Hi, My name is William Sang. I'm from the laboratory of Dr.Edgar Engelman in the Department of Pathology at Stanford University. I'm also a surgery resident at the University of California San Francisco.
Today I'm gonna show you how to establish an orthotopic mouse model of colorectal cancer. This model is useful for studying the natural progression of colorectal cancer and for testing new therapeutic agents against colorectal cancer. This is in contrast to the traditional subcutaneous tumor model.
The subcutaneous tumor model is less than ideal in that tumor cells growing under the skin of an altered phenotype and frequently fail to progress and to metastasize. In fact, tumor response to therapy can vary widely depending on whether the tumor cells are implanted in a subcutaneous versus an orthotopic location. Two techniques can be used to establish an orthotopic mouse model of colorectal cancer.
One technique involves injection of a colorectal cancer cell suspension into the SQL wall. The other technique involves transplantation of a piece of subcutaneous tumor onto cecum. Both techniques are similar and involve the following steps, one, cell or tumor preparation.
Two, mouse preparation, laparotomy and exposure of the cecum. Three injection of cells or transplantation of tumor, and four mouse abdominal wall closure and recovery. Let's get started.
So for the procedure today, we have a number of different instruments that we use. These are forceps, scissors, a regular needle holder, a Castro Viejo ne needle holder. And importantly for the procedure, we have a precut piece Of sterile gauze.
Colorectal cancer cells Are grown in culture and harvested when sub confluent, a single cell suspension is prepared in phosphate buffered saline and kept on Ice. A malice with a Previously established subcutaneous colorectal tumor is euthanized. The subcutaneous tumor is removed using sterile technique and divided into two to three millimeter pieces.
The tumor pieces are kept in phosphate buffered Saline on ice. In our laboratory, We use an inhaled anesthetic isof fluorine. Alternatively, one can use injectable anesthetics to achieve the same effect.
The depth of anesthesia is assessed using toe pinch. There should not be a withdrawal reflex upon toe pinch. Antibiotics may be given at this point.
The anesthetized mouse, which was previously shaved, is properly positioned. The abdomen is prepped three times with Betadine solution, and then the abdomen and the surgical site is draped in a sterile fashion. A small nick is made in the skin.
The abdominal wall musculature is grafted and lifted up. The abdominal cavity is injured and a single blade of the scissors is used to push the intraabdominal contents away. The incision is extended to two to three centimeters.
The secum with its blind ending pouch is identified and exteriorized. The secum is isolated from the rest of the mouse. Using a precut sterile gauze warm saline is used to keep the cecum Moist.
A 27 gauge Or finer needle is used to inject a 50 microliter volume of cells into the SQL wall. The needle is removed. The injection site is inspected to ensure no leakage in addition to injection of cells into the SQL wall, an alternative approach is to transplant tumor onto the cecum.
A figure of eight stitch is placed onto the cecum using a six oh or seven oh size suture. The seco wall is lightly damaged. Then the tumor piece is properly positioned.
The stitch is tied down. The secum is returned to the abdominal cavity. The Mouse abdominal wall is closed using three interrupted stitches using three oh or four oh size suture.
Alternatively, one can use a simple running stitch, postoperative analgesics, and a fluid bolus may be given. At this point, the mouse is allowed to recover from anesthesia with inhaled anesthetics. The mouse typically recovers from anesthesia within 30 seconds.
I've just shown you how to establish an orthotopic mouse model of colorectal cancer. The time to developing primary tumors and liver metastases will depend on the technique used, the cell line used and the mouse species used. This model is useful for studying the natural progression of colorectal cancer and for testing new therapies against colorectal cancer.
So that's it. Thanks for watching and good luck with your Experiments.
