The subcutaneous tumor experiment is to transplant the tumor into the subcutaneous tissue of the same or xenogeneic animals to form a tumor mass. The growth of the tumor in the animal body is observed to evaluate the proliferation ability of tumor cells/tissues in the animal body. It is generally used for screening and testing drugs that inhibit tumor growth (cell proliferation). It has now become a common method for detecting tumorigenic potential and evaluating new anticancer drugs in vivo.
1. Experimental Principle
Subcutaneous tumor-bearing experiment is to inoculate tumor cells into the subcutaneous part of the animal's flank or back, and use the tumor cells to absorb nutrients in the body for cell proliferation, forming visible and measurable tumor tissue. By observing and monitoring the formation of tumor tissue, the proliferation ability of tumor cells in the animal and the feasibility of anti-tumor drug therapy are evaluated.
2. Experimental steps
2.1 Selection of tumor cell lines and mouse strains
In the selection of tumor cell lines and mouse strains, the following aspects need to be considered:
a. Different tumor cell lines have different tumor formation rates, and cell lines with high tumor formation rates need to be selected;
b. Select human or mouse cell lines according to the purpose of the experiment. Generally, mouse cell lines are more likely to form tumors, and human cell lines are closer to human tumor growth;
c. The selection of mouse strains should be based on the source of the cell line: human cell lines are xenotransplants, and immunodeficient mice should be selected, such as nude, NOD/scid, NSG, etc.; mouse cell lines are allogeneic transplants, and mice with the same source as the tumor cell line should be selected.
2.2 Tumor cell subculture and expansion
Take the tumor cells frozen in liquid nitrogen and resuscitate and expand them to ensure the vitality and good growth state of the tumor cells.
2.3 Calculate the cell inoculation amount
According to experience, generally 2 to 1 million mouse tumor cells are inoculated per mouse, while human tumor cells require 2 to 10 million per mouse. For details, please refer to relevant literature. Calculate and determine the number of cells in advance according to the total number of mice required for the experiment.
2.4 Prepare mice
Generally, 5-6W mice are selected. Animals less than 4W may be intolerant and die prematurely. Animals over 6W will have enhanced immunity and are less likely to form tumors. Only animals of the same sex are used in each batch of experiments. Usually, both males and females can be used, but they should be selected according to the tumor type and tumor cell line being studied (females should be selected for breast cancer, and males should be selected for prostate cancer, etc.)
2.5 Establishment of subcutaneous tumor-bearing model
Tumor cells in the exponential growth phase of in vitro culture are collected, routine cell counts are performed under a microscope, cell density is adjusted, and inoculated subcutaneously in healthy animals under sterile conditions. The inoculation site is generally selected from the site with rich blood vessels and easy operation, such as the armpit, groin, flank and neck back.
2.6 Experimental grouping and drug administration
Generally, 7-10 days after inoculation, when the tumor grows to about 1 cm, grouping and drug administration can be carried out according to the purpose of the experiment. Generally, subcutaneous tumors are mainly used for the detection of anti-tumor drugs, so the groups are often the control group, positive drug group, low, medium and high dose drug group.
2.7 Observation of changes in mouse weight and tumor volume
Return the mice to the original breeding cage and continue to feed them. Regularly observe the status of the mice and the growth of the tumor, measure the tumor size with a vernier caliper 1-2 times/week, and record the change curve of the tumor volume and mouse weight.
3. Example of results
Colon cancer cells (HCT116) were infected with lentivirus shSeptin4 and then inoculated into the axilla of nude mice. The tumor volume was measured every 3 days. After 4 weeks, the tumor was dissected, separated, and weighed for analysis.
4. References
[1] Berne, S. , Majaemaar, Frange, R. , et al. (2019). APS8 Delays Tumor Growth in Mice by Inducing Apoptosis of Lung Adenocarcinoma Cells Expressing High Number of α7 Nicotinic Receptors. Marine Drugs, 16(10), 367.
[2] Hai-Bo, Q. , Li-Yi, Z. , Chao, R. , Zhao-Lei, Z. , Wen-Jing, W. , & Hui-Yan, L. , et al. (2013). Targeting cdh17 suppresses tumor progression in gastric cancer by downregulating wnt/β-catenin signaling. Plos One, 8(3), e56959.
[3] X Zhao, Feng, H. , Wang, Y. , Wu, Y. , & Cao, L. . (2020). Septin4 promotes cell death in human colon cancer cells by interacting with bax. International journal of biological sciences, 16(11), 1917-1928.