Reagents and antibodies
Rabbit monoclonal antibodies against MMP9, MMP2 were purchased from Cell Signaling Technology (Danvers, MA, USA).
Cell culture
The mouse lewis lung cancer cells were obtained from Guang’ anmen Hospital China academy of Chinese medical sciences and then cultured in DMEM medium, which is supplemented with 10 % fetal bovine serum, 1×105 u·L− 1 penicillin, and 100 mg·L− 1 streptomycin (Gibco, USA). The cell line was maintained in a humidified atmosphere at 37 °C and 5 % CO2.
Preparation and quality control of Sijunzi Tang
SJZ was extracted from a mixture of 4 herbs including Panax ginseng C.A.Mey., Atractylodes macrocephala Koidz., Poria cocos (Schw.) Wolf and Nardostachys jatamansi DC. by refluxing extraction method. Its quality control was applied by detecting content, heavy metals, pesticide residues and aflatoxin according to Pharmacopoeia of the People's Republic of China. The detailed preparation and quality control method has been previously reported[15].
Animals and Tumor model
Animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, with the approval of animal ethics committee which belongs to Chinese Academy of Medical Sciences Cancer Hospital (Beijing, China). Age-and sex-matched C57BL/6J mice (male, 6–8 weeks old, 16–20 g) were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All rodents used in the study were housed under standardized light- and temperature-controlled conditions with free access to food and water. To establish spontaneous lung metastatic models, 1×106 LLC cells were injected subcutaneously in the shaved right lateral axilla of mice.
Treatment regime
84 mice were randomly divided into four groups with 21 mice in each group at each time point. The four groups of mice were administered the following dosing regimen once a day by intragastric administration (a) saline solution; (b) gefitinib (50 mg·kg− 1); (c) SJZ (25.74g·kg− 1); (d) gefitinib + SJZ. Gefitinib was suspended in 0.5% CMC-NA solution. Mice were sacrificed separately on 7, 14, 21 days.
Flow cytometry assay
Mouse peripheral blood was collected in sodium Heparin blood collection tube. Erythrocyte cells were harvested, washed, and resuspended in 100 µL PBS solution at a density of 1×106 cells. Mouse lung were also collected, shredded and digested with collagenase. The density of lung cell suspension is also 1×106 cells. All the cells were labeled by BD Pharmingen™ APC-Cy™7 Rat Anti-Mouse CD45 antibody (557659, BD Biosciences, San Jose, CA, USA). For neutrophils and monocytes analysis, BD Horizon™ BV711 Rat Anti-CD11b (563168, BD Biosciences, San Jose, CA, USA), PE-Cy™7 Rat Anti-Mouse Ly-6G (560601, BD Biosciences, San Jose, CA, USA) and BV605 Rat Anti-Mouse Ly-6C (563011, BD Biosciences, San Jose, CA, USA) were used. For detection of dendritic cells, Ms CD11c BUV395 HL3(563011, BD Biosciences, San Jose, CA, USA) and BV650 rat anti-mouse I-A/I-E (743873, BD Biosciences, San Jose, CA, USA) were used. For identification of Lymphocytes, lung cells and peripheral blood cells were incubated with BUV496 Hamster Anti-Mouse CD3e (612955, BD Biosciences, San Jose, CA, USA) and BUV737 Rat Anti-Mouse CD19 (564296, BD Biosciences, San Jose, CA, USA). Besides, the surface receptors of c-Kit, CXCR1, CCR2 and VEGFR2 on immune cells were also identified by PE anti-mouse CD117 (c-Kit) Antibody (105807, BD Biosciences, San Jose, CA, USA), PE Rat Anti-Mouse CD181 (CXCR1) (566383, BD Biosciences, San Jose, CA, USA), Alexa Fluor® 647 Mouse anti-Human CD192 (CCR2) (561744, BD Biosciences, San Jose, CA, USA) and BV421 Rat Anti-Mouse FLK-1 Clone Avas 12α1 (RUO) (562941, BD Biosciences, San Jose, CA, USA). After incubation, cells were washed with PBS and subjected into the FACS Canto™ II flow cytometer (BD Biosciences, San Jose, CA, USA).
HE staining
H&E staining was performed as described[16]. Lungs were inflated with 10% formalin and embedded in paraffin. Subsequently, 5-µm sections were cut for hematoxylin/eosin staining.
TUNEL staining
Cell apoptosis in tumor tissues were detected using the TUNEL method. The assay was carried out with Roche TUNEL assay kit according to the manufacturer's protocols. The tumor tissues of mice were first immersed in 10% neutral formalin. After dehydration with graded ethanol (70%-100%) and degrease disposing with xylene, the paraffin embedded tumor tissues were made for further use. Sections were deparaffinized, re-hydrated and incubated with 20 mg/ml proteinase K for 20 min at room temperature. The TUNEL reaction mixture was prepared as follows: 50 µL TdT enzyme, 450µL Biotin-dUTP. The slides were incubated in the mixture for 60 min at 37 °C and then incubated in the 50 µl converter-POD for 30 min at 37 °C. And DAB solution was applied to the sections for 10 min at 15–25℃. Images of the cells were captured using a microscope.
Immunohistochemistry
5 µm sections of the paraffin embedded femur were incubated at 60℃ for 24 h and then followed by defatting in xylene and hydrating with graded ethanol (100%-70%). After successively incubating with antigen retrieval solution and 3% H2O2 for 30 min, the slides were rinsed and incubated with the primary antibody (MMP-2, MMP9) overnight at 4℃. For the negative controls, the primary antibodies were replaced by non-immunized goat serum. The next day, the slides were rinsed and incubated with the second antibody for a period of time followed by DAB and haematoxylin staining, respectively. Quantification of positive cells was examined in at least five random fields from each section.
Cytokine measurement
Mouse testing samples were obtained by centrifugation from tumor tissue homogenate at 10000 rpm for 15 min. After protein quantification to 10 pg·mL− 1, the samples were stored in -80℃ environment. Cytokines (ENA-78, Eotaxin, G-CSF, GM-CSF, GROα, IFN-γ, IFNα, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15/IL-15R, IL-17A, IL-18, IL-22, IL-23, IL-27, IL-28, IL-31, IP-10, LIF, MCP-1, M-CSF, MCP-3, MIP-1α, MIP-1β, MIP-2, RANTAS, TNF-α) in tumor issue were tested by ProcartaPlex™ Mouse Cytokine&Chemokine Panel.
Statistical analysis
Numerical data are presented as mean ± SD. All data analyses were performed by Graph Pad Prism (version8.0, Graph Pad Software, Inc.). Between-group analyses were conducted using one-way ANOVA and unpaired t-Test.