Orthotopic tumor experiment, also known as orthotopic transplantation model experiment, is to inoculate tumor tissue/cells into the primary tumor tissue and organ to generate tumors and form spontaneous metastases, which can better simulate the whole process of tumor growth, development, invasion and metastasis. Compared with the subcutaneous tumor-bearing model, the orthotopic transplantation model can simulate the tumor microenvironment similar to the primary tumor site, which can better predict the effect of drugs and has certain clinical significance.
1. Experimental Principle
The interaction between tumor cells and stromal cells and tissue-specific immune cells will affect tumor growth, differentiation and drug sensitivity. Open surgery is used to expose the animal tissues and organs corresponding to the primary tumor, and tumor tissues or cells are implanted into the organs. By visualizing the in vivo tumor growth-related methods, such as IVIS bioimager, in situ imaging can be formed to gain a deeper understanding of the biological characteristics of tumor growth, distribution and metastasis.
2. Experimental Steps
2.1 Tumor Cell Recovery, Subculture and Amplification Culture
Take the tumor cells frozen in liquid nitrogen and recover, amplify and culture them to ensure the vitality and good growth state of the tumor cells.
2.2 Calculate the Cell Inoculation Amount
Refer to relevant literature and calculate the number of cells in advance according to the total number of mice required for the experiment.
2.3 Selection and Grouping of Mice
Generally, 7-8W healthy mice of the same sex are selected, and most of them are immune-deficient nude mice. If the tissue block inoculation method is selected, subcutaneous tumor-bearing mice need to be prepared. Grouping is carried out according to the purpose of the experiment.
2.4 Construction of orthotopic transplantation model
2.4.1 Cell inoculation method
After anesthetizing the mice, surgical procedures were used to expose the primary tissue and organs of the tumor. The diluted tumor cells were directly inoculated under the serosa of the organs, the anatomical position of the organs was restored, and the serosa was sutured.
2.4.2 Tissue block inoculation method
First, the tumor cells were subcutaneously inoculated to make a subcutaneous tumor model. When the subcutaneous tumor grew to about 1 cm, tumor-bearing mice with good growth and no ulceration were selected. The tumor nodules were completely peeled off under aseptic operation in the clean bench, and the blood stains were washed off with saline. The tumor nodules were cut open, the necrotic tissue in the center was removed, and the tumors were cut into small rice-like tumors with a diameter of about 1 mm. After anesthetizing the mice, the limbs were fixed, and the primary tissue and organs of the tumor were exposed by surgical procedures. The prepared tumor tissue of about 1 mm was transplanted under the serosa of the organs, the anatomical position of the organs was restored, and the serosa was sutured.
2.5 Observation of tumor growth and mouse survival
The mice were returned to the original breeding cage and continued to be raised. After surgery, the animals were observed every day for complications and abnormal conditions (activity, neurological function, breathing, and animal appearance). The nude mice were killed by cervical dislocation when they developed cachexia and were in a dying state. The growth of the tumor in the tumor-bearing area was observed after dissection. Alternatively, IVIS bio-imaging devices were used to monitor tumor growth changes.
3. Example of experimental results
Breast cancer cells (4T1-luc2) were cultured and then injected orthotopically into the mammary gland of Balb/c mice. After 7 days, the tumor volume was measured every day with a vernier caliper, and the tumor size and metastasis were observed using a bioluminescence imaging system.
4. References
[1] He, M., Henderson, M. K. , Muth, S. , Murphy, A. , & Zheng, L. . (2020). Preclinical mouse models for immunotherapeutic and non-immunotherapeutic drug development for pancreatic ductal adenocarcinoma. Annals of Pancreatic Cancer, 3, 7-7.
[2] Zhang, G. L., Zhang, Y., Cao, K. X., & Wang, X. M. (2019). Orthotopic injection of breast cancer cells into the mice mammary fat pad. Journal of Visualized Experiments (143).
[3] Vinod N, Hwang D, Azam SH, &Van Swearingen AED, et al. (2021). Preparation of an Orthotopic, Syngeneic Model of Lung Adenocarcinoma and the Testing of the Antitumor Efficacy of Poly(2-oxazoline) Formulation of Chemo-and Immunotherapeutic Agents. Bio Protoc, 11(6):e3953.