the skin clips. If the mice are being following for survival, it may be necessary to suture the skin of the mice rather than using skin clips. Occasionally, the skin clips will fall off prior to the full healing of the skin wound, which could render the mice susceptible to infection. Suturing of the skin, separate from the peritoneum, will prevent this problem.
While this technique is invaluable for studying the metastatic process and metastatic disease, this model has a few limitations. First, the reproducible nature of this model makes it preferred over transgenic and orthotopic models for studying the metastatic process and the tumor microenvironment at metastatic sites. However, because of the nature of the model, only the final steps of the metastatic process, extravasation and colonization, can be studied. This limits the utility of this model for studying the full metastatic process and does not allow for investigation into the signals that stimulate intravasation of tumor cells. Second, while it is important to retain full immune function in these mice by retaining half of the hemispleen, analysis of lymphocytes in the liver include both lymphocytes from the normal liver as well as lymphocytes from the tumor microenvironment, which could result in a biased representation of the true immune status within the tumor microenvironment. The use of more aggressive tumor cell lines that lead to more extensive metastases to the liver could help overcome this limitation of the model. Additionally, if the metastases are visualized upon necropsy, it may be possible to dissect the metastases from the normal liver and analyze only the lymphocytes found in the metastases further circumventing this problem. In summary, while this model has several disadvantages, it also offers many advantages over other models and should therefore be used complement these models when studying metastatic disease to the liver.

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer related deaths in the United States1. The five year survival for patients with metastatic PDA is 2-12%1. Although adjuvant and neoadjuvant chemotherapy for pancreatic cancer is more effective today than ever, there still remains a desperate need for novel therapies.
The development of novel therapeutics necessitates reliable preclinical animal models. Although subcutaneous tumor models are straightforward to use, drawbacks include the inability of these tumors to metastasize and mimic the tumor progression and microenvironments seen in human cancers. Genetically engineered models, such as the genetically engineered mouse containing Kras and p53 knock-in oncogenic mutations (the KPC model)2 and the KPC model with a YFP lineage tracer (the PKCY model)3 allow for the study of naturally occurring tumors in immunocompetent mice. Additionally, the tumor microenvironment is unaltered and more closely resembles that seen in human tumor progression. Unfortunately, the timing of tumor development is variable within these models, which makes it difficult to study experimental therapies. Additionally, backcrossing of these mice requires a significant amount of time and financial commitment, which is not always feasible. Finally, implanted xenograft PDA mouse models necessitate immunocompromised mice, which have altered or absent tumor microenvironments. As a result, the efficacy of experimental therapies demonstrated in these preclinical models are often not reproducible in human clinical trials4.
This hemisplenectomy mouse model utilizes Panc02 tumor cells, which are a highly tumorigenic, methylcholanthrene induced pancreatic tumor cell line derived from C57BL/6 mice5,6. Additionally, other murine PDA cell lines derived from the KPC mice can be used in this model. Schulick et al. have previously developed a hemisplenectomy technique and created a syngeneic mouse model of colon cancer metastases7. This hemisplenectomy model (Figure 1) offers consistent and equal tumor inoculation in immunocompetent mice lending itself to the investigation of novel therapeutic agents7-10.

<table><tbody><tr><td>Cell culture media and components</td><td></td><td></td><td>will vary depending on cell line</td></tr><tr><td>Phosphate Buffered Saline</td><td>Gibco by Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>Curved Operating Scissor</td><td>Roboz Surgical Store</td><td>RS-6835</td><td></td></tr><tr><td>Micro Dissecting Graefe Forceps</td><td>Roboz Surgical Store</td><td>RS-5130</td><td></td></tr><tr><td>Needle holder (regular)</td><td>World Precision Instruments, Inc</td><td></td><td></td></tr><tr><td>3-0 or 4-0 suture</td><td></td><td></td><td>courtesy of dept of surgery, Johns Hopkins</td></tr><tr><td>Weck Horizon Open Ligating Clip Applier, small, angled</td><td>Teleflex</td><td>137085</td><td></td></tr><tr><td>Weck Horizon Open Ligating Clip Applier, medium, angled</td><td>Teleflex</td><td>237115</td><td></td></tr><tr><td>Weck Horizon medium ligating clips</td><td>Teleflex</td><td>002200</td><td></td></tr><tr><td>Weck Horizon small ligating clips</td><td>Teleflex</td><td>001200</td><td></td></tr><tr><td>Syringe, 1 cc, tb syringe, 3/8&quot; slip tip</td><td>Becton Dickinson</td><td>309625</td><td></td></tr><tr><td>Syringe, 1 cc, tb syringe, 5/8&quot; slip tip</td><td>Becton Dickinson</td><td>309597</td><td></td></tr><tr><td>9 mm Autoclip Applier</td><td>Mikron</td><td>427630</td><td></td></tr><tr><td>9 mm Autoclip</td><td>Becton Dickinson</td><td>427631</td><td></td></tr><tr><td>Heating pad</td><td>Sunbeam</td><td>CAT93</td><td></td></tr><tr><td>70% Ethanol</td><td></td><td></td><td>sold by numerous vendors</td></tr><tr><td>Ketamine Hydrochloride&amp;nbsp;</td><td>Hospira</td><td>22395DD</td><td></td></tr><tr><td>Xylazine hydrochloride</td><td>Sigma</td><td>X1251</td><td></td></tr></tbody></table>

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.

Panc02 tumor cells injected into the hemispleen (Figure 1) form liver metastases in 100% of the mice, while lung and peritoneal metastases are not observed if the technique is performed correctly. This technique has been performed on over one hundred mice using Panc02 cells in multiple, independent experiments, and reproducibly, all untreated mice die with liver metastases between 30 and 60 days following the hemisplenectomy procedure (Figure 2). To obtain statistical significance between the untreated and treated groups, ten mice per group are needed. However, this number will vary with respect to the cell line and treatment scheme used. When Panc02 cells are used in this model, multiple liver metastases can be observed upon necropsy (Figure 3).
KPC tumor cells, which are a syngeneic pancreatic tumor cell line derived from transgenic mice having tissue-specific Kras and p53 knock-in mutations11, can also be used in this model. Using these cells results in similar liver metastases (Figure 4). However, when evaluating liver metastasis formation using a different cell line, the number of cells injected should be titrated until all untreated mice develop liver metastases. Panc02 cells can be used as a positive control to ensure that the surgery was performed correctly. Importantly, while the extent of the formation of liver metastases and survival can vary depending on the cell line used, within one experiment, all untreated mice develop liver metastases at a similar burden and die from these metastases reproducibly within a short period of time. Therefore, normalization of tumor burden by ultrasound is not necessary when using this technique.
Failure to induce liver metastasis results in a normal appearing liver upon necropsy, which may indicate that the procedure was not performed properly. However, failed or delayed formation of metastases may result following treatment with therapies that suppress or inhibit metastatic growth (Figure 5), resulting in prolonged survival. Therefore, this model can be used to assess the effects of various therapeutic agents on tumor growth and the formation of metastases.
Because it is technically feasible to evaluate the liver by ultrasound, a small animal ultrasound (Figure 6)12 can be used to assess the in vivo formation of metastases in the liver. In addition, the formation of metastases can be further examined by histopathology following necropsy (Figure 7).
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/51677/51677fig1highres.jpg" src="/files/ftp_upload/51677/51677fig1.jpg" /> 
Figure 1. Schematic of the hemisplenectomy model, which is used to create pancreatic hepatic metastases with Panc02 tumor cells.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/51677/51677fig2highres.jpg" src="/files/ftp_upload/51677/51677fig2.jpg" /> 
Figure 2. Representative survival of C57BL/6 mice after undergoing Panc02 hemisplenectomy on day 0 is shown in the Kaplan-Meier curve (n=10). Mice injected with Panc02 tumor cells in this model will all die between 30 and 60 days following the procedure if anti neoplastic therapies are not administered.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/51677/51677fig3highres.jpg" src="/files/ftp_upload/51677/51677fig3.jpg" /> 
Figure 3. Gross examination of the liver following injection of Panc02 cells into the hemispleen reveals liver metastases (indicated by arrows). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51677/51677fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/51677/51677fig4highres.jpg" src="/files/ftp_upload/51677/51677fig4.jpg" /> 
Figure 4. Gross examination of the liver following hemispleen injection of KPC tumor cells into the hemispleen reveals liver metastases (black arrows) that replace the normal hepatic parenchyma (white arrow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51677/51677fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="6in" fo:src="/files/ftp_upload/51677/51677fig5highres.jpg" src="/files/ftp_upload/51677/51677fig5.jpg" /> 
Figure 5. Gross examination of the liver following effective therapeutic intervention. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51677/51677fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" fo:content-width="6in" fo:src="/files/ftp_upload/51677/51677fig6highres.jpg" src="/files/ftp_upload/51677/51677fig6.jpg" /> 
Figure 6. Liver ultrasonography using a Vevo 660 small animal ultrasound on a mouse with a large metastatic lesion in the liver (black arrow) following the hemisplenectomy procedure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51677/51677fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" fo:content-width="6in" fo:src="/files/ftp_upload/51677/51677fig7highres.jpg" src="/files/ftp_upload/51677/51677fig7.jpg" /> 
Figure 7. H&#38;E staining of paraffin embedded liver metastases formed by Panc02 cells in the hemisplenectomy model. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51677/51677fig7highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Preclinical Murine Model of Hepatic Metastases at JoVE.com

A Preclinical Murine Model of Hepatic Metastases

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

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

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Nanyang Technological University

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28.6K Views.  The Johns Hopkins University School of Medicine.  Een preklinische, muizenmodel van levermetastasen uitgevoerd via een hemispleen injectietechniek. 

Video: A Preclinical Murine Model of Hepatic Metastases

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 ...

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.

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

Cancer Research

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

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.

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 ...

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).

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.

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

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

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.

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  ...

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).

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

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.

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

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 ...

A Preclinical Murine Model of Hepatic Metastases

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