Orthotopic Mouse Model of Colorectal Cancer

Summary

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Abstract

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Protocol

I. Cell Preparation

1. Colorectal cancer cells are grown in culture and harvested when subconfluent.
2. A single cell suspension is prepared in phosphate buffered saline and kept on ice.

II. Tumor Preparation

1. A mouse with a previously established subcutaneous colorectal tumor is euthanized.
2. The subcutaneous tumor is removed using sterile technique and divided into 2-3 mm pieces
3. The tumor pieces are kept in phosphate buffered saline on ice.

III. Mouse Preparation

Note: In our laboratory we use inhaled isoflurane to an....

Discussion

Although mouse subcutaneous tumor models are easy to establish and monitor, it is clear that this model cannot replicate the original anatomic site of colorectal cancer. Due to the difference in microenvironment, colorectal cancer cells growing under the skin have been shown to change their phenotype and almost always fail to progress and metastasize ^1,2. In fact, tumor response to therapy can vary dramatically depending on whether cancer cells are implanted in an ectopic (subcutaneous) versus orthotopic locat.......

Materials

Material Name	Type	Company	Catalogue Number	Comment
Name	Company	Catalog Number	Comments
Cell culture media and components				will vary depending on cell line used
Phosphate Buffered Saline		BioWhitaker	17-512F	also sold by a number of other vendors
15 mL centrifuge tubes, polypropylene		Corning	430790
6 well plates with lid		Becton Dickinson Labware	353046
Iris forceps, straight, serrated tips		World Precision Instruments	15914	best to have a pair of these instruments
Iris dissecting scissors, straight, sharp		World Precision Instruments	14393
Needle Holder (regular)		World Precision Instruments	14109
Needle Holder (Castroviejo)		World Precision Instruments	14137
Cidex Plus		World Precision Instruments	7364	instruments should be autoclaved periodically, but may also be sterilized by soaking in Cidex
6-0 or 7-0 suture				for cecum; courtesy of Department of Surgery, UCSF
3-0 or 4-0 suture				for abd wall; courtesy of Department of Surgery, UCSF
Anesthetic machine / Isoflurane		J.A. Baulch and Associates	3206	also sold by a number of other vendors
Heating Pad, disposable, 6" x 6"		Prism Technologies	20419
Tape, orange, 13 mm x 13 m		Fisher Scientific	15901F	for restraining anesthetized mouse
Pro-Cord/Cordless Trimmer		Oster	78997-010	for shaving mouse fur
Betadine		Fisher Scientific	19-027132	may also be purchased at any medical supply store
2 x 2 gauze, 3-ply		Johnson and Johnson	7635	may also be purchased at any medical supply store
Sterile Field, Barrier, 16" x 29"		Johnson and Johnson	0905	may also be purchased at any medical supply store
Cotton tipped applicators, 6"		Fisher Scientific	14-960-3Q	may also be purchased at any medical supply store
Syringe, 10 cc, Luer lock tip		BD	309604
Syringe, 1 cc, tuberculin, slip tip		BD	309602
27 1/2 G needle		BD	305109
30 G needle		BD	305106