The overall goal of this approach is to model liver colonization by metastatic tumor clones generated by colorectal cancers, and to use this model to improve diagnostics and therapy of liver metastases. Clinical observations indicate the heterogeneity of metastatic disease. Our model provides an ability to capture this heterogeneity to detect genes which are associated with oligometastatic and polymetastatic disease.
And to use this knowledge for improvement of early detection and treatment of metastasis. The main advantage of our technique is that we use double-labeled monoclonal cell lines derived from patients with colorectal cancer, and can observe development of liver metastases both in vivo and ex vivo. The implications of this technique extend toward a therapy of metastatic disease.
It gives us the ability to test any potential drug with quantitative estimation of numbers of metastatic lesions and growth kinetics of each lesion. After generating luciferase tdTomato labeled HCT116 cells and preparing media, reagents, and instruments, according to the text protocol, plate the transpected cells at the following densities per well, in triplicate, in 96-well plates. Following the incubation of the cells for five hours, quantify the tdTomato fluorescent intensities using an IVIS system by starting the image software on the desktop.
Click the Initialize button to initialize the imaging time, medium binning, two for F/Stop, 535 for the excitation filter, and 580 for the emission filter. Place the 96-well plate on the imaging station, and click on Acquire to start imaging. After the image is acquired, in the Tool Palette, click ROI tools and select 12x8 from the slit icon.
Create 12x8 slits to cover the area of the signal on the image of the 96-well plate and click the Measurements tab. Observe a window with the table of ROI measurements with units of photons per second, per steradian, per square centimeter. Once the cells reach 70 to 80 percent confluency, approximately one hour prior to spleen injection, add 0.05 percent tripsin and 0.53 millimolar EDTA to the cells to dissociate them.
After incubating the cells for three to five minutes, use an equal volume of C-DMEM to neutralize the tripsin. Thoroughly pipette the cultures to avoid clumping, then use an automatic cell counter to count the cells. Freeze suspended cells in 1x DBPS at a concentration of 1.2 to 2 x 10 to the 6 cells per 100 microliters, pipetting thoroughly to avoid clumping.
Then, keep the cells on ice until injection, occasionally re-suspending them in the tube. After administering analgesics and anesthetizing a six to eight week old female athymic nude mouse according to the text protocol, identify the spleen as a purple area seen externally through the skin on the left flank, and make an eight millimeter left flank incision on the skin just above the organ. Next, lift up on the abdominal wall and make a small incision.
Allow air into the abdominal cavity so that the internal organs move away from the incision site. Then enlarge the abdominal wall incision to approximately five millimeters. The steps demonstrated for injection are critical for the procedure.
Leaking of tumor cells during injection can lead to extra-hepatic metastases. Injection should be performed slowly, over 30 to 60 seconds, with no more than 1.5 million cells to prevent portal venous thrombosis. Expose the spleen by using cotton swab to gently apply pressure around the incision.
If needed, the pancreatic fat can be gently manipulated to help with exposure, so as not to injure the spleen. Using a one milliliter syringe with a 27-gauge needle, over a period of 30 to 60 seconds, inject 100 microliters of cells into the tip of the exposed spleen. At the end of the injection, place a micro-clip on the spleen prior to removing the needle, to prevent leakage of the injected cell suspension, and leave it in place for five minutes.
Five minutes post-injection, use a hand-held cautery device to perform a splenectomy for hemostasis. Begin with the splenic hilum on the anterior side by pre-coagulating the proximal side of the vessels with adjacent fat tissue. Splenic bleeding can occur at the injection site.
The micro-clip is applied to the spleen to prevent tumor cell leakage and to control splenic bleeding. Methods of hemostasis can include cautery, holding pressure for three to five minutes, and suture ligation if necessary. Close the abdominal wall using a 5.0 absorbable braided suture and a single horizontal stitch.
Then, use a 4.0 non-absorbable mono-filament suture to close the skin, also with a single horizontal stitch. Monitor the animal post-operatively, according to the text protocol. To perform bioluminescent imaging, after anesthetizing mice according to the text protocol, intraperitoneally inject the animals with 150 microliters of previously prepared firefly luciferin.
Place the mice in an IVIS in a supine position with spacers in between the animals to minimize the signal to adjacent mice. Three minutes after luciferin injection, after initializing the IVIS, select luminescent, one second exposure time, and medium binning. Click the Acquire button to start imaging, ensuring that imaging is consistently performed three minutes after luciferin injection.
After the image is acquired, and an image window and tool palette appear, click ROI tools and select a number from one to five from the circle icon that corresponds to the number of mice imaged. TO define ROIs, circle the signal area of the luminescent signal on the abdominal cavity of the mouse, and then click measurements of the ROI as an arbitrary unit of radiance. Include an additional ROI circle for the background.
Four weeks post-injection, to carry out diffuse luminescent imaging tomography, or DLIT, after initializing the IVIS, select Imaging Wizard, Bioluminescence, and click Next. Select DLIT and click Next. Select Firefly Probes, and click Next.
Select Mouse for Image Subject, Auto for Exposure Parameter C-13 Centimeter for Field of View, and 0.5 Centimeter as Subject Type, and click Next. Then click the Acquire button to start imaging. After acquiring an image, select the DLIT 3D Reconstruction tab, in the Analyze tab, and click Reconstruct to generate the 3D reconstruction image.
Click Tools and 3D Animation. Then, in the 3D Animation window, select Spin CCW on Y-Axis. Click Record and Save to save the file into a mov format file.
To carry out ex vivo fluorescent imaging, after harvesting and dissecting livers from mice according to the text protocol, measure the fluorescent intensities for each tumor colony using the IVIS. By selecting Fluorescence, four seconds Exposure Time, Medium Binning, 2 for F/Stop, 535 for the excitation filter, and 580 for the emission filter. Click the Acquire button to start imaging.
After acquiring the image, locate the Image window in Tool Palette. Click ROI Tools, and select 1 from the circle icon. To define ROIs, circle the signal area of individual tumor colonies on the image.
Acquire an additional ROI circle of the background, and then click Measurements of the ROI as an arbitrary unit of radiance. Acquire an additional ROI circle of the background. Finally, carry out calculations according to the text protocol.
As shown here by bioluminescence, three weeks after spleen injection, HCT116 L2T clones P1 and P2 had a larger tumor burden, as compared to clones O1 and O2.These data indicated that clones O1 and O2 can mimic oligometastatic disease, while P1 and P2 represent aggressive wide-spread metastatic colonization of the liver. The quantification of ex vivo fluorescence of the liver, as of four weeks after spleen injection, confirmed that P1 and P2 had higher fluorescent intensities compared with O1 and O2 livers. Fluorescent labeling of tumor clones identified the number and sizes of individual metastatic colonies in the liver.
Overall, ex vivo fluorescent images were consistent with macroscopic findings, but small colonies hidden under the surface were better detected by fluorescent imaging. As depicted here, the numbers and sizes of metastatic colonies were counted and measured in each liver, and ex vivo fluorescence imaging was done on each metastatic colony for each monoclone. As shown in these graphs, the total number of metastases in P1 was higher as compared to P2, O1, and O2, while the average size of individual colonies was larger in P2 than in P1, O1, and O2.Our model provides an approach to identifying new persuasion genes associated with molecular heterogeneity of metastasis.
These genes can be used as targets for new approaches to metastasis treatment. Our model can be also used for validation of different protein-coding and non-coding genes which emerge from current large clinical databases but are not yet functionally defined in the context of metastatic disease. Generally, individuals new to this method will struggle, because of the delicacy of the animal surgery.
However, with practice, good results are achievable in a short amount of time. While attempting this procedure, it's important to remember to follow animal surgery guidelines, carefully control bleeding, and to prevent the leakage of tumor cell suspension.
