Name: In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model (Video) | JoVE Journal
Uploaded: 2016-04-12T14:00:00.000+00:00
Duration: 8 min 23 s
Description: Explore the dorsal root ganglia model for studying cancer cell-neuron interactions in pancreatic ductal adenocarcinoma. This reliable technique recapitulates the neural microenvironment to advance therapeutic development.

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5 minutes

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
<ol>
	<li>Euthanize the mouse using a CO2 chamber. Avoid cervical dislocation as it might cause damage to the ganglion roots due to shear forces. From here on, perform all steps under sterile conditions.</li>
	<li>Soak animal down with 70% ethanol. This step is important for sterilization and for preventing the hair from dragging through the inner organs.</li>
	<li>Leave the mouse to dry from ethanol. Prevent any contact of ethanol with the inner organs due to neurotoxicity.</li>
	<li>After drying the mouse, position it using pins in prone position (face down). Place the pins on each hindlimb and forelimb.</li>
	<li>Using forceps, make a midline incision extending craniocaudally from the posterior neck to the lumbar spine. Next, raise dermal flaps bilaterally using forceps and expose subcutaneous tissue.</li>
	<li>Palpate mouse skull until reaching the craniocervical junction. Using scissors perpendicular to the animal, cut through the cervical muscles and spine at the craniocervical junction (the 7th cervical vertebrae), using heavy forceps. Dissect the spine caudally and excise the spine with heavy forceps until reaching the lumbar level (hind limbs level).</li>
	<li>Perform a complete caudal transection at the lumbar spine level (the 5th lumbar vertebrae) using heavy forceps.</li>
	<li>Roll the mouse over (face up). There is no need to cover the incision since the DRG is extracted from lower vertebral levels and not the cervical.</li>
	<li>Cut down at the midline from the neck to the abdomen, retract the skin laterally and then cut to open the peritoneum.</li>
	<li>Remove the inner organs inside the peritoneum (liver, spleen, pancreas, stomach, and intestine) en bloc. Next, remove the retroperitoneal organs (i.e., kidneys and pancreas). Cranially, open the chest wall.</li>
	<li>Using forceps and a surgical blade, cut the ribs, leaving 5 mm of ribs bilaterally away from the vertebral column. At this point, the vertebral column is separated from the rest of the body.</li>
	<li>Wash the vertebral column from blood with cold (4 &#176;C) fresh phosphate buffered saline (PBS) twice.</li>
</ol>
2. Isolating the Dorsal Root Ganglia (DRG)
<ol>
	<li>Use a stereomicroscope with 4X magnification.</li>
	<li>Position the spine on a non-adherent non absorbing platform (Telfa/nylon). Position the spine face up&#160;at the same cranio-caudal orientation.</li>
	<li>Remove any spinal muscles and connective tissue. Use the ribs as a landmark for nerves as they leave the spine. Ribs also protect the nerves from the surgical tools. The DRG are located at the cervical, thoracic, and lumbar sites of the vertebrae.</li>
	<li>Next, using scissors cut the vertebral body in the midline and expose the spinal cord and DRG roots by gentle lateral retraction of the vertebral body.</li>
	<li>In the intercostal space, follow the peripheral nerve medially along the rib lateral to the DRG.</li>
	<li>Identify the DRG. It looks like the yolk of &#39;fried egg&#39; lying on the nerve.</li>
	<li>Cut the intercostal nerve distal to the DRG leaving 2-3 mm of nerve distal to the ganglia, to be used for retraction. This &#34;tail&#34; will be removed after harvesting.</li>
	<li>Grasp the nerve using forceps. Pinching the DRG body itself will cause neural cell damage and should be avoided.</li>
	<li>Approach the DRG proximally along its attachment to the spinal cord, via its anterior and posterior roots.</li>
	<li>Apply gentle retraction to the DRG by pulling the efferent nerve (intercostal nerve stump) laterally, and cut the anterior and posterior roots close to the DRG.</li>
	<li>Keep the DRG in a 35 mm Petri dish filled with fresh, ice cold DMEM supplemented with 10% fetal calf serum (FCS), 1% penicillin-streptomycin, 1% non-essential amino acids, and 1% Sodium pyruvate.</li>
</ol>
3. Implantation in the Petri Dish
<ol>
	<li>Perform the next steps on ice using pre-cooled pipette tips to prevent ECM solidification.</li>
	<li>Under a stereomicroscope at 2-4X magnification, place a 35 mm glass bottom petri dish on a paper grid. 
	IMPORTANT: Work on ice in order to keep the ECM in liquid state. Create a 1 mm diameter spot (approximately 20 &#181;l) of growth factor-depleted ECM at the center of the grid.</li>
	<li>Under direct visualization, place the DRG at the center of the ECM spot close to the bottom of the dish. 
	Note: The cancer cells used in this protocol are murine pancreatic cancer cells (KPC) established from freshly isolated tumor specimens from KPC mice as described elsewhere19.</li>
	<li>Harvest 40,000 cancer cells from confluent cultures, wash them once with PBS, and resuspend them in 40 &#181;l ECM on ice.</li>
	<li>Under direct visualization of the grid using a stereomicroscope measure 500 &#181;m at each direction from the DRG. At this point slowly seed 10,000 cells/10 &#181;l ECM. Use a 2-10 &#181;l precooled tip to inject the cells close to the dish bottom, avoiding their spread in the matrix or detachment of the matrix. (Figure 1).</li>
	<li>Leave the dish within a laminar flow hood for 10-15 min&#160;to solidify. Avoid ECM drying.</li>
	<li>Slowly add DMEM, prepared as previously mentioned, against the side wall of the plate. Add enough medium to cover the ECM (about 2 ml).</li>
	<li>Put the dish back to the incubator at 37 &#176;C.</li>
	<li>Replace the medium with fresh medium on the following day.</li>
	<li>Replace the medium every 2 days.</li>
</ol>
4. Data Acquisition, Time-lapse Videomicroscopy
<ol>
	<li>On day 7 after the implantation, take the dish for time lapse microscopy. Note that certain cells might require acquisition earlier due to faster migration.</li>
	<li>During the live imaging, place the dish in a closed environment at a mean temperature of 37 &#177;0.1 &#176;C and 5% CO2.</li>
	<li>Record the cells by using a charge coupled device camera placed on the microscope.</li>
	<li>Take digitized images every 10 min&#160;for up to 72 hr&#160;to follow cell locomotion from three to six different cells.</li>
	<li>Replace medium after 24 hr.</li>
	<li>Perform data acquisition and simple analysis using the microscope software.</li>
</ol>
5. Data Analysis
<ol>
	<li>For more advanced applications (i.e., cell tracking in 2D or 3D) use specialized software (such as Imaris 4D). Note: This software determines the xy coordinates of a cell at any given time, along with the distance from origin, and speed of motion.</li>
	<li>Calculate the mean velocity by measuring the travel distance between subsequent positions in 10 min&#160;intervals. Calculate the Forward Migration Index (FMI) which is the distance of the cell front from the neurite and describes the unidirectional movement of the cells toward the DRG.</li>
</ol>

<ol><li>Carter, R. L., Foster, C. S., Dinsdale, E. A., Pittam, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perineural+spread+by+squamous+carcinomas+of+the+head+and+neck%2C+a+morphological+study+using+antiaxonal+and+antimyelin+monoclonal+antibodies%5BTitle%5D)+AND+%22J+Clin+Pathol%22%5BJournal%5D)">Perineural spread by squamous carcinomas of the head and neck, a morphological study using antiaxonal and antimyelin monoclonal antibodies.</a> J Clin Pathol. 36, 269-275 (1983).</li>
<li>Beard, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perineural+invasion+is+associated+with+increased+relapse+after+external+beam+radiotherapy+for+men+with+low-risk+prostate+cancer+and+may+be+a+marker+for+occult%2C+high-grade+cancer%5BTitle%5D)+AND+%22Int+J+Radiat+Oncol+Biol+Phys%22%5BJournal%5D)">Perineural invasion is associated with increased relapse after external beam radiotherapy for men with low-risk prostate cancer and may be a marker for occult, high-grade cancer.</a> Int J Radiat Oncol Biol Phys. 58, 19-24 (2004).</li>
<li>Maru, N., Ohori, M., Kattan, M. W., Scardino, P. T., Wheeler, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prognostic+significance+of+the+diameter+of+perineural+invasion+in+radical+prostatectomy+specimens%5BTitle%5D)+AND+%22Hum+Pathol%22%5BJournal%5D)">Prognostic significance of the diameter of perineural invasion in radical prostatectomy specimens.</a> Hum Pathol. 32, 828-833 (2001).</li>
<li>Ceyhan, G. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pancreatic+neuropathy+and+neuropathic+pain--a+comprehensive+pathomorphological+study+of+546+cases%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Pancreatic neuropathy and neuropathic pain--a comprehensive pathomorphological study of 546 cases.</a> Gastroenterology. 136, 177-186 (2009).</li>
<li>Ceyhan, G. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neurotrophic+factor+artemin+promotes+pancreatic+cancer+invasion%5BTitle%5D)+AND+%22Ann+Surg%22%5BJournal%5D)">The neurotrophic factor artemin promotes pancreatic cancer invasion.</a> Ann Surg. 244, 274-281 (2006).</li>
<li>Takahashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perineural+invasion+by+ductal+adenocarcinoma+of+the+pancreas%5BTitle%5D)+AND+%22J+Surg+Oncol%22%5BJournal%5D)">Perineural invasion by ductal adenocarcinoma of the pancreas.</a> J Surg Oncol. 65, 164-170 (1997).</li>
<li>Zhu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nerve+growth+factor+expression+correlates+with+perineural+invasion+and+pain+in+human+pancreatic+cancer%5BTitle%5D)+AND+%22J+Clin+Oncol%22%5BJournal%5D)">Nerve growth factor expression correlates with perineural invasion and pain in human pancreatic cancer.</a> J Clin Oncol. 17, 2419-2428 (1999).</li>
<li>Hirai, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perineural+invasion+in+pancreatic+cancer%5BTitle%5D)+AND+%22Pancreas%22%5BJournal%5D)">Perineural invasion in pancreatic cancer.</a> Pancreas. 24, 15-25 (2005).</li>
<li>Mitchem, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+tumor-infiltrating+macrophages+decreases+tumor-initiating+cells%2C+relieves+immunosuppression%2C+and+improves+chemotherapeutic+responses%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Targeting tumor-infiltrating macrophages decreases tumor-initiating cells, relieves immunosuppression, and improves chemotherapeutic responses.</a> Cancer Res. 73, 1128-1141 (2013).</li>
<li>Kelly, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Attenuated+multimutated+herpes+simplex+virus-1+effectively+treats+prostate+carcinomas+with+neural+invasion+while+preserving+nerve+function%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Attenuated multimutated herpes simplex virus-1 effectively treats prostate carcinomas with neural invasion while preserving nerve function.</a> FASEB J. 22, 1839-1848 (2008).</li>
<li>Dai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+survival+in+perineural+invasion+of+pancreatic+cancer%2C+an+in+vitro+approach%5BTitle%5D)+AND+%22Hum+Pathol%22%5BJournal%5D)">Enhanced survival in perineural invasion of pancreatic cancer, an in vitro approach.</a> Hum Pathol. 38, 299-307 (2007).</li>
<li>Ayala, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer-related+axonogenesis+and+neurogenesis+in+prostate+cancer%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">Cancer-related axonogenesis and neurogenesis in prostate cancer.</a> Clin Cancer Res. 14, 7593-7603 (2008).</li>
<li>Ayala, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stromal+antiapoptotic+paracrine+loop+in+perineural+invasion+of+prostatic+carcinoma%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Stromal antiapoptotic paracrine loop in perineural invasion of prostatic carcinoma.</a> Cancer Res. 66, 5159-5164 (2006).</li>
<li>Ceyhan, G. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+invasion+in+pancreatic+cancer%2C+a+mutual+tropism+between+neurons+and+cancer+cells%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">Neural invasion in pancreatic cancer, a mutual tropism between neurons and cancer cells.</a> Biochem Biophys Res Commun. 374, 442-447 (2008).</li>
<li>Bapat, A. A., Hostetter, G., Von Hoff, D. D., Han, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perineural+invasion+and+associated+pain+in+pancreatic+cancer%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">Perineural invasion and associated pain in pancreatic cancer.</a> Nat Rev Cancer. 11, 695-707 (2011).</li>
<li>Ketterer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reverse+transcription-PCR+analysis+of+laser-captured+cells+points+to+potential+paracrine+and+autocrine+actions+of+neurotrophins+in+pancreatic+cancer%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">Reverse transcription-PCR analysis of laser-captured cells points to potential paracrine and autocrine actions of neurotrophins in pancreatic cancer.</a> Clin Cancer Res. 9, 5127-5136 (2003).</li>
<li>Gil, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nerve-sparing+therapy+with+oncolytic+herpes+virus+for+cancers+with+neural+invasion%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">Nerve-sparing therapy with oncolytic herpes virus for cancers with neural invasion.</a> Clin Cancer Res. 13, 6479-6485 (2007).</li>
<li>Gil, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves%5BTitle%5D)+AND+%22J+Natl+Cancer+Inst%22%5BJournal%5D)">Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.</a> J Natl Cancer Inst. 102, 107-118 (2010).</li>
<li>Weizman, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophages+mediate+gemcitabine+resistance+of+pancreatic+adenocarcinoma+by+upregulating+cytidinedeaminase%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Macrophages mediate gemcitabine resistance of pancreatic adenocarcinoma by upregulating cytidinedeaminase.</a> Oncogene. 33, 3812-3819 (2014).</li>
</ol>

This research was supported by the ICRF Barbara S. Goodman endowed research career development award (2011-601-BGPC) and a grant from the US-Israel Binational Science Foundation. No other financial disclosures from the authors are to be made.

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.

<table><tbody><tr><td>&lt;strong&gt;Equipment:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Operating microscope</td><td>Leica</td><td>M205</td><td></td></tr><tr><td>Time Lapse System</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Forceps</td><td>Sigma-Aldrich</td><td>F4142&amp;nbsp;</td><td></td></tr><tr><td>Surgical blade</td><td>Sigma-Aldrich</td><td>Z309036</td><td></td></tr><tr><td>Scissors</td><td>Sigma-Aldrich</td><td>S3271</td><td></td></tr><tr><td>35 mm Petri dishes, glass bottom</td><td>de groot</td><td>60-627860</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>70% ethanol</td><td>Sigma</td><td></td><td></td></tr><tr><td>Cold PBS</td><td>Biological industries</td><td>02-023-1A</td><td></td></tr><tr><td>DMEM</td><td>Biological industries</td><td>01-055-1A</td><td></td></tr><tr><td>FCS</td><td>Rhenium</td><td>10108165</td><td></td></tr><tr><td>Penicillin and streptomycin</td><td>Biological industries</td><td>01-031-1B</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Biological industries</td><td>03-042-1B</td><td></td></tr><tr><td>L-Glutamine</td><td>Biological industries</td><td>03-020-1B</td><td></td></tr><tr><td>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

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 ...

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

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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Neuroscience

In this video, we describe the method to co-culture dorsal root ganglia and cancer cells on chick chorioallantoic membrane or CAM to study perineural invasion.

Chick Chorioallantoic Membrane or CAM Tumor Model: An In Ovo Method to Simulate Perineural Invasion of Tumor Cells

In this video, we demonstrate the procedure for developing an in vitro perineural cancer cell invasion model by co-culturing mouse dorsal root ganglion and neurotropic cancer cells. The model can be utilized to conduct cancer assays, producing rapid and reproducible results.

Establishing a Three-dimensional Co-culture Model of Murine Dorsal Root Ganglia and Cancer Cells: An In Vitro Model to Study Perineural Cancer Cell Invasion

In this video, we demonstrate an in vitro method for co-culturing DRG and cancer cells to study neurotropism and neural invasion.

Modeling Cancerous Neural Invasion In Vitro: A Method to Co-culture Dorsal Root Ganglia and Cancer Cells

In this video, we develop an in vivo mouse model for perineural invasion by injecting pancreatic cancer cells into the sciatic nerve. This model helps quantify the extent of invasion and study the interactions between cancer cells and nerves.

Murine Perineural Invasion Model: Generating an In Vivo Model to Simulate Perineural Invasion in Mouse Sciatic Nerve

This video demonstrates a method to develop dorsal root ganglia or DRG explant culture and DRG-dissociated cell models using isolated mouse DRG tissues. These models help to study various mechanisms with neuron-glial interactions in a controlled environment.

Generating Dorsal Root Ganglion Explant and Dissociated Cell Culture

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

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

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

This protocol describes methods for establishing whole mount and dissociated cultures of mouse Dorsal Root Ganglia (DRG) and their use to assess tumor-nerve interactions.

Establishing In Vitro Models of Dorsal Root Ganglia Culture: Complementary Approaches for Investigating Cancer-Nerve Crosstalk

Explore the dorsal root ganglia model for studying cancer cell-neuron interactions in pancreatic ductal adenocarcinoma. This reliable technique recapitulates the neural microenvironment to advance therapeutic development.

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

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