Our protocol overcomes the limited bioluminescence imaging window by including a GFP tag which also facilitates the detection and quantification of micrometastasis by PCR analysis. Because the GFP tag circumvents the limited bioluminescence acquisition window, more subjects can be imaged without the signal loss and the potential for false negative results is limited. This study will be useful to researchers for the evaluation of therapeutic efficacy of anti-cancer agents.
This methodology provides a useful model for researchers interested in EMT, drug resistance, and cancer stem cell mechanisms related to the metastatic process as well as for anti-cancer drug development. After confirming a lack of response to pedal reflex, swab the left fourth mammary gland with alcohol and use fine rat-tooth forceps to lift the fourth mammary gland slightly. Making sure the tip is bevel up, insert a 25 gauge needle attached to a one milliliter syringe containing 100 microliters of cell basement membrane matrix mix and slightly retract the plunger to make sure the needle does not contact any blood vessels.
For comparative results, the tumor cell injection site must be consistent among all the recipient animals. If no blood enters the syringe, slowly inject the entire volume of solution into the mammary fat pad. A round raised mass will appear under the skin.
Wait 10 to 15 seconds for the basement membrane matrix to harden before removing the needle and allow the xenograft to grow freely without interruption for seven to 10 days prior to performing a tumor size measurement. Then use calipers to measure the size of the xenograft in all of the mice and determine the tumor volume using the equation as indicated. Before imaging the study animals, image three additional tumor bearing mice for 40 minutes at two-minute intervals using the parameters as indicated to obtain the luciferin kinetics for the study.
Use the peak measured bioluminescence signal to define the optimal image acquisition time window for all of the subsequent time points. To perform metastasis imaging, cover the primary tumor with a sleeve cut from a black glove and place an anesthetized mouse into the scanner then image the luciferin signal using the same parameters as just demonstrated with the ventral side of the mouse facing the camera for lung metastasis imaging and the dorsal side of the mouse facing the camera for brain metastasis imaging. Using a scanner and data analysis software, draw a region of interest over the imaged area to ensure that the entire organ of interest is covered to allow evaluation of the metastatic burden and quantify the bioluminescence output as the total flux and photons per second.
Immediately after bioluminescence signal quantification, harvest the brain and lung tissue and quickly rinse the organs in PBS to remove any superficial blood stains. Place the organs on a low auto fluorescence black plastic plate and transport the samples to a multi-spectral fluorescence scanner equipped with spectral unmixing capability for GFP detection. To acquire multi-spectral GFP images of the extracted organs, scan the organs from 500 to 720 nanometers with step size of 10 nanometers.
To obtain tissue auto fluorescence profile, scan excised organs of uninjected control mice. Additionally, image GFP positive tissues such as primary tumor to acquire auto fluorescence mixed GFP profile. Process these spectra as per the manufacturer's protocol to establish a spectral library with pure GFP and auto fluorescence profiles.
After confirming accuracy of the spectral library and separating GFP signal from tissue auto fluorescence background, utilize the same library to unmix all images on study. For DNA extraction after snap freezing, place the whole brain in a five milliliter homogenizing tube containing two milliliters of DNA lysis buffer and use a homogenizer set to 50 to homogenize the tissue. When the tissue has been fully homogenized, transfer one milliliter of the lysate to a DNAse and RNAse free two milliliter microcentrifuge tube and isolate the DNA according to the manufacturer's protocol.
Add 500 microliters of 100%ethanol to the isolate and mix the sample by inversion. After three minutes at room temperature, use a pipette to transfer the wool-like DNA precipitate to a new two milliliter DNAse and RNAse free tube. Let the sample air dry for one minute before adding one milliliter of 75%ethanol to the tube and inverting the tube three to six times to wash the sample.
Discard the ethanol after the last inversion before washing the DNA two more times with fresh ethanol per wash as just demonstrated. After the last wash, air dry the sample for 10 seconds before dissolving the DNA in 52 microliters of eight millimolar sodium hydroxide. Transfer a two microliter aliquot of DNA to a spectrophotometer vial and quantify the concentration of DNA in the sample using an absorbance ratio between A260 and 280 according to the formula.
Adjust the DNA concentration of the sample to 50 nanograms per microliter with additional eight millimolar sodium hydroxide and design the primer pair specific to the exogenous green GFP sequence in-house using an open-source primer design web portal for the real-time PCR detection of the GFP tag and the metastatic cells that have invaded and colonized the distal sites of the tumor. Then use a fast real-time PCR reagent and a real-time PCR machine that supports a fast real-time PCR protocol for real-time quantitative PCR analysis of the sample. For the detection of metastatic breast cancer cells in the lung, use dissecting scissors to open the chest cavity and cut past the heart and thymus to expose the trachea.
Insert a 22 gauge needle attached to a 10 milliliter syringe containing Bouin solution into the trachea and depress the plunger until the collapsed lungs become swollen with approximately two milliliters of the solution. Remove the syringe and needle once lungs have been inflated and transfer the whole lung into a 15 milliliter tube containing fresh Bouin solution. Gently invert the tube a few times before leaving the tissue to soak for 24 hours at room temperature.
The next day, rinse the lung with water and place the sample into a tube of 70%ethanol. Then use a dissecting microscope to count the number of metastatic white patches and nodules on the surfaces of the lungs. Tumor volume measurement by caliper is a well-established method for assessing treatment efficacy.
To obtain comparable signals between study groups, a pre-imaging BLI kinetics study must be conducted to determine the best imaging timeframe. Due to the superior signal-to-background ratio, whole body in vivo BLI is quite sensitive in detecting low level metastasis signals compared to the GFP approach. However, due to the stable nature of the GFP molecule, researchers have sufficient time to process the carcass prior to capturing the GFP signals from target organs in dual reporter studies.
This real life example demonstrates how caliper tumor size measurements can distort the data interpretation as this xenograft lost most of the viable tumor cell content while still maintaining its large mass and shape. Molecular detection by real-time PCR can also provide convincing evidence of brain metastasis in the orthotopic breast cancer model using exogenous GFP DNA sequencing as the transfected GFP DNA sequence does not naturally exist in humans or rodents. The implantation site should be physiologically and anatomically related to cancer malignancy.
Both tumor gene and protein expression can be evaluated at primary and distal sites and metastatic cells can be isolated for further characterization.
