Name: in vitro modeling of cancerous neural invasion: the dorsal root ganglion model
Uploaded: 2016-04-12T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model at JoVE.com

Edith Suss-Toby is thanked for her assistance in the time-lapse microscopy and image analysis. Nofar Rada is thanked for the artistic work.

Four- to six-week-old female C57BL/CJ mice (Harlan, Jerusalem, Israel) were used in the experiment according to the Association for Assessment and Accreditation of Laboratory Animal Care specifications. All experimental procedures were done in accordance with Institutional Animal Care and Use Committee and the Department of Agriculture regulations.
1. Harvesting the Spinal Cord
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	<li>Euthanize the mouse using a CO2 chamber. Avoid cervical dislocation as it might cause damage t...

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This article presents an in vitro model that recapitulates the cancerous microenvironment in the neural niche, the DRG model. The video demonstrates all the steps starting from recognizing anatomical landmarks such as the DRG in the mouse, its extraction, and finally, its culturing in ECM. Co-culturing the DRG alongside with cancer cells is also presented. There are no other models for in vitro perineural invasion research described in the literature making this model essential for studying the perineur...

Solid tumors disseminate in three main ways: direct invasion, lymphatic spread, and hematogenic spread. However, there is a fourth means of cancer spread that is frequently disregarded, dissemination along nerves. Cancerous neural invasion (CNI) is a well-known route of cancer spread, especially in cancers of the head and neck,1 prostate2, 3 and pancreas.4-8 CNI occurs in more than 80% of individuals with pancreatic adenocarcinoma, leading to retroperitoneal tumor spread through celiac ganglion nerves. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS).9 This finding suggests that the perineural microenvironment can be exploited by cancer cells, providing factors that support malignant growth.
One of the few in vitro models for CNI research is the dorsal root ganglia (DRG)/cancer cell model. This model is frequently used for studying the paracrine interaction between neural stroma and cancer cells.10-18 In this model, mouse or human cancer cell lines are grown in extracellular matrix (ECM) adjacent to preparations of freshly dissociated cultured DRG.
This video article shows the application of in vitro CNI in pancreatic ductal adenocarcinoma.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:417px;">Equipments:</td>
			<td style="width:121px;"></td>
			<td style="width:108px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Operating microscope</td>
			<td>Leica</td>
			<td>M205</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tiime Lapse System</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F4142&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical blade</td>
			<td>Sigma-Aldrich</td>
			<td>Z309036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3271</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35mm petri dishes, glass bottom</td>
			<td>de groot</td>
			<td>60-627860</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">70% ethanol</td>
			<td>sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cold PBS</td>
			<td>Biological industries</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Biological industries</td>
			<td>01-055-1A</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Rhenium</td>
			<td align="right">10108165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin and streptomycin</td>
			<td>Biological industries</td>
			<td>01-031-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Pyruvate</td>
			<td>Biological industries</td>
			<td>03-042-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Biological industries</td>
			<td>03-020-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Growth factor depleted matrigel</td>
			<td>Trevigen</td>
			<td>3433-005-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro modeling of cancerous neural invasion: the dorsal root ganglion model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

Using video microscopy imaging, the DRG can be seen sprouting neurites 5-7 days after implantation while the cancer cells migrating away from their colonies toward the DRG. By the 7th day after the implantation, the cancer cells come in contact with the neurites (Figure 2).
The Forward migration index of the pancreatic cancer cells used in the protocol is 3-4 fold higher than that of other cell lines ...

Watch this Scientific Journal Video about In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model at JoVE.com

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

The overall goal of this model is to recapitulate the cancerous neural microenvironment. This enables exploration of cancer cell neuron interactions. This method can help answer key questions in the tumor microenvironment field, such as interaction between tumor cell and neurons and the mutual influence of each set.The main advantage of this technique is that it is reliable, easy to perform and address both cellular and cerebral factors in the neural niche. The implications of this technique extend towards therapeutic development and pharmacological targeting of key factors in the neural remodeling and invasion process throughout cancer development. After euthanizing the animal in a CO2 chamber, soak the fur with 70%ethanol and allow it to dry.Pin the mouse in a prone position, and then make a midline incision extending craniocaudally from the posterior neck to the lumber spine. Next, use forceps to raise the dermal flaps bilaterally and expose the subcutaneous tissue. Palpate the mouse skull until the craniocervical junction is identified.Use scissors angled perpendicular to the animal to cut through the cervical muscles and spine at the craniocervical junction. Then, dissect the spine caudally and use scissors to perform a complete caudal transection at the fifth lumbar vertebrae. Next, position the mouse in a supine position, and after making an incision along the midline from the neck to the abdomen, retract the skin laterally and then cut open the peritoneum.Cranially, open the chest wall with scissors. After removing the peritoneal and retroperitoneal organs, use forceps and a surgical blade to cut the ribs, leaving approximately five millimeters of ribs protruding from the vertebral column. Remove the vertebral column from the rest of the body.and wash twice with cold PBS. Position the spine facing up in the same craniocaudal orientation on a nonadherent absorbing platform under a stereo microscope equipped with 4X magnification. While looking through this stereo microscope, remove any spinal muscles and connective tissue.Use the ribs as landmarks for nerves, as they leave the spine. The DRG are located at the cervical, thoracic, and lumbar vertebrae. Use scissors to cut the vertebral body at the midline, creating a window to the spinal cord, and then gently retract the vertebral body to expose the spinal cord and the DRG roots.Next, using spring scissors, remove the upper rib to expose the DRG. Follow the peripheral nerve medially along the rib lateral to the DRG. Identify the DRG, which can be described as looking like the yolk of a fried egg lying on the nerve.Cut the intercostal nerve distal to the DRG leaving two to three millimeters of nerve distal to the ganglia to be used for retraction. Carefully grasp the efferent nerve using forceps without pinching or damaging the DRG. Now apply gentle retraction to the DRG by pulling the nerve laterally.Then cut the anterior and posterior roots close to the DRG. Transfer the isolated DRG to a 35-millimeter petri dish filled with ice-cold supplemented DMEM. Working in a laminar flow hood, place a 35-millimeter glass bottom petri dish on a paper grid on ice.Using a precooled pipette tip, dispense approximately 10 microliters of growth-factor-depleted ECM at the center of the grid. While viewing the dish through a stereo microscope under 2 to 4X magnification, place the DRG at the center of the ECM close to the bottom of the dish. After harvesting and washing 40, 000 cancer cells of interest from confluent cultures resuspend the pellet and 40 microliters of ECM on ice.Once again, view the grid through the stereo microscope, measure 500 microns from the DRG, and dispense 10 microliters of the cell suspension at this point. Repeat in all directions from the DRG. Leave the dish in the laminar flow hood for 10 to 15 minutes to solidify but not dry out.Once the ECM has solidified, slowly add supplemented DMEM by pipetting against the sidewall of the plate. Add approximately two milliliters of media, enough to cover the ECM. The addition of DMEM should be done very slowly by pipetting against the sidewall of the plate to avoid detachment of the DRG from the plate bottom.Set the plate in the tissue culture incubator, and follow the instructions in the written portion of the protocol to maintain the culture. This micrograph shows the DRG at the top of the field of view and cancer cells at the bottom of the field of view on day zero after seeding. Here, the same culture is shown seven days after seeding.As indicated by the arrows, cancer cells migrate along the DRG neuron. This histogram shows the DRG nerve invasion index of different types of cancer cells. It can be seen that Kpc 989 and MiaPaCa cells have a higher nerve invasion index than QLL2 or NIH3T3 cells.This coordinate graph depicts the migration path of a Kpc cancer cell in contact with the nerve shown in red and a QLL2 cell shown in purple. The X and Y coordinates are shown. This figure shows an analysis from distance of origin for migrating QLL2 cancer cells with axonal contact.Here the distance of origin is shown for Kpc cancer cells. In each case, the direction of migration was towards the nerve ganglion. While attempting this procedure, it is important to remember to prepare more ganglia than required since the check rate is not 100%After it's development, the DRG motive paved the way for researchers in the field of cancer research to explore the neural niche within the tumor microenvironment.After watching this video, you should have a good understanding of how to recognize anatomical landmarks such as the DRG in the mouse, extract the DRG, and finally, how to culture it in DCM. Coculturing of the DRG alongside with cancer cells is also presented.

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