Preparation of MSJZD
MSJZD is composed of ginseng (15 g; the place of origin is Gansu, China; purchased from Hangzhou huadong traditional Chinese medicine yinpian co., Ltd., Zhejiang, China), atractylodes (12 g; the place of origin is Zhejiang, China; purchased from Zhejiang qianwang traditional Chinese medicine co. Ltd., Zhejiang, China), Poria cocos (15 g; the place of origin is Anhui, China; purchased from Zhejiang zoli baicao decoction piece co. Ltd., Zhejiang, China), honey-fried licorice root (6 g; the place of origin is Xinjiang, China; purchased from Hangzhou huadong traditional Chinese medicine yinpian co., Ltd., Zhejiang, China), zedoary (12 g; the place of origin is Guangxi, China; purchased from Quzhou nankong traditional Chinese medicine co., Ltd., Zhejiang, China), and fruit of five-leaf akebia (15 g; the place of origin is Hubei, China; purchased from Zhejiang yingte traditional Chinese medicine yinpian co. Ltd., Zhejiang, China). These herbs were immersed in distilled water for 2 h, decocted, filtered, condensed to 0.77 g/mL with a routine method, and kept at 4℃ for use.
Establishment of the mouse model of breast cancer
Ten 8-week-old female Balb/c mice weighing 20 ± 2 g (purchased from the Laboratory Animal Center of Zhejiang Chinese Medical University) were maintained in a specific-pathogen-free room at constant temperature (24°C ± 2°C) and humidity (55% ± 5%) under a 12/12-h light/dark cycle. Luciferase-expressing Luc-4T1 breast cancer cells derived from the epithelium of female Balb/c mice (Caliper Life Sciences, Hopkinton, MA) were inoculated into the right mammary fat pad of anesthetized mice at a dose of 5 × 106 cells/mL in 0.1 mL of normal saline per animal. After inoculation for 24 h, the mice were randomly divided into the MSJZD group and control group (n = 5 per group). The mice in the MSJZD group were gavaged with 0.77 g/mL MSJZD once daily for 35 days. The mice in the control group were administered the same volume of normal saline. On day 35, the mice were sacrificed by cervical dislocation, and the tumors were harvested and weighed. The animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University.
In vivo image analysis
To detect tumor growth and metastasis, in vivo imaging study was performed on days 15, 22, 29, 32, and 35. The mice were intraperitoneally injected with 1.5 mg/10 g body weight of fluorescent DiD oil. Six hours after the injection, the mice were anesthetized via isoflurane inhalation and imaged using a Caliper Life Sciences in vivo imaging system (MA, USA). By means of living imaging software (version 4.3.1, Xenogen, Alameda, USA), image display analysis was conducted and data were collected by drawing regions of interest on the subcutaneous tumors. By calculating the number of photons emitted from the tumor site of the mice, the fluorescence intensity (radiance) was measured.
Macrophage quantification by flow cytometry
After 35 days, TAMs were analyzed by flow cytometry based on the amount of M1/M2 macrophages. For cell sorting, the mouse tumor cells were harvested by trypsinization with 0.25% trypsin-ethylenediaminetetraacetic acid (Invitrogen; Thermo Fisher Scientific, Inc.) and then re-suspended in pre-warmed (37°C) Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) containing 2% fetal bovine serum. The density of the cells was adjusted to 1 × 106 cells/mL and the cells were passed through 40-µm cell strainers (BD Falcon; BD Biosciences, San Jose, CA, USA) to obtain single cell suspensions. To characterize the infiltration of M1 and M2 macrophages, the cells were stained for 20 min at room temperature with the following antibodies: phycoerythrin anti-mouse F4/80 (BioLegend, San Diego, CA), rabbit anti-human inducible nitric oxide synthase (iNOS, eBioscience, San Diego, CA, USA), and mouse anti-human CD206 (BioLegend). The cells were then subjected to flow cytometry on a BD FACSCantoII apparatus (BD Biosciences) and the data were analyzed using the CellQuest software.
The measurement data obtained from at least three independent assays in each experiment were expressed as the mean ± standard deviation. Statistical differences between two groups were compared through one-way analysis of variance. All statistical analyses were conducted using the SPSS Statistics 17.0 software (IBM, Armonk, NY, USA). Statistical significance was indicated when P < 0.05.