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>

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J., 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=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+the+diameter+of+perineural+invasion+in+radical+prostatectomy+specimens.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+neuropathy+and+neuropathic+pain--a+comprehensive+pathomorphological+study+of+546+cases.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurotrophic+factor+artemin+promotes+pancreatic+cancer+invasion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+by+ductal+adenocarcinoma+of+the+pancreas.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor+expression+correlates+with+perineural+invasion+and+pain+in+human+pancreatic+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+in+pancreatic+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+tumor-infiltrating+macrophages+decreases+tumor-initiating+cells,+relieves+immunosuppression,+and+improves+chemotherapeutic+responses.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+multimutated+herpes+simplex+virus-1+effectively+treats+prostate+carcinomas+with+neural+invasion+while+preserving+nerve+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+survival+in+perineural+invasion+of+pancreatic+cancer,+an+in+vitro+approach.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-related+axonogenesis+and+neurogenesis+in+prostate+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+antiapoptotic+paracrine+loop+in+perineural+invasion+of+prostatic+carcinoma.">Stromal antiapoptotic paracrine loop in perineural invasion of prostatic carcinoma.</a> Cancer Res. 66, 5159-5164 (2006).</li><li>Ceyhan, G. 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D., Han, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+and+associated+pain+in+pancreatic+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+transcription-PCR+analysis+of+laser-captured+cells+points+to+potential+paracrine+and+autocrine+actions+of+neurotrophins+in+pancreatic+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve-sparing+therapy+with+oncolytic+herpes+virus+for+cancers+with+neural+invasion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+mediate+gemcitabine+resistance+of+pancreatic+adenocarcinoma+by+upregulating+cytidinedeaminase.">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 perineural niche microenvironment in vitro.
The protocol presented in the video has two critical steps. First, care should be taken when grasping the nerve adjacent to the DRG body (step 2.7 in the protocol). Grasping or pinching the DRG might cause irreversible mechanical or ischemic damage to the DRG preventing it from growing and sprouting when seeded in the Petri dish. The second critical step is the implantation of the cancer cells in the Petri dish inside the ECM. It is important to seed the cells slowly and with caution, to avoid the floating of cells and their spread over the Petri dish. The aim of the implantation is to localize the cells in one place, to facilitate tracking their movement (distance and direction).
Once established the model microenvironment can be modified according to the tested hypothesis. For instance, adding a third cell culture to the Petri dish (for example, non-cancerous cells) enables the researcher to compare the neurotropic migration capabilities of different cells. Furthermore, the researcher can apply different conditions (i.e., temperature, humidity, soluble factors, etc.) and examine their effect on the cells invasion ability.
The DRG model enables the researcher to study the interaction between cancer cells and nerves. It is also used for time lapse experiments in which morphological changes in the perineural niche are demonstrated in a temporal fashion. Furthermore, the neuroinvasive cells can be subjected to various treatments and their effect on the interaction with neurites of the ganglia can be assessed.
Not every cell line is suitable for the DRG model. They should be cancer cells with the intrinsic ability to invade through nerve and to degrade ECM. Furthermore, note that each cancer cell type has different invasion characteristics and so the time the cells are expected to make contact with the DRG neurites varies. For example, The KPC cells we used need about 7 days to invade through the ECM toward the DRG whereas MiaPaca cells (human pancreatic adenocarcinoma cells) need only 72-96 hr&#160;to make that contact.
A limitation of this model includes the possible incompatibility with human cells. Due to its use of mouse DRG, the mouse-human protein-protein interaction cannot always be demonstrated when human cancer cells are used. Whenever planning to use this model with the two different species, the homology of the examined protein should be tested and if there is high homology the assay is supposed to be suitable for evaluating protein-protein interactions. As well, it should kept in mind that the DRG assay as proposed in this protocol represents the neural microenvironment including other cells, not only neurons (i.e., Schwann cells, fibroblasts, macrophages, etc.). Hence, specific cell populations cannot be tested specifically.

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

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 (QLL2, B16F)&#160;(Figure 3a). Figure 3b presents a representative X and Y coordinate graph, depicting the migration path of a KPC cancer cell in contact with the nerve; time-lapse videomicroscopy analysis showed differences in the forward movement but not velocity between the KPC cells and non-invading cells (Figure 3c-d).
<img alt="Figure 1" src="/files/ftp_upload/52990/52990fig1.jpg" /> 
Figure 1: Schematic Illustration of the Protocol Steps. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52990/52990fig2.jpg" /> 
Figure 2: Cancer Cell Invasion Along the Neurons of the DRG. (a) DRG (top) and cancer cells (bottom) on day 0 after seeding&#160;(5X magnification).&#160; (b) DRG and cancer cells on day 7 after seeding&#160;(5X magnification). (c) Cancer cells migrate along the DRG neuron (arrows). All bars represent 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52990/52990fig3.jpg" /> 
Figure 3: Dorsal Root Ganglion (DRG) Neurons Induce CNI. (a) Nerve invasion index of MiaPaCa2 cancer cells , KPC cells, QLL2, and NIH3T3 cells. (b) A coordinate graph depicting the migration path of a KPC cancer cell in contact with the nerve (red) and QLL2 cell (purple). The Y and X coordinates are shown. (n = 12-20 in each group). (c) Analysis of distance from origin of migrating QLL2 cancer cells with axonal contact and (d) KPC cancer cells (n = 20). The direction of migration was constantly toward the nerve ganglion. P values in (a) were calculated by two-sided Student t test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

in-vitro-modeling-cancerous-neural-invasion-dorsal-root-ganglion

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.0K Views.  Rambam Medical Healthcare Campus. This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

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

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

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

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.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Cancer Research

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.

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.

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

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

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

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

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

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.

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

Medicine

Advanced In Vitro Model for Studying Neuronal Hypersensitivity in DRG Cultures

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

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

Author Spotlight: In Vitro and In Vivo Models to Enhance Chronic Pain Treatment Efficacy

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.

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

Bioengineering

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

Source: Huyett, P. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55043" target="_blank">A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma.</a> J. Vis. Exp. (2017)
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.

Ustanowienie trójwymiarowego modelu kohodowli mysich zwojów korzenia grzbietowego i komórek rakowych: model in vitro do badania inwazji okołonerwowych komórek rakowych

Source: Huyett, P. et al. A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma. J. Vis. Exp. (2017)
In this video, we demonstrate the ...

JoVE Encyclopedia of Experiments

Head and Neck Cancer

Assays & techniques

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

Source: Deborde, S. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56857" target="_blank">An In Vivo Murine Sciatic Nerve Model of Perineural Invasion.</a> J. Vis. Exp. (2018)
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.

Mysi model inwazji okołonerwowej: generowanie modelu in vivo do symulacji inwazji okołonerwowej w nerwie kulszowym myszy

Source: Deborde, S. et al. An In Vivo Murine Sciatic Nerve Model of Perineural Invasion. J. Vis. Exp. (2018)
In this video, we develop an in vivo mouse ...

Cancers of the Nervous System

In vivo studies

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

Source: Na&#39;ara, S. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/52990" target="_blank">In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model.</a> J. Vis. Exp. (2016)
In this video, we demonstrate an in vitro method for co-culturing DRG and cancer cells to study neurotropism and neural invasion.

Modelowanie rakowej inwazji neuronalnej in vitro: metoda wspólnej hodowli zwojów korzenia grzbietowego i komórek rakowych

Source: Na&#39;ara, S. et al. In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model. J. Vis. Exp. (2016)
In this video, we ...

Generating Dorsal Root Ganglion Explant and Dissociated Cell Culture

Source: Fornaro, M. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56757">Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection.</a> J. Vis. Exp. (2018).
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.

Generowanie eksplantatu zwoju korzenia grzbietowego i zdysocjowanej hodowli komórkowej

1. Explant culture of DRG from Adult Mouse

	Place the collected DRG in a 35 mm Petri dish containing 3 mL of ice-cold serum-free media (SFM).
	Transfer ...

Neuronal Culture Techniques

Primary Neuronal Culture

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

To prepare the semi-solid matrix droplets, transfer one glass well-bottom plate from ice onto an ice block under the operating microscope. Place the tip of a no more than 10-microliter pipette directly onto the glass at a 45-degree angle and carefully dispense a 1.5-microliter droplet of matrix onto the glass. Slowly move the pipette tip away from the glass as the matrix becomes engaged with the plate.
Deposit one droplet of matrix onto each of the four corners of the plate and transfer the plate to a room temperature surface for about 1 minute. When the matrix has slightly stiffened, return the plate to the ice block and use closed microscopic forceps to scoop the DRG gently with the left hand.
Using the right hand, carefully transfer the nerve tissue to the tip of a 21G needle and use the needle to gently insert the DRG into the middle of the matrix droplet. The DRG should release easily into the matrix for central positioning with the needle.
After inserting a DRG piece into each of the four droplets, place the plate into a 37 degrees Celsius incubator for 3 minutes. When all of the DRG have been embedded, hold the plates one at a time at a slight angle and slowly add 4 milliliters of DMEM supplemented with 10% FBS to the plates such that the medium only gradually comes into contact with the matrix-DRG units.
Return the plates to the incubator for another 48 to 72 hours. When the neurites reach at least 75% of the way to the edge of the matrix, aspirate the medium from the wells without removing the matrix-DRG units. Then, using a 200-microliter pipette with a smooth trigger, place two drops of 3 times 10 to the fifth cells per milliliter of medium over each matrix DRG assay and return the plates to the incubator. After one hour, gently add 4 milliliters of DMEM with 10% FBS along the sidewall of the glass-bottom plate and return the plates to the incubator until their next microscopic evaluation.

Ustanowienie trójwymiarowego modelu kohodowli zwojów korzenia grzbietowego myszy i komórek rakowych: model in vitro do badania inwazji okołonerwowych komórek rakowych

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

Podsumowanie

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Rozdziały w tym wideo

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

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

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

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

Ustanowienie trójwymiarowego modelu kohodowli mysich zwojów korzenia grzbietowego i komórek rakowych: model in vitro do badania inwazji okołonerwowych komórek rakowych

Mysi model inwazji okołonerwowej: generowanie modelu in vivo do symulacji inwazji okołonerwowej w nerwie kulszowym myszy

Modelowanie rakowej inwazji neuronalnej in vitro: metoda wspólnej hodowli zwojów korzenia grzbietowego i komórek rakowych

Generowanie eksplantatu zwoju korzenia grzbietowego i zdysocjowanej hodowli komórkowej

Ustanowienie trójwymiarowego modelu kohodowli zwojów korzenia grzbietowego myszy i komórek rakowych: model in vitro do badania inwazji okołonerwowych komórek rakowych