1Department of Surgery, University of California, San Francisco - UCSF, 2Department of Pathology, Stanford University School of Medicine
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Tseng, W., Leong, X., Engleman, E. Orthotopic Mouse Model of Colorectal Cancer. J. Vis. Exp. (10), e484, doi:10.3791/484 (2007).
I. Cell Preparation
II. Tumor Preparation
III. Mouse Preparation
Note: In our laboratory we use inhaled isoflurane to anesthetize the mouse; alternatively, one can use injectable anesthetics to achieve the same effect
V. Exposure of the Cecum
VI. Injection of Cells into the Cecal Wall
VII. Transplantation of Tumor onto the Cecum
Note: In addition to injecting cells into the cecal wall, an alternative approach is to transplant tumor onto the cecum
VIII. Mouse Abdominal Wall Closure and Recovery
Note: With inhaled anesthetics this typically takes 30 seconds
IX. Results - Primary Tumor and Liver Metastasis
Primary Tumor - shown in situ (A) and with evidence of neovascularization (B); on H&E staining, tumors are locally invasive (C)
Liver Metastases - shown ex vivo (D)
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 location 3. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. The two techniques to establish this model have unique advantages and disadvantages. Injection of a cell suspension into the cecal wall introduces colorectal cancer cells which have been previously growing in an in vitro environment and are arguably homogeneous. Cells may have reduced invasive or metastatic potential after several passages in culture. Transplantation of a piece of subcutaneous tumor introduces a more heterogeneous population of cancer cells that have been established in vivo. However, tumors contain stromal cells and frequently have necrotic portions which may affect the consistency of this model (personal communication, I. Fidler, MD Anderson). From a technical standpoint, our laboratory has found that injection of a cell suspension into the cecal wall is more difficult and carries the risks of intraluminal injection and leakage post-injection. Interestingly, an orthotopic mouse model of rectal cancer has been described that does not require mouse laparotomy 4. Mice are anesthetized and the rectal mucosa is prolapsed with digital pressure. A small volume of colorectal cancer cells in single cell suspension is injected submucosally. Primary, invasive rectal cancers develop in mice as early as one week after injection; however, none of the mice develop liver metastases and the authors did not comment on the frequency of lung metasases.
Department of Surgery, University of California, San Francisco
Raymond Shaheen, M.D.
|Cell culture media and components||will vary depending on cell line used|
|Phosphate Buffered Saline||BioWhittaker||17-512F||also sold by a number of other vendors|
|15 mL centrifuge tubes, polypropylene||Corning||430790|
|6 well plates with lid||BD Biosciences||353046|
|Iris forceps, straight, serrated tips||World Precision Instruments, Inc.||15914||best to have a pair of these instruments|
|Iris dissecting scissors, straight, sharp||World Precision Instruments, Inc.||14393|
|Needle Holder (regular)||World Precision Instruments, Inc.||14109|
|Needle Holder (Castroviejo)||World Precision Instruments, Inc.||14137|
|Cidex Plus||World Precision Instruments, Inc.||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 Professional Products||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 & Johnson||7635||may also be purchased at any medical supply store|
|Sterile Field, Barrier, 16" x 29"||Johnson & 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 Biosciences||309604|
|Syringe, 1 cc, tuberculin, slip tip||BD Biosciences||309602|
|27 1/2 G needle||BD Biosciences||305109|
|30 G needle||BD Biosciences||305106|
1. Heijstek, M.W., Kraneburg, O., Borel Rinkes, I.H. Mouse models of colorectal cancer and liver metastases. Dig Surg. 22, 16-25 (2005).
2. Kobaek-Larsen, M., Thorup, I., Diederichsen, A., Fenger, C., Hoitinga, M.R. Review of colorectal cancer and its metastases in rodent models: comparative aspects with those in humans. Comp Med. 50, 16-26 (2000).
3. Wilmanns, C., Fan, D., O’Brian, C.A., Bucana, C.D., Fidler, I.J. Orthotopic and ectopic organ environments differentially influence the sensitivity of murine colon carcinoma cells to doxorubicin and 5-fluorouracil. Int J Cancer. 52, 98-104 (1992).
4. Kashtan, H., Rabau, M., Mullen, J.B., Wong, A.H.C., Roder, J.C., Shiptz, B., Stern, H.S., Gallinger, S., Intra-rectal injection of tumor cells: a novel animal model of rectal cancer. Surg Oncol. 1, 251-256 (1992).