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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Schwann+cells+facilitate+cancer+progression+in+nerves.">How Schwann cells facilitate cancer progression in nerves.</a> Cell Mol Life Sci. 341 (177-186), 1236361-1236416 (2017).</li><li>Abiatari, I., 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=Consensus+transcriptome+signature+of+perineural+invasion+in+pancreatic+carcinoma.">Consensus transcriptome signature of perineural invasion in pancreatic carcinoma.</a> Mol Cancer Ther. 8 (6), 1494-1504 (2009).</li><li>Ayala, G. 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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 ...

The overall goal of this surgical procedure is to create an in vivo model of perineural invasion by injecting cancer cells into the sciatic nerve of mice. Perineural invasion is the invasion of nerves found in many cancer types This method can help answer key questions on the interdependent relationship between cancer and nerve cells, which may promote cancer progression and invasion. The main advantage of this technique is that it provides an in vivo model for perineural invasion in which both the cancer cells and the nerve micro-environment can be manipulated and in which an invasion can be assessed using histological, clinical, or radiographic endpoints.The implications of this technique extend toward therapy for perineural invasion because the host mice can be treated with candidate therapeutic agents. Though this method can provide insight into molecular mechanism of pancreatic cancer invasion, it can also be applied to other cancer cell types. The host mice can also be genetically modified, allowing the study of the effect of the nerve micro-environment on cancer progression.Demonstrating the procedure will be Yasong Yu and Andrea Marcadis, members from my laboratory. Begin by harvesting subconfluent Panc02-H7 cells from a T225 flask with four milliliters of 0.25%Trypsin for five minutes at 37 degrees Celsius. Collect the cells in a 15-milliliter centrifuge tube and centrifuge at 900 times g at 4 degrees Celsius for five minutes.Wash the cells by re-suspending the cell pellet in one milliliter of PBS by pipetting up and down at least twice. Then transfer the suspension to a 1.5-milliliter microcentrifuge tube on ice and centrifuge again at 900 times g at four degrees Celsius for five minutes. Following centrifugation, discard the supernatant without disturbing the pellet.Keep the pelleted cells on ice until injection. On the day before the surgery, remove the fur along the length of femur on the dorsal side of a eight-week old male or female C57 Black 6J mice with either a surgical clipper or chemical hair removal agent. On the day of the surgery, disinfect the stage, anesthetize the mice using 2%isoflurane in an induction chamber, and apply vet ointment to the eyes to prevent dryness under anesthesia.Inject weight-appropriate amount of analgesic subcutaneously, and transfer a mouse to a nosecone in the surgical area. Heat support is provided throughout the surgical procedure. Place the anesthetized mouse on its ventral side and gently secure each limb with hypoallergenic tape to create mild tension in the limb to be injected.Clean the injection site with Betadine swab, then again with 70%alcohol. Repeat this process two more times and make sure that no loose hair remains on the surgical field. As a final step, apply Betadine solution to the surgical area.After donning sterile gloves and placing the sterile drape, use a dedicated sterile tool to apply toe pinch prior to making the incision under the drape, then use sterile small scissors to make a one-centimeter incision about two millimeters below and parallel to the femur. Retract the skin with forceps laterally to expose the muscles underneath. Separate the gluteus maximus and biceps femoris muscles along a fascial plane and expose the sciatic nerve underneath.Free the nerve from the surrounding muscles using blunt dissection. Draw three microliters of cancer cells from the pellet into a sterile 10-microliter syringe. Place a small metal spatula underneath the nerve at the point of injection for support.While visualizing with a dissecting microscope, keep the needle parallel to the nerve and insert it into the nerve. Be careful not to puncture through the back of the nerve. Slowly inject the cells into the nerve over five seconds.A formation of a bulb in the injection area indicates a good injection. Leave the needle in place for three seconds to minimize backflow before removing the needle gently. Proper injection of the cancer cells in the sciatic nerve is critical.If the cells are injected outside of the nerve, the consistency of the invasion will suffer. However, with practice and experience, this is very rare. After injection, return the nerve to its original position and cover the nerve with the overlying muscles.Treat the mice with proper analgesia and then close the skin with 5-0 nylon sutures. Place the mouse alone in a clean cage for observation and provide heat support during recovery until it has regained sufficient consciousness to maintain sternal recumbency. On post-operative day seven, place the euthanized mouse on its ventral side and stabilize the distal limbs using pins.After removing the skin on the dorsal side of the injected limb and torso, use blunt dissection to expose the sciatic nerve deep to the muscles. To access the nerve at the spinal cord region, separate the ilium and the sacrum by inserting closed scissors in the narrow area where the sciatic nerve is located, then open the scissors while holding the mouse. Carefully dissect the sciatic nerve distally to the end of the femur and proximally to the spinal cord.Careful handling is essential, as invaded nerves are extremely fragile and prone to breaking under tension or forceful handling. Be delicate and maintain the integrity of the sciatic nerve during the dissection. Harvest the nerve by first cutting its distal end.Carefully lift the nerve while freeing it from adjacent tissue. Cut the nerve at the proximal end as close as possible to its exit from the spinal column. Use a Vernier caliper to estimate the gross length of invasion.Begin processing by embedding the dissected nerve in O.C.T.compound. Place nerves longitudinally and as flat on the bottom of the mold as possible. Indicate on the cassette the proximal and distal side of the nerve by marking the letter P and D, then place the embedded nerves on top of dry ice.Section samples using a cryostat microtome at five-micron thickness and place sections on glass slides. If possible, fit two nerve sections per slide. Indicate proximal side of the nerve.After staining the slides with H&E and obtaining images with a slide scanner, use imaging software to quantify the length of invasion. Injection of the sciatic nerve with Panc02-H7 cancer cells at the site indicated resulted in infiltration of the nerve up to 14 millimeters proximal to the spinal cord as shown here. NCAM knockout mice are mice in which the adhesion molecule, Neural Cell Adhesion Molecule 1, is deficient.The infiltration of Panc02-H7 is less pronounced in these mice. Quantification of length and area of invasion showed that invasion was significantly reduced in NCAM knockout mice. Once mastered, this procedure can be done in 30 minutes if it is performed properly.Since its development, this technique has allowed researchers in the field of cancer biology to explore the relationships between cancer cells and nerve cells in perineural invasion. After watching this video, you should have a good understanding of how to utilize the sciatic nerve model to study perineural invasion, including the injection of cancer cells into the sciatic nerve and the extraction and processing of the invaded nerves. Thanks for watching and good luck with your experiments.

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