The overall goal of this procedure is to establish a mouse spontaneous metastasis breast cancer model that allows the quantification of primary tumor breast cancer growth as well as the visualization and measurement of the breast cancer metastasis kinetics.This method can help answer key questions in the field of spontaneous cancer metastasis, such as how can altering tumor-associated genes affect cancer growth and metastasis?The main advantage of this technique is that this model mimics human breast cancer development, making it ideal for testing the efficacy of therapeutic agents.On the day of the experiment, harvest an overnight culture of 4T1 luciferase tumor cells, and pellet the cells by centrifugation.Remove the supernatant by vacuum, then wash the cells in 10ml of PBS, and re-suspend the pellet in 10ml of fresh PBS for counting.Next, dilute the cells to 2x10^5 cells/ml concentration in HBSS, and output the suspension into 1.5ml sterile microcentrifuge tubes on ice.Then, restraining the mouse by hand, label the mammary glands of a 6-8 week-old female BALB/c mouse with a black marker, and use an electronic hair trimmer to shave the hairs surrounding the number 3 nipple.Proceed to re-suspend the cells by 2-3 inversions of the microfuge tube, then carefully aspirate 50
