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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Semi-solid Matrix Droplets (&#60; 1 min per plate)

<ol>
	<li>Place a pre-chilled glass well-bottom plate on an ice block underneath the operating microscope. Make sure the aliquot of matrix remains on the ice at all times.</li>
	<li>Place a 1.5 &#181;L droplet of matrix in each of the four corners of the glass-bottom plate with a 2 &#181;L or 10 &#181;L micropipetter, leaving a distance at least as great as the droplet itself from the edge of the glass well.
	<ol>
		<li>Place the tip of the pipette directly onto the glass at a 45-degree angle. Slowly pipette the matrix. Slowly move away from the glass bottom after the matrix is engaged on the plate. 
		NOTE: The surface tension between the matrix and glass should create a perfect hemisphere each time.</li>
		<li>Stop pipetting just before the tip is empty because inadvertent injection of air into the matrix droplet makes the diameter of the matrix droplet far greater and is difficult to remove.&#160;&#160;&#160;&#160; 
		NOTE: Using a second hand to stabilize the pipetting hand facilitates more accurate placement.</li>
	</ol>
	</li>
</ol>
2. Insertion of DRG into Semi-solid Matrix Droplets (&#60; 2 min per plate)
<ol>
	<li>Leave the plate with the matrix droplets briefly at room temperature (~1 min). This slightly stiffens the matrix, making it easier for the precise placement of the DRG.</li>
	<li>Scoop (do not grasp) the DRG gently with closed microscopic forceps in the left hand. Again, a dark background against the small, white DRG facilitates visualization. Transfer the DRG to the tip of a 21G needle in the right hand. This transfer will leave residual media on the forceps.</li>
	<li>Gently insert the DRG into the middle of the matrix droplet using the 21G needle (Figure 1). Most of the time, the DRG will easily release into the matrix and then can be positioned centrally with the needle.
	<ol>
		<li>If the DRG sticks to the needle, use the microscopic forceps to push the DRG off of the needle and into the matrix droplet. Wick away excess media from the microscopic forceps with a lab wipe.&#160; 
		NOTE: Introducing media to the matrix will alter the diameter, volume, and consistency of the assay. An ice block with color or colored writing provides background contrast, which eases visualization of this delicate process.</li>
	</ol>
	</li>
	<li>After the plate has all four DRGs, perform a final inspection to ensure that the DRG is in the center of the matrix droplet. Make adjustments as needed with the 21G needle.</li>
	<li>Transfer the completed plate to a 37 &#176;C incubator. This will solidify the semi-solid matrix and fix the DRG in position.</li>
	<li>Repeat this process for all of the DRGs. Place blank matrix droplets without a DRG as the negative control.</li>
	<li>Add 4 mL of DMEM with 10% FBS media to each glass-bottom plate after completing all plates and exposing them to the 37 &#176;C incubator for at least 3 min. Place the plate at a slight angle and slowly add the medium, such that it gradually comes into contact with the matrix-DRG units.&#160; 
	NOTE: If the medium is added too quickly or vigorously, the matrix and/or DRG can become dislodged.</li>
	<li>Store the assays in the 37 &#176;C incubator for the next 48&#8211;72 h. 
	NOTE: Periodic examination of the assays will show circumferential outgrowth of neurites towards the matrix edge (Figure 2). When the neurites are greater than three-fourths of the way to the matrix, it is appropriate to plate the cells.</li>
</ol>
3. Preparation of the Head and Neck Cancer Cells
NOTE: Cell lines other than head and neck squamous cell carcinoma cells can be used in this experimental design.
<ol>
	<li>Maintain squamous cell carcinoma cell lines in DMEM with 10% FBS in preferred flasks or culture dishes in a 37 &#176;C incubator. To prepare cells for experimentation, suction all the medium from the culture flask or dish and wash it twice with PBS. Suspend the cells by adding an appropriate amount of 0.025% trypsin for 5 min, followed by DMEM with 10% FBS. Pipette 4 mL of the cell and media mixture into 6 mL culture dishes.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: A 6 mL culture plate provides more than enough cells, even when less than 50% confluent.</li>
	<li>Expose the HNSCC cell lines to different conditions 24 h prior to staining and plating on the DRG assay. Re-dose the condition once the cells are plated to maintain a consistent environment. 
	NOTE: Add antibodies, growth factors, cytokines, or other molecules to the cell culture dishes 1&#8211;2 days following mouse dissection and implantation of the DRG into the matrix.</li>
	<li>Add fluorescent cell stains 1 h before plating the cells (2&#8211;3 days after the DRG harvest and implantation into the matrix).&#160;&#160;&#160;&#160;&#160; 
	NOTE: The particular stains used here pass freely through the cell membrane; however, after reacting with intracellular thiol groups, the stain remains within the cell and is passed on to daughter cells. The staining greatly facilitates visualization within the assays. These temporary stains are ideal for these assays because the fluorescence is present for 2&#8211;3 days, which is the timeframe of these experiments. It also allows for the use of many different colors and obviates the need for creating fluorescent protein-transfected cell lines.
	<ol>
		<li>Prepare 10 &#181;M of dye solution by mixing 2 &#181;L of 10 mM stock cell stains in 2 mL of serum-free DMEM for every cell condition. Warm this to 37 &#176;C before adding it to cells.</li>
		<li>Remove the culture medium within the 6 mL plate, leaving the adherent HNSCC cells on the plate. Add 2 mL of phosphate-buffered saline (PBS) and remove 2 mL of the 10 &#181;M cell stain/serum-free DMEM added to each plate.</li>
		<li>Return the 6 mL culture plate with the cell stain to the 37 &#176;C incubator for 40 min.</li>
		<li>After 40 min, aspirate the medium. Add 2 mL of PBS and then aspirate it. Add 1 mL of 0.025% trypsin to each plate and replace them in the 37 &#176;C incubator.</li>
		<li>Add 2 mL of DMEM with 10% FBS after 3&#8211;5 min and transfer the suspended cells to a 15 mL conical tube. Spin the cells and resuspend them in 1 mL of DMEM with 10% FBS.</li>
		<li>Count the cells with a hemocytometer or automated cell counter and then add DMEM with 10% FBS to create a final cell concentration of 300,000 cells per mL.</li>
	</ol>
	</li>
</ol>
4. Plating Head and Neck Cancer Cells
<ol>
	<li>Remove the glass-bottom plates with the matrix-DRG assays from the incubator. 
	NOTE: Again, they are appropriate when neurites are at least 75% of the way to the edge of the matrix, which generally occurs after 2&#8211;3 days. This is best seen using at least a 10X objective.</li>
	<li>Aspirate the medium within the glass-bottom plate. Completely aspirate the medium within the glass well without removing the matrix-DRG units.</li>
	<li>Draw up 200 &#181;L of the 300,000 cells/mL medium using a 200 &#181;L pipette. Place two drops of the cells over each matrix-DRG assay. Perform this step for each matrix droplet, creating four repeats of each cell condition on one plate. 
	NOTE: The hemispherical shape of the matrix allows the cells to settle in a ring along the periphery of the assay (Figure 3a, Figure 3b). Finding a pipette with a smooth trigger makes the placement of uniform drops far easier.</li>
	<li>Confirm similar cell numbers and distribution of the various cell conditions under 4X microscopy.</li>
	<li>Place the glass-bottom plates into the 37 &#176;C incubator for approximately 60 min. 
 NOTE: Although there is only a small volume of cells and media (~150 &#181;L), the assays do not dry out until several hours of incubator time have elapsed.</li>
	<li>After 60 min, gently add 4 mL of DMEM with 10% FBS along the sidewall of the glass-bottom plate.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Over a period of time, the cells partially adhere to the plate bottom, and the method of adding the medium does not disturb the cells. Different cell types may take more or less time to begin to adhere to the glass-bottom plate.</li>
	<li>Replace any exogenous drugs or growth factors at this time to maintain the previous concentration levels.</li>
	<li>Return the assays to the 37 &#176;C incubator, except when they are examined microscopically. Quantify the results of this assay by microscopic imaging. Briefly, count the strands of perineural invasion within four quadrants using a 4X microscopic image.&#160;&#160; 
	NOTE: A microscope with a 4X objective and fluorescence capabilities is adequate for photo-documenting the assays. The size of the assays fits nicely within the field of a 4X objective, which is detailed enough to capture the individual areas of perineural invasion. Perineural invasion becomes apparent within 24 h, but beyond 48 h, the integrity of the assays begins to weaken, as the tumor cells divide along the periphery of the matrix and invade. Beyond 48 h, there is also a substantial efflux of fibroblasts from the DRG, which obscures visualization using brightfield microscopy. Therefore, it is advisable to photo-document the results with 4X microscopy at least twice during this window (e.g., at 24 and 48 h after plating the tumor cells). Be mindful that these are 3-dimensional assays, and it is difficult to completely image the entire assay. Most perineural invasion occurs in a plane from the bottom of the plate, where the cells are embedded in the matrix slightly above the bottom of the plate. Because this plane is close to horizontal, the vast majority of neurites with tumor cells are captured in a single-level image at 4X.</li>
</ol>

<table><tbody><tr><td>DMEM/F-12 50/50 Mix with L-glutamine &amp;amp; 15 mM HEPES</td><td>Corning Cellgro</td><td>10-090-CV</td><td>Manassas, VA</td></tr><tr><td>Fetal bovine serum</td><td>Atlanta biologicals</td><td>S11150</td><td>Flowery Branch, GA</td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Life Technologies Corporation</td><td>25200056</td><td>Grand Island, NY</td></tr><tr><td>Phosphate buffered Saline 1x</td><td>Corning</td><td>21-040-CM</td><td>Manassas, VA</td></tr><tr><td>Matrigel hESC-Qualif Mouse</td><td>Corning Incorporated</td><td>354277</td><td>Bedford, MA</td></tr><tr><td>Gamma Irradiated 35 mm glass bottom culture dishes</td><td>MatTek Corporation</td><td>P35G-1.5-14-C</td><td>Ashland, MA</td></tr><tr><td>SteREO Discovery.V8 Operating Microscope</td><td>Carl Zeiss Microimaging</td><td>495015-0021-000</td><td>Thornwood, NY</td></tr><tr><td>Schott ACE I light source</td><td>Schott</td><td>A20500</td><td>Germany</td></tr><tr><td>CellTracker</td><td>Life Technologies Corporation</td><td>C2925</td><td>Carlsbad, CA</td></tr><tr><td>BD PrecisionGlide Needle 21 G</td><td>BD</td><td>305195</td><td>Franklin Lakes, NJ</td></tr></tbody></table>

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.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20487/20487fig1.jpg" /> 
Figure 1. DRG-matrix assay. Correct placement of the dorsal root ganglion within the matrix droplet, shown with brightfield microscopy at 4X. Scale bar represents 1 mm.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/20487/20487fig2.jpg" /> 
Figure 2. Outgrowth of neurites. Neurites are absent on day 0 immediately after placing the DRG in the matrix droplet (A). By day 1 (B), neurite outgrowth is apparent at 10X on brightfield microscopy. Neurite growth continues on day 2 (C) and day 3 (D); however, note the efflux of the fibroblasts. Scale bar represents 0.1 mm.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/20487/20487fig3.jpg" /> 
Figure 3. DRG-matrix assay with FaDu. Head and neck squamous cell carcinoma line FaDu added peripherally around an assay on day 3 (A), shown in green fluorescent, and brightfield microscopy at 4X Progressive neural invasion is seen on day 4 (B) and day 5 (C). Scale bar represents 1 mm.

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 Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma. J. Vis. Exp. (2017)
In this video, we demonstrate the ...

Perineural invasion or PNI refers to the migration of cancer cells along the nerves and their subsequent invasion into the perineurium - a suitable microenvironment for cancer growth and metastasis.
To mimic&#160; PNI in vitro, begin by adding a drop of extracellular matrix or ECM suspension onto a culture dish such that it takes the shape of a hemisphere. Now, incubate the culture dish to reinforce the structural and functional features of the 3D scaffold.
Next, carefully place a pre-harvested dorsal root ganglion or DRG, a cluster of sensory neurons, at the center of the semisolid ECM droplet. Incubate to embed the DRG within the ECM droplet.
Thereafter, add a suitable growth media and incubate. The nutrient supplemented ECM favors the sprouting of DRG into small nerve projections called neurites that outgrow radially towards the circumference of the droplet.
Remove the spent medium to prep the ECM droplet for subsequent assay. Seed fluorescently stained adherent cancer cells over the droplet. Due to the hemispherical shape, cancer cells roll over and settle along the droplet periphery.
Incubate the culture dish to allow for cancer cell-neurite interaction. When viewed under the fluorescence microscope, the cancer cells can be seen tracking alongside the neurites and migrating towards the DRG, confirming perineural invasion.

establishing-three-dimensional-co-culture-model-murine-dorsal-root

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Cancer Research

Head and Neck Cancer

Assays & techniques

1.3K 조회수. 출처: Huyett, P. et al. 두경부 편평 세포 암종의 신경 주위 침습 모델. J. Vis. 특급 (2017년) 이 비디오에서는 마우스 등뿌리 신경절과 신경성 암세포를 공동 배양하여 체외 신경주위 암 세포 침입 모델을 개발하는 절차를 보여줍니다. 이 모델은 암 분석을 수행하여 빠르고 재현 가능한 결과를 생성하는 데 사용할 수 있습니다. 

비디오: 쥐 등뿌리 신경절과 암세포의 3차원 공동 배양 모델 구축: 신경주위 암세포 침입을 연구하기 위한 체외 모델 - 개념

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.

Vivo에서 Murine 좌 골 신경의 모델 Perineural 내 습

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

연구

JoVE Journal

암 연구학

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.

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.

등쪽 뿌리 신경절 모델 : 암성 신경 침략의 생체 외 모델링에서

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 ...

신경과학

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.

두경부암의 신경성 침윤을 평가하는 생체내 병아리 융모난토암 막

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 ...

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

내레이션 대본

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