This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. This model recapitulates advanced colorectal cancer metastatic disease. The orthotopic colorectal cancer model presented here recapitulates the clinical scenario of advanced intestinal tumors and metastatic disease in colorectal cancer patients that cannot be studied using patient-derived organoids or patient-derived xenograft models.
From a euthanized mouse, extract the tumor by carefully removing it from the skin and surrounding non-tumor tissue using scissors and forceps. Place the harvested tumors in PBS at four degrees Celsius. Using a blade, dissociate the tumors in a 10-centimeter culture plate containing one-millimeter of medium.
Place the homogeneous dissociated sample into a 15-milliliter conical tube. Then, add the medium to a final volume of five milliliters. Then, add the digestion medium to the tube.
Place the tube at a 45-degree position inside a cell culture incubator for one hour at 37 degrees Celsius. During the one-hour incubation, mix the solution every 15 minutes with a five-milliliter pipette. After incubation, add five milliliters of medium and mix well.
Then, sort the solution with a 100-micrometer cell strainer using a sterile 50-milliliter tube. Centrifuge the sorted cells at 500g for 10 minutes at room temperature. Aspirate the supernatant and resuspend the pellet in three milliliters of 1x RBC Lysis Buffer solution.
Incubate for 10 minutes at room temperature. Then, add three milliliters of medium and mix the sample. Centrifuge at 500g for 10 minutes at room temperature and aspirate the supernatant.
Resuspend the pellet with five to 10 milliliters of medium, and use a cell counter to calculate the total number of cells. After centrifuging the cells at 500g for 10 minutes at room temperature, resuspend in 10 milliliters of PBS. Repeat the centrifugation and wash with 10 milliliters of PBS.
Resuspend the pellet to obtain a concentration of 20 times 10 to the six cells per milliliter and mix well to obtain a homogeneous cell suspension. Next, prepare a 29g syringe for cecum injection. Load 50 microliters of the tumor cell suspension into the syringe, and ensure that no air bubbles remain in the syringe.
Place the syringe on ice. First, sterilize the surgical site by spraying with disinfecting detergent and wiping. Then, depilate the abdomen using a mouse hair removal machine.
Place the mouse in a supine position and disinfect the abdomen by scrubbing with chlorhexidine or povidone-iodine. Make a one-centimeter longitudinal incision over the lower abdomen using surgery scissors. Carefully separate the skin to the sides to expose the peritoneum.
Then, make a 0.5 to one-centimeter incision in the peritoneum membrane, sufficient to exteriorize the cecum. Using a precut sterile gauze, carefully isolate the cecum from the mouse. Moisten the cecum was saline solution throughout the procedure.
Immobilize the cecum by carefully holding it with forceps, then introduce the needle superficially into the cecum wall. Slowly, inject the entire 50 microliter volume of tumor cell suspension. This process takes around 10 seconds.
After injection, slowly remove the needle from the cecum and apply gentle pressure on the injection site with a cotton tip applicator. Clean the cecum with saline solution to remove any debris and return the cecum back to the abdomen of the animal. Close the peritoneum using 5/0 sutures and then close the skin of the abdomen using 5/0 sutures.
Orthotopic CRC-PDX tumors were observed in the intestine of mice implanted with patient-derived cancer cells. Histological analysis by hematoxylin and eosin staining on the cecum showed the presence of tumor cells. The histological analysis also revealed lung metastasis and liver metastasis in the implanted mice.
Mice carrying orthotopic tumors were treated with either the vehicle, testing drug, or the chemotherapy drug irinotecan. Analysis of micro CT scanning images showed that compared to the vehicle, the testing drug induced a reduction of the tumor volume, which was further reduced in combination with irinotecan treatment. This model will help to study the biology of the metastasis in colorectal cancer tumors.
