Name: a model for perineural invasion in head and neck squamous cell carcinoma
Uploaded: 2017-01-05T13:00:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma at JoVE.com

This work was supported in whole by funding from the NIH through the R21 grant, "Mechanisms of Perineural Invasion in Head and Neck Cancer" and the NCI T32 training grant, "Post-Doctoral Research Training in Head and Neck Oncology (2T32CA060397-21)." Thank you to Richard Steiman, MD, PhD and lab staff.

1. Preparation of Culture Medium and Dishes (10 min)
<ol>
	<li>Add 100 &#181;L of Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) to the wells of a 96-well V-bottom plate.</li>
	<li>Remove a pre-aliquoted-vial of approximately 100 &#181;L of semisolid matrix from the 20 &#176;C freezer and place it directly on ice. 
	NOTE: Do this prior to the dorsal root ganglion (DRG) harvest because the semisolid matrix takes approximately 30 min to reach liquid state on ice, which is necessary for the subsequent steps. Failure to keep the semisolid matrix on ice at all times will result a poor-quality matrix droplet.</li>
	<li>Label glass-bottom cultures plates with permanent ink, creating unique identifiers in each of the four corners on the undersurface of the plate. Place the plates right-side up on ice to cool the plate surface. 
	NOTE: Each mouse yields 32 - 40 DRG, so label them from 1 to 40. It is not necessary to pre-chill pipet tips, as chilled tips will warm over the course of placing the matrix droplets, potentially introducing differences in matrix consistency.</li>
</ol>
2. Dissection of Murine DRG (45 min)
<ol>
	<li>Euthanize an athymic nude mouse as per specific laboratory protocols, such as through the use of a CO2 chamber and a thoracotomy.</li>
	<li>Set up the dissection area and microscope. Use the clean, sterilized, and flat underside of polystyrene containers for 15- or 50-mL conical tubes. 
	NOTE: Additionally, they are suitable for use as the dissecting surface, as they are inexpensive, disposable, and allow for fixation of the spine using pins or needles. Only use sterilized instruments and needles.</li>
	<li>Under natural vision, make a longitudinal incision along the spinal column from the root of the tail to the head of the mouse with a pair of straight or curved fine scissors.</li>
	<li>Use the same scissors to transversely divide below the sacral spine. Dissect both sides of the spine all the way up to the skull base using the scissors. At this point, ensure that the cervical spine is transected cranially as far as possible with scissors.</li>
	<li>Observe the cervical end of the spinal column under the operating microscope at low power. The white spinal cord is apparent in the middle of a bony ring, which is surrounded by variable amounts of paraspinal muscles and soft tissue.</li>
	<li>Split the dorsal or superior aspect of the first 2 - 3 vertebral bodies with microscopic spring scissors. Using the spring action of the scissors, open this initial bony cut to ensure that the vertebral body dissection occurred at the midline. Continue in small increments towards the sacral spine, and then bisect the spine by completing the identical cuts on the ventral or inferior aspect of the vertebral bodies. 
	NOTE: Failure to ensure the midline dissection of the bony spinal column will result in a lower DRG yield.</li>
	<li>At this point, the spinal column is divided into two halves. Place one hemi-spine aside, with the spinal cord in place and facing down on a sterile plate. 
	NOTE: Leaving the spinal cord in place will prevent the DRGs from drying out, as the DRGs are deep to the spinal cord.</li>
	<li>Secure either end of the other hemi-spine with two 18-gauge needles on the polystyrene dissecting platform. Starting at the cervical end (which is apparent because the spine is narrower, the DRG are closer to one another, and there are rib insertions), gently peel back the spinal cord from about 4 vertebral levels.</li>
	<li>Observe the sensory nerves (usually two) connecting the DRG. In the area where the nerves insert to the DRG, gently grasp the surrounding fascia with microscopic forceps. Dissect and trim this fascia and other nerve tissue with the microscopic spring scissors to free up the DRG. Gentle retraction will bring the DRG out of its position within the bony spine. 
	NOTE: Increasing the microscope power at this step is beneficial. Crush injury to the DRG will significantly limit the growth of neurites. This is avoided by never grasping the DRG directly, but rather by grasping the surrounding fascia.</li>
	<li>Cut the peripheral nerve (the DRG generally has one peripheral nerve, which is deep relative to the dissecting view) with the microscopic spring scissors to release the DRG. 
	NOTE: It is easiest to ascertain where the nerve ends and the DRG begins while on tension, so making this cut as close as possible to the DRG at this point is ideal. Once this distal branch is cut, the proximal branches are trimmed as well.</li>
	<li>Trim the DRG of any stray nerve fibers or fascial attachments with the microscopic spring scissors. After adequate isolation, place the DRG into the room-temperature medium within the 96-well V-bottom plate. 
	NOTE: Placing a dark background underneath the plate makes visualizing the sub-mm white DRGs easier. Only place one DRG in each well; this way, they can be accounted for more easily in the subsequent steps.</li>
	<li>Repeat this process all the way down one hemi-spine and then the other. Each side yields 16 - 20 DRGs, totaling 32 - 40. 
	NOTE: If there are fewer DRGs than this, the dissection of the spine needs to be carried out further cephalad and/or caudally. The DRGs become increasingly less well defined as one proceeds caudally.</li>
</ol>
3. Preparation of Semisolid Matrix Droplets (&#60; 1 min per plate)
<ol>
	<li>Remove one glass well-bottom plate from ice and place it on an ice block underneath the operating microscope. Make sure the aliquot of matrix remains on 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 pipet 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. 
		NOTE: Using a second hand to stabilize the pipetting hand facilitates more accurate placement.</li>
	</ol>
	</li>
</ol>
4. Insertion of DRG into Semisolid 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 21-gauge 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 21-gauge 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. 
		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 21-guage needle.</li>
	<li>Transfer the completed plate to a 37 &#176;C incubator. This will solidify the semisolid 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. 
	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 - 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>
5. 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. 
	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 - 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 - 3 days after the DRG harvest and implantation into the matrix). 
	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 - 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 - 5 min and transfer the suspended cells to a 15-mL conical tube. Spin the cells and re-suspended 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>
6. 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 - 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 pipet. 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 pipet with a smooth trigger makes 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. 
	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 make 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. 
	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 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>

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The authors have no competing financial interests.

Critical Steps within the Protocol
The most important steps within this protocol are the precise dissection and extraction of the dorsal root ganglia. Proper transection of the spinal column and a midline-longitudinal division into two hemi-spines are critical to obtaining large numbers of DRG. During the dissection of individual DRGs, the ganglion should never be handled directly, but rather the surrounding fascia should be grasped with the microscopic forceps. Failure to do ...

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the US, with 10,000 deaths per year nationally and 300,000 deaths per year worldwide1. The overall prognosis for HNSCC has remained unchanged at 50% for the past several decades. Perineural invasion (PNI) is one of the most prominent pathological features that portend a poor prognosis in patients with HNSCC. Unfortunately, PNI is a frequent occurrence in HNSCC and can be found in up to 40% of HNSCC patients2,3. 
PNI is the process by which malignant cells track along nerves to adjacent tissues, allowing for higher rates of local and distant spread. Accordingly, PNI-positive HNSCC tumors have higher rates of locoregional recurrences and distant metastases, resulting in lower overall survival compared to HNSCC patients without PNI4-8.
Although the treatment of patients with PNI is typically maximized by employing surgery, radiation, and chemotherapy, the overall survival rates of these patients are still decreased by up to 40% compared to patients without PNI9-11. Thus, it is clear that the current treatment modalities for HNSCC are ineffective in improving the adverse prognosis associated with PNI. The approach of developing targeted therapy against PNI in HNSCC has been hindered by the poor understanding of the factors that regulate this process. This is, in part, a consequence of the lack of a consistent in vitro model for the study of PNI in HNSCC. 
In recent years, several groups have been utilizing an in vitro model for studying PNI in predominantly pancreatic and prostate cancers12-19. This model uses the neurites generated from dorsal root ganglia isolated from mice or rats as a surrogate for large-nerve invasion. The dorsal root ganglia are fixed in a factor-depleted semisolid matrix, which is a solubilized basement membrane protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. This matrix allows for the outgrowth of the neurites and the tracking of single cancer cells along these neurites. Described here is the adaption of this model for the examination of PNI in HNSCC.

<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&amp;nbsp;</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&amp;nbsp;</td><td>Life Technologies Corporation</td><td>C2925</td><td>Carlsbad, CA</td></tr><tr><td>BD PrecisionGlide Needle 18 G and 21 G</td><td>BD</td><td>305195</td><td>Franklin Lakes, NJ</td></tr><tr><td>Premium Microdissecting Tweezer</td><td>Harvard Apparatus</td><td>60-3851</td><td>Holliston, MA</td></tr><tr><td>Premium Fine Operating Standard Scissors</td><td>Harvard Apparatus</td><td>52-2789</td><td>Holliston, MA</td></tr><tr><td>Premium Spring Scissors</td><td>Harvard Apparatus</td><td>60-3923</td><td>Holliston, MA</td></tr><tr><td>Dressing Forceps</td><td>Harvard Apparatus</td><td>72-8949</td><td>Holliston, MA</td></tr><tr><td>Athymic nude mice (002019)</td><td>Jackson Laboratory</td><td>002019</td><td>Bar Harbor, ME</td></tr></tbody></table>

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.

After the dissection of the DRG and the placement within the matrix droplet, the appearance of the assay should resemble Figure 1. Note that the DRG is not perfectly round, but it is centered within the matrix droplet. This allows for the outgrowth of neurites in 360 degrees, shown partially in Figure 2. Be aware that certain parts of the DRG send out neurites faster and in greater numbers than others, typically corresponding to where the efferent and aff...

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

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, ...

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Research

JoVE Journal

Cancer Research

10.6K Views.  University of Pittsburgh Medical Center. 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.

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Medicine

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 ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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.

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 ...

Neuroscience

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 ...

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

Source: Schmidt, L. B. et al. <a href="https://www.jove.com/video/59296" target="_blank">The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.</a> J. Vis. Exp. (2019)
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.

Source: Schmidt, L. B. et al. The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer. J. Vis. Exp. (2019)
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JoVE Encyclopedia of Experiments

Head and Neck Cancer

In vivo studies

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/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.

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 ...

Assays & techniques

Murine Sciatic Nerve Model of Perineural Invasion or PNI: Extraction and Processing of Sciatic Nerve to Understand Invasion by Cancer Cells

Source: Deborde, S. et al. <a href="https://www.jove.com/video/56857" target="_blank">An In Vivo Murine Sciatic Nerve Model of Perineural Invasion.</a> J. Vis. Exp. (2018)
In this video, we describe the procedure to establish a murine model of perineural invasion by injecting cancer cells into the sciatic nerve. The model can help gain an understanding of the magnitude of nerve invasion for subsequent assessment of anti-cancer drugs.

Source: Deborde, S. et al. An In Vivo Murine Sciatic Nerve Model of Perineural Invasion. J. Vis. Exp. (2018)
In this video, we describe the procedure to ...

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

Summary

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Chapters in this video

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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

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

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

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

Murine Sciatic Nerve Model of Perineural Invasion or PNI: Extraction and Processing of Sciatic Nerve to Understand Invasion by Cancer Cells