2.4. 4T1-Luc Lung Metastasis Model Establishment

On the basis of a previous study [29, 30], 4T1-luc cells were harvested and suspended at the concentration of 2 × 10⁷/mL in PBS. Then, 50 μl cell suspension (1 × 10⁶) was steadily injected into the fourth intramammary gland fat pad of female BALB/c mouse at a consistent speed. According to our experience on the regularity of lung metastasis, the TBP and PIP are both important in lung metastasis, and 20 days ± 2 days is nearly the lowest requirement for successful lung metastasis, 27 days ± 2 days is nearly the longest time for control the volume of the breast tumor so as not to affect the free movement of mice. In addition, 15 days ± 2 days or 1 day were additionally time for circulating breast tumor cells successfully metastasize and grow in the lung. Therefore, four experimental groups based on these parameters were set up. According to the TBP, the mice were divided into two groups (27 days ± 2 days and 20 days ± 2 days). According to the PIP (15 days ± 2 days, 1 day), the two groups were further divided into 4 total subgroups: TBP 25 + PIP 17, TBP 29 + PIP 1, TBP 22 + PIP 13, and TBP 18 + PIP 1. A normal group was also set up.

According to the TBP, breast tumors were removed as follows: first, a mouse was fixed on a paraffin table in the supine position with inhaled anesthesia (oxygen : isoflurane = 98 : 2). Next, importantly, the mouse was disinfected with 70% alcohol, and ophthalmic forceps were used to lift the upper right corner of the tumor, which was removed carefully with surgical scissors from the left side of the mammary fat pad. According to the radical principle, tumor-free principle, and aseptic principle, attention was paid to clearly detach the blood vessels, completely eliminate the tumor capsule, and thoroughly disinfect the work space and animal, respectively. Notably, complete separation and proximal ligation of the main nutrient vessel were key to reducing bleeding (Figure 1). Ultimately, the incision was sutured closed using 5-0 nonabsorbable surgical sutures, and the animals were observed daily.

Complete separation and proximal ligation of the main nutrient vessel.