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

The overall goal of this procedure is to produce a mouse model of oral cancer that allows the visualization and quantification of tumor invasion in the tongue. This is accomplished by first generating oral squamous carcinoma cells for two photon imaging by Lentiviral infection of life Act M cherry and stable clonal selection. Next orthotopic xenograft tumors are produced in nude mice by injection of life Act M cherry labeled cells into the tongues of anesthetized mice.

Then tumor containing tongues are imaged by two photon microscopy. Ultimately, results can be obtained that show quantified local oral tumor invasion within the tongue muscle through two photon imaging of fresh tongue tissue and subsequent computer aided three dimensional reconstruction of primary tumors and regionally invaded cellular groups. The main advantage of this technique over existing methods like immunohistochemistry or bioluminescent imaging, is that tumorous cell invasion can be quantitatively measured in three dimensions.

The implications of this technique extend towards therapeutic intervention oral cancer because it provides a model system for the analysis of proteins involved in tumor invasion, as well as the assessment of preclinical compounds on anti invasive therapeutic strategies. Generally, individuals new to this technique will struggle as injection of the tumor cells into the tongue and preparation of the tongue for two photo microscopy is technically demanding, requiring skill and practice. Visualization of this method is critical as the tumor injection in two photon steps are difficult to learn and require specialized skill To begin culture.

Human head and neck tumor cell lines in complete medium consisting of DMEM supplemented with 10%FBS, 1%penicillin streptomycin, and 1%non-essential amino acids. In order to transfer the LIFE Act M cherry coating sequence into the PLL 7.0 lentiviral vector site directed mutagenesis is first required to introduce three silent mutations into the SBF one recognition site in the parent mCherry CD NA.Next PCR amplify the Modified Life Act mCherry sequence with flanking ECO R one SBF one sites and sub clent into the PLL 7.0. To generate a PLL 7.0 LIFE Act M cherry construct following a lentiviral expression system culture, the packaging cell line 2 9 3 T 17 to 40%confluence in complete medium.

Using Cal Foss transfect the cells with the PLL 7.0 LIFE Act M cherry PS PACS two and P-V-S-V-G vectors in a three to two to one ratio respectively. After 24 hours, replace the medium with fresh, complete medium, collect and replenish the medium every 12 hours for 72 hours, and store the collected medium at four degrees Celsius to produce head and neck cell lines with stable life Act M cherry expression. Spin the collected medium at 2000 RPM for 10 minutes at four degrees Celsius, add one milliliter of the clarified medium containing the virus to the OSC 19 or US CCC one cells for 12 hours.

Rinse the cells, then add an additional one milliliter of virus for another 12 hour period. Culture the cells in medium containing 200 milligrams per milliliter of pur mycin for two weeks To select resistant colonies, visually screened surviving colonies for life. Act m cherry expression by fluorescence microscopy trypsin eyes, individual positive colonies using sterile three millimeter cloning discs.

Maintain positive cells in medium containing 200 milligrams per milliliter of pur mycin until frozen back, or used for orthotopic injection to generate an orthotopic tumor xenograft trypsin life Act. M cherry expressing tumor cells then centrifuge the culture and resuspend about 2.5 times 10 to the fourth cells in 50 microliters of complete medium. Load the tumor cells into a one milliliter syringe attached to a 27 gauge half inch needle.

Next anesthetize, eight week old female athymic fox. One nude mice with a combination of 80 milligrams per kilogram of ketamine and 10 milligrams per kilogram of xylazine. Once the animal stop moving, apply eye lubrication ointment and check for responsiveness by pressing a fingernail into the footpad.

Maintain the mice on a heating pad between 37 and 40 degrees Celsius. Using sterile forceps, gently grasp the tip of the tongue and pull it out of the oral cavity. Slowly inject the cells into one side of each tongue to create a bulbous mass in the tongue center.

Inject the mice with 2.1 milligrams per kilogram of yohi being and return them to the heating pad to monitor them during recovery from anesthesia. Place mice in sterile cages containing a soft transgenic dough diet. Weigh the mice every two to three days and monitor them visually for tumor onset.

To prepare mouse tongues for ex vivo imaging, euthanize mice harboring tumors at different time points. Using carbon dioxide inhalation, extract the tongues and rinse them with one XPBS. Then using monofilament sewing thread from a local hobby shop and a size eight sewing needle.

Attach the tongue to one side of a conventional paraffin tissue embedding cassette. Once the tongue is immobilized, submerge the entire cassette assembly in one XPBS. Immediately process the tongues using two photon microscopy to image the tongues using two photon microscopy.

Submerge the tongue cassettes in a 60 millimeter dish containing one XPBS. Secure the dish in a custom design holder on a retractable cantilever arm positioned under the objective of the two photon microscope. Place a 40 x 0.8 numerical aperture water dipping objective lens directly on or over a visible tumor lesion.

Image the tongue by two photon microscopy with the titanium sapphire laser with an intensity of 60 milliwatts and an input wavelength of 755 nanometers. To optimize the M cherry signal, collect serial one micrometer laser scanning images at one micrometer incremental depths over a total tissue depth between 15 and 100 micrometers. With this configuration, tumors up to one millimeter in tissue depth can be analyzed.

Use scan image to capture images. Scan image generates a two channel output of raster scan patterns to control XY galvan metric scan mirrors and at the same time captures a maximum four channel signal Input simultaneously from photo multiplier tubes through a data acquisition board. Scan image collects Z stack images by controlling the Z axis of the objective and collects time-lapse images in a single or cyclic mode.

Save the images in a single TIF file with 16 bit depth. Using the Amira software render tumor image stacks into a single three dimensional image by opening the TIF file containing the set of Zs stack images. Use the TEX function to generate a three-dimensional rendering.

A large primary tumor image with several smaller dissociated invasive groups or igs will likely be present in the rendered image. To measure the tumor volume, define the threshold of primary tumor area and each slice of the image stack correct for and eliminate background fluorescence. Repeat the thresholding procedure for each invasive group.

Once the primary tumor and all the igs are selected, determine the volume measurement as well as the X, Y, and Z tumor OID coordinates for the primary tumor. In order to calculate distances of igs from the central tumor point, calculate the tumor invasive index or ti using the ti equals N NT times VT times dt. Where NT equals the total number of igs in the image VT equals the total volume of all the igs and DT equals the total distance traveled by all invasive groups from the center of the primary tumor.

Three dimensional renderings with greater topographical detail can be generated by importing the original 16 bit monochrome tip files into Nikon NIS elements within the software. Create an ND file from an image stack. Then manually calibrate the document to specify the pixel size for higher quality renderings.

Add additional slices in the Z plane. This figure shows an example of the raw data acquired from a two photon image NIS elements or a mirror. Software can be used to reconstruct the raw data of tongue tumors, which is shown in this figure.

This panel shows an example of the three dimensional rendering using the TEX function in the Amira software. Here is an example of thresholding of an individual two photon section from the image stack to identify invasive groups from the primary tumor as well as the primary tumor center or oid. After each threshold section is analyzed, Amira quantifies the volume and distance traveled by each invasive group from the Centro.

These data can be graphically demonstrated and used to obtain the numeric value for the complete level of tumor cell invasion. A attributable to the primary site shown here are representative images of tumors as visualized by conventional pathological immunohistochemistry labeling compared to a three dimensional image of a similar tumor using the described protocol Once mastered from the point of tongue extraction to the final 3D rendering, this technique can be done in about two hours if properly performed while attempting this procedure, take care not to puncture the tongue through with the needle, thereby losing tumor cells and impeding the tumor take Following this procedure. Testing of therapeutic compounds or cells with manipulated protein levels can be conducted in order to determine their efficacy on oral cancer invasion.

In an in vivo setting, Don't forget working with human tumor cells modified with lentivirus is hazardous precautions such as proper PPE and working in a BSL two certified laminar flow hood should always be taken while working with injected mice.