CTC Isolation from a Whole Blood Sample: A Method to Obtain CTCs from Murine Colorectal Cancer Model
Transkript
– Circulating Tumor Cells, or CTCs, are the cancer cells that stem from the primary tumor and enter the blood circulation. Most CTCs can survive in the bloodstream without attaching to the surface; therefore, we can detect them directly from the blood of most cancer patients.
CTCs in the blood can indicate the tumor's spread inside the body, hence identifying them helps understand cancer progression. Begin by taking an anesthetized colorectal cancer mouse model and obtain blood by cardiac puncture. Transfer the blood into a conical tube containing density gradient medium and centrifuge to separate blood components.
Discard the topmost layer containing plasma and collect the peripheral blood mononuclear cell fraction, or PBMC, present at the interface in a separate tube. Next, add the anti-EpCAM antibody to PBMC cells and observe under a fluorescent microscope. Circulating CTCs express a cell marker known as Epithelial Cell Adhesion Molecule or EpCAM. Therefore, anti-EpCAM antibodies can help identify EpCAM positive CTCs.
Collect the CTCs and store them for further downstream analysis. In the following protocol, we will identify the EpCAM-expressing CTCs from the peripheral mononuclear cell fraction of blood of colorectal cancer mouse model.
– To isolate the circulating tumor cells at the appropriate experimental endpoint, fill 15 milliliter conical tubes with 5 milliliters of density gradient medium per tube and carefully transfer whole blood samples collected from tumor-bearing animals onto the density gradient layers.
Separate the cells by centrifugation and carefully recover the interface containing the mononuclear cells. Pipette the mononuclear cells into a new 15 milliliter tube for a second centrifugation, followed by two washes in PBS. After the second wash, resuspend the pellets in 200 microliters of PBS supplemented with EDTA and add four microliters of anti-EpCAM antibody to each sample for a 20 minute incubation on ice in the dark.
Next, use a hydrophobic barrier pen to draw an approximately 1 centimeter circle in one sterile 6 centimeter Petri dish per sample, and add 700 microliters of picking buffer to each circle, followed by 50 microliters of cell suspension. Use a microscope to check the density of the cells and allow the samples to settle for about five minutes. Then, screen the drop of cells for EpCAM positivity and use the micromanipulator to transfer the cells of interest into 50 microliters of the appropriate buffer for the intended subsequent downstream analysis.
