Tumor metastasis refers to the phenomenon that cancer cells break away from the primary lesion and invade surrounding tissues, eventually overcoming the distant tissue microenvironment and achieving metastasis. The metastatic tumor experiment is a method of studying the process of tumor metastasis in vivo using a tumor metastasis animal model, which can better reveal the process of tumor metastasis, the biological function of genes, and the role of signaling pathways.
1. Experimental Principle
Tumor cells have a certain ability to invade and metastasize. Injecting tumor cells into the blood system of animals can better simulate the situation where tumor cells break away from the primary lesion and enter the blood circulation. By observing the formation of metastatic lesions in distant organs (lungs, liver, etc.), the metastatic ability of tumor cells can be judged. Commonly used inoculation methods are tail vein injection and left ventricular injection.
2. Experimental steps
2.1 Preparation of cell suspension
Pre-inoculate cells into culture dishes and culture them in an incubator at 37 ℃ and 5% CO2. When the cells in the logarithmic growth phase grow to 80%-90%, prepare cell suspension by trypsin digestion. Count the cells in the cell suspension and adjust the cell density.
2.3 Selection and grouping of mice
Generally, 4-6W healthy mice of the same sex are selected, and most of them are immunodeficient mice. Experimental grouping is carried out according to the purpose of the experiment.
2.4 Construction of tumor metastasis model
2.4.1 Tail vein injection method
On the clean bench, fix the mouse in the mouse holder, press the base of the mouse tail, wipe the tail with an alcohol cotton ball to disinfect and expand the blood vessels, use a 1 mL syringe to absorb the cell suspension, and insert the needle at the middle third of the tail. Pay attention to controlling the speed to prevent embolism in the mouse. 0.1 mL per mouse, that is, 1×106 cells. Different cell inoculation amounts may be different, and preliminary experiments are needed to explore.
2.4.2 Left ventricle injection method
After anesthesia, supine fixation, and alcohol cotton ball disinfection, the mouse was gently pressed on the chest, and the needle was inserted between the second and third ribs on the left side of the sternum (close to the sternum). When there were bubbles in the syringe, the needle entered the left ventricle; the needle was inserted 3-5 mm, and bright red blood was observed to spurt into the needle tube, and the surface of the blood returned rose and fell with the heart rate of the nude mouse, which was used as the standard for entering the left ventricle; the injection was completed within 10 s; after the injection, the needle was slowly withdrawn, and the cotton swab pressed the injection point; the nude mouse was placed on the warming table to wake up; the inoculated nude mice were placed in an SPF environment and closely observed.
2.5 Observation of tumor growth and metastasis and mouse survival
After tumor transplantation, the general condition and activity of mice in each group were carefully observed. When mice showed obvious signs of exhaustion such as weight loss, hunched back, and mental depression, the mice were killed by cervical dislocation. Mice were killed and dissected regularly to observe whether there were metastatic lesions in organs such as the lungs, liver, and lymph nodes, and the number and size of metastatic lesions in each part were counted. Tumor metastasis can also be regularly observed through in vivo imaging methods.
3. Example of experimental results
Luciferase-labeled MDA-MB-231 cells were injected into mice via the tail vein, and the tumor lung metastasis was observed using a live imaging system at 1 hour and 24 days. Dissection at 28 days showed that MDA-MB-231 cells formed metastatic foci in the lungs.
4. References
[1] Gomes, C. A., Junior, C. S., do Valle, J. A., de Assis Lopes, C. T., Barra, A., Gomes, C. C., ... & Gomes, F. C. (2012). Cancer metastasis. Cell Dev Biol, 1(106), 2.
[2] Thies, K. A. , Knoblaugh, S. E. , & Sizemore, S. T. . (2020). Pathological analysis of lung metastasis following lateral tail-vein injection of tumor cells. Journal of Visualized Experiments(159).
[3] E., Heller, M., A., Hurchla, & J., et al. (2011). Hedgehog signaling inhibition blocks growth of resistant tumors through effects on tumor microenvironment. Cancer Research, 72(4).