Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

The overall goal of this microsurgical technique is to inject colorectal tumor cells into the cecal wall to induce reproducible and metastatic colorectal tumors in mice. This method can answer key questions in the field of circulating tumor cell biology, its main advantages are the highly reproducible and uniform tumors, allowing reliable stage prediction and circulating tumor cell prediction. Begin by using atraumatic forceps to carefully exteriorize the cecum of a six to eight week old immunocompromised mouse.

Placing the blind end of the cecum on the abdomen so that the pouch points cranially. For the intra-cecal injection, mount a standard one milliliter syringe equipped with a 30 gauge canella and load it with the tumor cells onto a micro injection pump that is mounted on a micro manipulator. Next, use the atraumatic forceps to carefully grasp the tip of the cecum and use a second forceps, moistened with warm saline to gently smooth the cecum with downward strokes.

Now move the animal under a binocular surgical microscope and with the canella directly parallel to and above the cecum, use two atraumatic forceps to carefully stretch the tissue at both ends of the exteriorized end of the cecum. Slowly pull the cecum over the canella, taking care not to perforate the entire belt of wall or the serosa beyond the initial point of penetration and place the canella above the blood vessels and under the thin translucent membrane inside belt of wall. When the canella is in place, use a footswitch to inject 20 microliters of cells over a period of 20 seconds.

Between the thin translucent serosa lining, above the intramural blood vessels and the muscularis. When all of the cells have been injected, carefully remove the canella and place a dry swap under the cecum. To prevent artificial peritoneal dissemination, lyse any leaked cells with a thorough distilled water rinse and gently return the cecum to the abdominal cavity.

Close the abdominal wall with rapidly absorbable running sutures, in the skin with surgical wound clips. Then place the mouse on a heating mat set to 38 degrees celsius with monitoring until full recumbency. To isolate the circulating tumor cells, at the appropriate experimental end point, fill 15 milliliter conical tubes with five 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 tube washes in PBS.

After the second wash, re-suspend the pellets in 200 microliters of PBS, supplemented with EDTA and add four microliters of anti amp camp antibody to each sample, for 20 minute incubation on ice in the dark. Next, use a hydrophobic barrier pen to draw an approximately one centimeters circle in one sterile six 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 amp camp positivity and use the micromanipulator to transfer the cells of interest into 15 microliters of the appropriate buffer for the intended subsequent downstream analysis. The use of colorectal cells in this model, reliably results in more abund mice within 35 days of tumor cell injection, with the primary tumors measuring about 10 millimeters in size, and with liver, and lung metastasis, and circulating tumor cells, almost invariably present. Further, the circulating tumor cells can be easily isolated for downstream analysis.

Once mastered and if performed properly, this technique can be performed within 10 minutes, facilitating the formation of uniform and metastatic tumors that can both be used for therapeutic and molecular studies. Limitations of the model include, its dependency on cell lyse, which have their own limitations, as well as the immunodeficiency of the mice, both of which, can be overcome by the use of mutant colorectal cancer cells. Due to its simplicity, this model maybe a valuable addendum to the currently available models of colorectal cancer.