Name: an in vivo murine sciatic nerve model of perineural invasion
Uploaded: 2018-04-23T13:00:00
Description: Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

The authors acknowledge the technical services provided by the molecular cytology facility and the animal facility of Memorial Sloan Kettering Cancer Center. This work was supported by NIH grants CA157686 (to R.J. Wong) and P30 CA008748 (Memorial Sloan Kettering Cancer Center support grant).

All of the procedures with animal subjects were approved by the Institutional Animal Care and Use Committee at Memorial Sloan Kettering Cancer Center.
1. Preparation of the Cancer Cells
<ol>
	<li>Harvest sub-confluent Panc02-H7 cells with 0.25% trypsin for 5 min at 37 &#176;C. Collect the cells in a 15 mL centrifuge tube. 
	NOTE: The cells are grown in T-225 flask, which contains about 12 x 106 cells per flask at 80% confluency and 4 mL trypsin/ flask is used.</li>
	<li>Centr...

<ol>
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In this protocol we describe an in vivo murine model of perineural invasion that allows for the quantification of sciatic nerve invasion by pancreatic cancer cells. This model enables the study of molecular mechanisms of nerve invasion. Successful experiments using this technique require a careful approach to three critical steps in the process: 1) the injection of cancer cells (steps 2.7, 2.8), 2) the extraction of invaded nerves (step 3.4), and 3) processing of harvested nerves (step 4.1).
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Nerves form a specific tumor microenvironment that stimulates cancer growth and migration1,2,3. Perineural invasion (PNI) is the process through which cancer cells invade in and around the nerves. It may be considered as a unique route of metastasis since cancer invasion extends away from the sites of origin along nerves. PNI is found in several cancer types including pancreatic, prostate, head &#38; neck, salivary, cervical, and colorectal cancers with an incidence ranging from 22% to 100%1,2. PNI is associated with pain and correlates with poor prognosis and worse survival rates1,2.
Developing models of perineural invasion is essential to elucidate the cellular and molecular mechanisms of this process, and to test candidate therapeutic agents to decrease PNI. In vitro methods of studying interactions between cancers and nerves include the co-culture of cancer cells with nerve explants4, with dorsal root ganglions5,6,7, or with specific cells from the nerve microenvironment such as Schwann cells7. In vivo approaches, however, are more physiologically relevant, include the use of cancer mouse models in which cancer has been induced or transplanted and have the advantage of accounting for the entire nerve microenvironment. In orthotopic models of pancreatic or prostate cancer, PNI has been reported8,9,10 and the incidence of PNI may be recorded, but because of the small size of the nerves in those organs, it is difficult to see the entire nerve and therefore to quantify the extent of PNI. The model we describe here is an in vivo model of PNI in which cancer cells are injected into the sciatic nerve of mice through a simple surgical procedure11. The heterotopic transplant invades within the nerve toward the spinal cord. The length of the nerve invasion from the site of injection to the spinal cord may be measured, as well as the volume of the cancer within the nerve. Importantly, the invaded nerve can also be collected for a variety of assays including microscopic, and molecular analyses. A variety of cancer cells can be tested, and the host mice that have been genetically modified or treated with specific compounds may be used as well. This powerful assay allows for the cancer cells and the host microenvironment to be modified for investigation into the mechanisms of PNI.

<table border="0" cellpadding="0" cellspacing="0" style="width:973px;" width="973">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Mouse</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;">Number and age variable depending on experimental needs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture media (PBS, Trypsin, and DMEM+10% FBS)</td>
			<td>Any</td>
			<td></td>
			<td>Steps 1.1, 1.2, 1.3.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Conical centrifuge tube, 50 mL</td>
			<td>Falcon</td>
			<td>352098</td>
			<td>Step 1.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microcentrifuge tube 1.5 mL</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td>Step 1.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electric razor</td>
			<td>WAHL</td>
			<td>9962</td>
			<td>Step 2.1. Can be substituted with commercial hair removal agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane, 250 mL</td>
			<td>Baxter</td>
			<td>1001936060</td>
			<td>Step 2.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hypoallergenic surgical tape</td>
			<td>3M Blenderm</td>
			<td>70200419342</td>
			<td>Step 2.3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Betadine Swapsticks</td>
			<td>PDI</td>
			<td>SKU 41350</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Webcol Alcohol Preps</td>
			<td>Covidien</td>
			<td>5110</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile surgical tools (scissors and forceps)</td>
			<td></td>
			<td></td>
			<td>Steps 2.4, 2.5, 3.3, 3.4, 3.5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">10 &#956;L Hamilton syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Steps 2.7, 2.8</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Steel Micro spatula</td>
			<td>Fisher Scientific</td>
			<td>S50823</td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td></td>
			<td></td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bupivacine, 1 g</td>
			<td>Enzo Life Sciences</td>
			<td>BML-NA139-0001</td>
			<td>Step 2.9. Reconstitute to 0.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5-0 Nylon suture</td>
			<td>Ethicon</td>
			<td>698H</td>
			<td>Step 2.9</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek O.C.T. Compound</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Step 4.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek Cryomold Molds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Step 4.1</td>
		</tr>
	</tbody>
</table>

an in vivo murine sciatic nerve model of perineural invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

This method describes the surgical implantation of pancreatic cancer cells into the murine sciatic nerve to create an in vivo model of quantifiable nerve invasion.&#160;Figure 1 illustrates the anatomical location of the sciatic nerve and the site of injection. Figure 2 shows the two sciatic nerves of a nude mouse. A PBS injected nerve (left) may be compared to a nerve injected with MiaPaCa-2 cancer cells (right). The ne...

Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

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