Metastasis is a leading cause of cancer-related deaths. This phenomenon occurs when cancer cells detach from the primary tumor, go into the blood circulation to finally reach a distant target organ. Once these detached cells go into the blood, we call them secondary tumor cells.
They represent a variable material because on one hand, they are the main culprit of metastasis formation and in other hand, they are easier than tumor biopsies. Indeed, they can be collected by a simple blood puncture. Despite the low number of these cells in the blood sample, we are the first to establish several circulating tumor cell line from the blood of patient with greater concern using the 3D controlled condition.
This cell line can be used at any commercial cell line for routine experiment. For example, for genetic drug screening, to protein of interest, but also for in vivo experiment to test the tumor initiation capacity and their metastatic potential. CTC, circulating tumor cell lines, should be cultured in 3D conditions in hypoxia.
Once CTC has formed spheres, we can put it in the medium and centrifuge it for five minutes at 300 RCF. Discard the supernatant and add 500 microliters of Accumax. We suspend one and incubate the solution 20 minutes at 37 degrees.
Then wash dissociated spheres with PBS and pellet the cells. Resuspend the pellet with a freshly supplemented DMEM/F12 and see the cells at the density of five cells per microliter in a new 24 Low Attachment plate. Centrifuge CTCs spheres eliminate the supernatant and wash the pellet twice with PBS.
At 500 microliter of PFE mix well and incubate the tube on ice for 20 minutes. Then, centrifuge the fixed spheres and wash the pellet twice with PBS, eliminating the maximum volume of PBS and add 20 microliter of formed HistoGel. Take it to suspension and make a droplet on the culture vase, let it dry for 10 minutes and detach it using a scalpel.
You can now process for any desired in order to CTCs targeting a desired protein of interest. Collect CTCs and disassociate the spheres with Accumax as previously described. Eliminate the Accumax and resuspend the pellet with three mL of blocking buffer and transfer the suspension through a 14 micrometers cell strainer to obtain single cell solution.
Count the cells and dispatch into tubes and add a volume of 200, 000 cells, centrifuge the tubes and discard the supernatant. According to the antibody data sheet, add in a tube, the desired volume of the antibody and in another tube its isotype and continue the steps following manufacturer's guidance. The remaining tube will not be labeled and the use of a viability market is strongly suggested in this experiment.
On the cytometer, start with the non-labeled tube to set the voltage in the negatively labeled area. Following this, place the isotype tube. The isotype would serve as a negative control to distinguish between the positive signal and non-specific signal.
And finish the experiment with the labeled cells with the antibody of interest where you should see a shift in the positively labeled gate, if the protein of interest is expressed. This suspend dissociated CTCs in supplemented medium at the density of 200, 000 cells per mL. In an Ultra Low attachment 96, well plate, seal the volume of 50 microliter and incubate the plate 24 hours in hypoxia.
The following day, add 50 microliter of different concentration of the tested drug and add the same volume of DMEM/F12 only to further determine the base of cellular viability and incubate the plate for another 48 hours. At day three, reconstitute the agent and add 70 microliter of the mix into each well. Put the plate on an orbital shaker for 20 minutes and then measure the luminescence.
This assay is a useful tool to test a large number of drugs in order to evaluate their effects on CTC's cell growth. In Eppendorf, resuspend cells in 100 microliter of PBS pinch the skin on the back of the mouse and insert the needle beneath the skin. Inject slowly the whole volume in the same spot and monitor tumor growth every second day and sacrificed the mice if the tumor exceeded a size of 1.5 cubic centimeters.
Prior to the surgery, inject subcutaneously a pain reliever, at the dorsal ventral side of the mouse, do a small incision below the 10th rib. Gently lift the spleen out and lay it on a sterile gauze. Using an insulin syringe, inject 50 microliter of resuspended CTCs in PBS and wait for three minutes without removing the needle.
Ligate the arterial vessel from one side and the venal vessel from the other side. Then remove the spleen by cutting directly above the two ligatures. Stitch the abdominal peritoneum and the skin and disinfect well the area.
The aim of this experiment is to test the capacity of CTCs derived from colorectal cancer patient to colonize the liver by inducing, visible metastatic foresight. Isolating and establishing of the clinic tumor cell line is an emergent tool for both fundamental and translational studies. For example, if you have a gene of interest where the in vivo studies demonstrated that this gene is involved in migration and invasion capacity of colorectal cancer cell lines, knocking out this gene in circulating tumor cell lines prior to the injection in the spleen of mice, to let them colonize the liver might be a good tool to confirm your result in vivo.
For translational studies, our main perspective of isolating circulating tumor cell lines is to use this precious material in personalized medicine. From patient's blood samples, we would isolate and amplify CTCs in culture for two or three weeks in order to have enough material to screen the panel of drugs that is available and assess its cytotoxicity to finally attribute
