Supported by a subproject of NIH grant P20 RR16440 and a bridge grant from the West Virginia University Office of Research and Graduate Education to S. Weed. Technical assistance from L. Lopez-Skinner during the early phases of project development is gratefully acknowledged. The authors are also grateful for technical assistance and OSC19 cells from J. Myers and M. Younes (Department of Head and Neck Surgery, M.D. Anderson Cancer Center, Houston, TX); P. Turner and K. Secrest (West Virginia University Department of Pathology Tissue Bank) for histological processing and procedures, R. Wysolmerski (West Virginia University, Department of Neurobiology and Anatomy) for the LifeAct-mCherry construct and J. Bear (University of North Carolina) for the pLL7.0 lentiviral vector. The use of the West Virginia University Microscopy Imaging Facility (supported by NIH grant P20 RR16440 and the Mary Babb Randolph Cancer Center) and its non-linear optical microscopy laboratory (NLOM; supported by a joint collaboration between the West Virginia University Center for Neuroscience and the West Virginia University Department of Physics/West Virginia Nanoscience Initiative) is also gratefully acknowledged. The NLOM is supported in part by NIH grant P30 RR031155 to the Center for Neuroscience.

1. Cell Lines, Vector Construction and Lentiviral Production 
<ol>
	<li>Human head and neck tumor cells lines (OSC19 or UMSCC1) were cultured in complete media consisting of DMEM (Cellgro cat # 50-003-PB) supplemented with 10% fetal bovine serum FBS (Hyclone cat # SH30070.03), 1% penicillin/streptomycin (Cellgro cat #30-002-CI), and 1% non-essential amino acids (Cellgro cat # 25-025-CI).</li>
	<li>To transfer the LifeAct-mCherry coding sequence into the pLL7.0 lentiviral vector, the Sbf1 recognition site in the parent mCherry cDNA was altered by introducing three silent mutations into the recognition sequence using site-directed mutagenesis (Stratagene cat # 200518-5). The resulting modified LifeAct-mCherry sequence was then PCR amplified with flanking EcoR1/Sbf1 sites and subcloned into pLL7.0 to generate the pLL7.0-LifeAct-mCherry construct.</li>
</ol>
2. pLL7.0-LifeAct-mCherry Virus Production
<ol>
	<li>Viral production was conducted according to the Lentiviral Expression Systems manual (System Bioscience version 2-051018).</li>
	<li>The packaging cell line 293T/17 cells (ATCC cat # CRL-11268) was grown to 40% confluence in the same complete media used for HNSCC lines.</li>
	<li>Cells were transfected with the pLL7.0-LifeAct-mCherry, psPAX2, and pVSV-G vectors in a 3:2:1 ratio, respectively using CalPhos ( Clontech cat # 631312).</li>
	<li>After 24 hours, the initial media from the transfection was replaced with fresh media.</li>
	<li>Media was then collected and replenished every 12 hours for 72 hours and stored at 4&deg;C.</li>
</ol>
3. Production of Head and Neck Cell Lines with Stable LifeAct-mCherry Expression
<ol>
	<li>The collected media was spun at 2000 rpm for 10 minutes at 4&deg;C.</li>
	<li>One ml of clarified media containing virus was directly added to OSC19 or UMSCC1 cells for 12 hours. Cells were then rinsed, and an additional one mL of virus was added for another 12 hour period.</li>
	<li>Cells were treated with media containing 200mg/ml puromycin for two weeks to select resistant colonies.</li>
	<li>Surviving clones were screened visually for LifeAct-mCherry expression by fluorescence microscopy. Individual positive colonies were typsinized using sterile 3mm cloning discs (Fisher cat # 0790710A).</li>
	<li>Positive cells were maintained in media containing 200mg/ml puromycin until frozen back or used for orthotopic injection.</li>
</ol>
4. Orthotopic Tumor Xenograft Formation
<ol>
	<li>All animal procedures were conducted in accordance with a protocol (09-0821) approved by the West Virginia University Animal Care and Use Committee.</li>
	<li>LifeAct-mCherry expressing tumor cells were trypsinized, centrifuged and 2.5 x 104 cells were resuspended in 50 &mu;L complete media.</li>
	<li>Tumor cells were loaded into a one ml syringe attached to a 27 &frac12; gauge needle.</li>
	<li>Female athymic Fox1nu/nu mice 8 weeks of age (Harlan Laboratories) were anesthetized with combination of 80mg/kg ketamine and 10mg/kg xylazine. Anesthetized mice were maintained between 37-40 &deg;C on a heating pad.</li>
	<li>Using sterile forceps, the tip of the tongue was gently grasped and carefully pulled out of the oral cavity.</li>
	<li>Cells were slowly injected into one side of each tongue to create a bulbous mass in the tongue center, avoiding the lingual arteries.</li>
	<li>Mice were injected with 2.1mg/kg yohimbine and returned to the heating pad where they were monitored for 2-3 hours during recovery from anesthesia.</li>
	<li>Once revived, mice were placed into sterile cages containing a soft transgenic dough diet (Bioserve cat # S3472).</li>
	<li>Mice were weighed every 2-3 days and monitored visually for tumor onset.</li>
</ol>
5. Preparation of Mouse Tongues for Ex-vivo Imaging
<ol>
	<li>Mice harboring tumors at different time points (typically 2-4 weeks post-injection) were euthanized by carbon dioxide inhalation.</li>
	<li>Tongues were extracted, rinsed with 1X PBS and attached to one side of a conventional paraffin tissue embedding cassette (StatLab Cat #H104,) using monofilament sewing thread from a local hobby shop and a size 8 sewing needle.</li>
	<li>Once the tongues were immobilized, the entire cassette assembly was placed in a 30mm tissue culture dish and immersed in 1X PBS.</li>
	<li>Processed tongues were immediately used for two-photon excitation microscopy.</li>
</ol>
6. Imaging of Tongue Tumors with Two-photon Microscopy
<ol>
	<li>Tongue cassettes were submerged in a 60mm dish containing 1X PBS secured in a custom designed holder on a retractable cantilever arm (Chamber Shuttle, Siskiyou instruments) positioned under the objective of an upright microscope (Moveable Microscope (MOM); Sutter Instruments).</li>
	<li>A 40X/0.8NA water dipping objective lens was placed directly on or over visible tumor lesions. Tongues were imaged by two-photon microscopy with the Ti:sapphire laser (Mira, Coherent) intensity at 60 mW and input wavelength of 755 nm to optimize the mCherry signal.</li>
	<li>Serial 1mm laser scanning images were collected at 1 &mu;m incremental depths over a total tissue depth between 15 and 100 &mu;m (depending on tumor volume). Images were captured using ScanImage, an open source program based on the MATLAB platform that was developed by the Karel Svoboda laboratory (Janelia Farms, HHMI). ScanImage generates a two-channel output of raster scan patterns to control the x/y galvanometric scan mirrors, and at the same time captures a maximum four-channel signal input simultaneously from photomultiplier tubes (PMTs) through a data acquisition board (PCI-6610S, National Instruments). The PMT signals are amplified by low noise current preamplifiers (SR570, Stanford Research System) before feeding into the NI-DAQ board for display on the monitor screen. ScanImage collects z-stack images by controlling the z-axis of the objective and collects time-lapse images in a single or cycle mode. Images were saved in a single TIFF file with 16 bit depth.</li>
</ol>
7. Image Analysis using Amira Software 
<ol>
	<li>Amira imaging for tumor quantification: In Aimra software, open the TIFF file containing the set of z-stack images.</li>
	<li>Highlight the file name, select and apply the Voltex function to generate a three dimensional rendering. A large primary tumor image with several smaller, dissociated invasive groups (IG) of collectively invaded cells is typically apparent in the rendered image (see Figure 6A).</li>
	<li>To select the primary tumor mass, select "Open Data", then "Labeling", then "Label Field".</li>
	<li>Thresholding the primary tumor- select all z-stack images and scroll through the images in the z-plane to ensure inclusion of only the primary tumor mass within three dimensions. This is typically the largest sized image in the field and often appears segmented as the imaged is traversed through the z-plane.</li>
	<li>Use the magic wand/threshold function to correct background fluorescence and eliminate it from the image without discarding any of the tumor signal. Highlight the "Inside File" label and click the &oplus; button. Select the "All Slices" check box. This produces a colored border around the thresholded primary tumor area in every z-stack increment. </li>
	<li>To select IGs, choose the "New" function. Select a single IG and ensure it is not associated with the primary tumor by scrolling through the z-plane.</li>
	<li>Select "All Slices" and click the &oplus; button. Rename the file (ex: IG 1)</li>
	<li>Repeat the thresholding procedure in 7.4 and the highlighting step in 7.5 for the determined IG. Select an outline color different than the color used to denote the primary tumor mass.</li>
	<li>Repeat as necessary for each additional IG throughout the image, scanning through the z-plane to ensure all IGs are denoted. Using different colors for each identified IG aids in future identification on the image.</li>
	<li>Once the primary tumor and all IGs are selected, select "Segmentation" from the pulldown menu, then select "Material Statistics". This provides the volume measurement as well as the X, Y and X tumor core coordinates for the primary tumor in order to calculate distances of IGs from the central tumor point.</li>
	<li>With the volumes calculated, determine the distance for each IG from the central core of the primary tumor. Select "Segmentation", then "Material Statistics". This step calculates all measurements into pixels. Convert pixels to micrometers based on the calibration of the microscope objective and any additional increases in magnification (i.e.; zoom functions). For the microscope and settings used in these experiments, the 40X objective was used with no zoom, giving a calibration of 1 pixel = 0.298 &mu;m. Import data into an Excel spreadsheet, which gives parameters for the primary tumor in three dimensions (X1, Y1, Z1) and for each IG (ex: X2, Y2, Z2 for the first IG).</li>
	<li>The invaded distance in microns for each IG from the center of the primary tumor is calculated using the formula &radic;((X2-X1)2 + (Y2-Y1)2 + (Z2-Z1)2).</li>
	<li>The tumor invasive index (TI) is calculated using the formula TI = NT x VT x DT, where NT = the total number of IGs in the image, VT = the total volume of all IGs, DT = total distance traveled of all invasive groups from the center of the primary tumor.</li>
</ol>
8. Three dimensional renderings with Nikon NIS-Elements Software 
<ol>
	<li>Three dimensional renderings with greater topographical detail can be generated by importing the original 16-bit monochrome TIFF files of the entire tumor image into the Nikon NIS-Elements software package (Nikon, Melville, NY). Select "File", then "ND", then "Create ND File from File Sequence". Select the TIFF z-series image stack and specify the appropriate step size.</li>
	<li>Calibrate the ND document in the xy plane. Specify the size of one pixel using a manual calibration.</li>
	<li>Choose the "Volume View". Use the "HQ" function to calculate additional slices in the z-plane for higher quality. In the "3D Renderer Settings", use the "Advanced Renderer" with a quality of "Ultra High Details" and "Full Resolution". Choose "Alpha Blending" to accentuate the tumor surfaces.</li>
	<li>Optimize the three-dimensional image for presentation. Zoom in on the tumor and associated IGs and crop as needed. Rotate the image in the xyz planes and adjust the LUTs.</li>
	<li>Capture the image for presentation. Select "Edit- Create View Snapshot (8 bit RGB)". Name and save file accordingly.</li>
</ol>
9. Representative Results
<img src="/files/ftp_upload/2941/2941fig1.jpg" alt="Figure 1" /> 
Figure 1. Overall schematic illustrating key steps in orthotopic tongue tumor production and in situ two photon imaging.
<img src="/files/ftp_upload/2941/2941fig2.jpg" alt="Figure 2" /> 
Figure 2. Injection of LifeAct-mCherry expressing OSC19 cells into the mouse tongue.
<img src="/files/ftp_upload/2941/2941fig3.jpg" alt="Figure 3" /> 
Figure 3 Resected tumor-containing mouse tongue prepared for two photon imaging.
<img src="/files/ftp_upload/2941/2941fig4.jpg" alt="Figure 4" /> 
Figure 4. Orientation of a tumor-containing tongue in position
on a two photon microscopy ready for imaging.
<img src="/files/ftp_upload/2941/2941fig5.jpg" alt="Figure 5" /> 
Figure 5. Representative screen shot from ScanImage
demonstrating raw data acquisition of the initial two-photon image.
<img src="/files/ftp_upload/2941/2941fig6.jpg" alt="Figure 6" /> 
Figure 6. Image analysis and quantification of tumor invasion
in an orthotopic OSC19 tumor. Representative screen shot images of
Amira Voltex rendering (A) and a single thresholded z-section (B) with
the primary primary tumor outlined in purple, invasive group 1 (IG-1)
outlined in blue, IG-2 outlined in red and IG-3 outlined in green as
an example of the identification procedure. Arrowheads denote IGs in
(A) and (B). C. Plots of volume versus invaded distance for eight
individual IGs used to calculate the tumor invasion index (Ti).
<img src="/files/ftp_upload/2941/2941fig7.jpg" alt="Figure 7" /> 
Figure 7. Representative images of tumors and invaded tumor
groups from this protocol compared with images from a conventional IHC
approach. A. Paraffin section of an entire mouse tongue harboring a
UMSCC1 orthotopic tumor. Immunohistochemical staining was conducted
using conventional procedures with a primary antibody against the
HNSCC-specific cell marker emmprin (Zymed; cat # 34-5600) at 1:1000
dilution and visualized by using the OmniMap DAB anti-Rb detection kit
(Ventana cat # 760-149) followed by iron hematoxylin staining. Images
encompassing the total tongue area were collected individually at 4X
magnification on an Olympus ZX70 Provis microscope with an Optronics
MicroFire CCD camera and reconstructed using the StereoINvestigator
imaging package (MBF Bioscience). The tumor is evident at the tongue
tip. An enlarged region showing the invasive front and individual
tumor cell groups (arrowheads) is shown in (B). C. Nikon
NIS-Elements rendering of a representative OSC19 tumor. Enhanced
contour detail and clear visualization of IGs (arrowheads) is evident.
 Bars in all images = 100 &mu;m.

<ol>
	<li>Sano, D., Myers, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograft+models+of+head+and+neck+cancers.">Xenograft models of head and neck cancers.</a> Head Neck Oncol. 1, 32-32 (2009).</li><li>Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+cancer+in+the+head+and+neck+region.">Animal models of cancer in the head and neck region.</a> Clin Exp Otorhinolaryngol. 2, 55-60 (2009).</li><li>Myers, J. N., Holsinger, F. C., Jasser, S. A., Bekele, B. N., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthotopic+nude+mouse+model+of+oral+tongue+squamous+cell+carcinoma.">An orthotopic nude mouse model of oral tongue squamous cell carcinoma.</a> Clin Cancer Res. 8, 293-298 (2002).</li><li>Sano, D., Myers, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis+of+squamous+cell+carcinoma+of+the+oral+tongue.">Metastasis of squamous cell carcinoma of the oral tongue.</a> Cancer Metastasis Rev. 26, 645-662 (2007).</li><li>Sano, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+combination+anti-endothelial+growth+factor+receptor+and+anti-vascular+endothelial+growth+factor+receptor+2+targeted+therapy+on+lymph+node+metastasis:+a+study+in+an+orthotopic+nude+mouse+model+of+squamous+cell+carcinoma+of+the+oral+tongue.">The effect of combination anti-endothelial growth factor receptor and anti-vascular endothelial growth factor receptor 2 targeted therapy on lymph node metastasis: a study in an orthotopic nude mouse model of squamous cell carcinoma of the oral tongue.</a> Arch Otolaryngol Head Neck Surg. 135, 411-420 (2009).</li><li>Kupferman, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrkB+induces+EMT+and+has+a+key+role+in+invasion+of+head+and+neck+squamous+cell+carcinoma.">TrkB induces EMT and has a key role in invasion of head and neck squamous cell carcinoma.</a> Oncogene. 29, 2047-2059 (2010).</li><li>Ammer, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saracatinib+Impairs+Head+and+Neck+Squamous+Cell+Carcinoma+Invasion+by+Disrupting+Invadopodia+Function.">Saracatinib Impairs Head and Neck Squamous Cell Carcinoma Invasion by Disrupting Invadopodia Function.</a> J Cancer Sci Ther. 1, 52-61 (2009).</li><li>Rosenthal, E. L., Kulbersh, B. D., King, T., Chaudhuri, T. R., Zinn, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorescent+labeled+anti-epidermal+growth+factor+receptor+antibody+to+image+head+and+neck+squamous+cell+carcinoma+xenografts.">Use of fluorescent labeled anti-epidermal growth factor receptor antibody to image head and neck squamous cell carcinoma xenografts.</a> Mol Cancer Ther. 6, 1230-1238 (2007).</li><li>Gleysteen, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labeled+anti-EGFR+antibody+for+identification+of+regional+and+distant+metastasis+in+a+preclinical+xenograft+model.">Fluorescent labeled anti-EGFR antibody for identification of regional and distant metastasis in a preclinical xenograft model.</a> Head Neck. 30, 782-789 (2008).</li><li>Bhirde, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+killing+of+cancer+cells+in+vivo+and+in+vitro+with+EGF-directed+carbon+nanotube-based+drug+delivery.">Targeted killing of cancer cells in vivo and in vitro with EGF-directed carbon nanotube-based drug delivery.</a> ACS Nano. 3, 307-316 (2009).</li></ol>

The authors have nothing to disclose.

Orthotopic mouse models have proven useful for studying many aspects of head and neck cancer1,2. We have combined a well established orthotopic system of SCOT3 with two-photon microscopy imaging of mCherry-labeled cells as a system to study the early events of head and neck tumor cell invasion. In this procedure, we have noted that cells can leak from the site of tumor injection, especially in mice six weeks or younger due to insufficient tongue size. We use older mice to avoid this issue. The larger tongue size with older mice also aids in avoiding rupture of a lingual artery and excessive hemorrhaging from the tongue. Tumor take is greatly enhanced when the tongue dramatically swells at the site of initial injection. This swelling subsides within one-two hours as the injected fluid is absorbed. Tumor growth appears evident one-two weeks post injection with the indicated injected cell number as a small white bump on the tongue surface. We also note that at the 40X objective used in our studies presented here, we cannot distinguish individual tumor cells but do identify IGs as invasive cell clusters that recapitulate the typical mode of HNSCC invasion.
To date this model has been extensively used for testing the role of specific molecules as well as several anti-tumor drugs on SCOT growth an invasion, with efficacy measured by cervical lymph node metastasis monitored using IHC or bioluminescent methods4-7. Tumors formed in this system manifest close to the tongue surface (Figure 7), allowing the application of two-photon microscopy to image entire tumors in situ as invasive multi-cellular clusters. The procedure can also be utilized to visualize tumor invasion with cellular resolution. Two-photon microscopy has been previously utilized to study experimental treatments for head and neck cancer in orthotopic8,9 and xenograft8,10 models. However, there are two major differences between these reports and our protocol. First, these studies use extracellular labels to target/identify head and neck tumor cells, possibly limiting detection only to tumor cells with ample access to the circulation. Second, invasion of tumor cells close to the primary site that likely recapitulates early metastatic activity was not assessed as an experimental parameter. Our protocol provides the ability to directly quantify tumor cell invasion at any point during tumor progression in mouse tongues. While the method described here describes the procedure using dissected tongues, we are currently in the process of adapting this method to image tumor invasion in live mice for use in combination with bioluminescence to simultaneously monitor early invasion, local lymph node involvement and distant metastasis in the same animal. Alterations to the protocol for in vivo imaging require designing of an appropriate stage for positioning and maintaining mice during imaging, as well as a practical system to properly irrigate the oral cavity of anesthetized mice during the imaging procedure. Once optimized, these adaptations will provide the ability to study the role of potential pro-invasive molecules and the testing anti-invasive compounds on local invasion and more distant metastatic involvement in animals over extended time periods.

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.

Watch this Scientific Journal Video about Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma at JoVE.com

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

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 ...

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19.0K Views.  Mary Babb Randolph Cancer Center, West Virginia University. 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.

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

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current &#39;gold standard&#39; method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...