Clinical samples collection
In this study, a total of 37 BCa patients' tumor tissues and paired adjacent normal tissues were collected. All of these patients were firstly diagnosed with BCa, without previous history of cancer-related diseases or treatment. The clinical tissues were stored at -80°C in a refrigerator.
This research has been approved by the ethics committee of Anhiu No.2 Provincial People’s Hospital and complied with the Declaration of Helsinki. All patients have signed written informed consent and voluntarily joined the study.
Cells and culture
Mice BCa cell line (4T1) was commercially provided by the American Type Culture Collection (ATCC, Manassas, Virginia, USA). The cell line was cultured at 37°C, 5% CO2 in dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS).
Animals and xenograft tumor model
Animal study has been approved by the animal ethics committee of Anhiu No.2 Provincial People’s Hospital. Female Balb/c mice (n = 20, 5 weeks old) were purchased from Shanghai Laboratory Animal Center of Chinese Academy of Sciences (Shanghai, China). Mice were housed in a (22 ± 1)°C and 12-h day/night cycle room. Food and water was freely available. Mice were randomly divided into 4 groups: Vehicle group, STING agonist group, Atezolizumab group and STING agonist + Atezolizumab group. For each group, 5 mice were included. The treatment of mice in each group was as follows:
Firstly, 4T1 cells were collected when the confluence researched to about 85%. Phosphate-buffer saline (PBS) was used to disperse 4T1 cells to a density of 1 × 107 cells/mL. Then 4T1 cells suspension (100 µL) was injected into the mammary fat pad of mice in each group. Five days after 4T1 cells injection, STING agonist (25 µg) was injected into the tumor mass of mice in STING agonist group. At the same time point, mice of PBS group were injected with a vehicle of PBS. Mice of Atezolizumab group were injected intraperitoneally with Atezolizumab (200 µg). Meanwhile, mice of STING agonist + Atezolizumab group were not only injected with STING agonist (25 µg) into the tumor mass, but also injected intraperitoneally with Atezolizumab (200 µg). The injection frequency of STING agonist and Atezolizumab was 2 times a week and injected for 2 weeks.
Furthermore, on the 1st, 7th and 14th day after injection of PBS, STING agonist, Atezolizumab or both STING agonist and Atezolizumab, peripheral blood of mice in each group was collected from orbit and stored at -80°C in a refrigerator. The peripheral blood collection time was 4 h after injection.
From the 5th day of 4T1 cells injection, the tumor volume was measured every 3 days with the following formula: V = 0.5 × length × width2. On the 29th day after 4T1 cells injection, mice were anesthetized by 2% isoflurane and then killed through cervical dislocation. The xenograft tumor mass was collected from mice and stored at -80°C in a refrigerator [18, 19]. Additionally, lung, liver, brain-cortex and kidney of mice were also collected and stored at -80°C in a refrigerator.
Anti-IFNART treatment of 4T1 cells
When reached to about 85% confluence, 4T1 cells were harvested and dispersed into DMEM containing 10% FBS (1 × 106 cells/mL). A total of 1 mL cells suspension was contained per well. After that, STING agonist was added into 4T1 cells to a final concentration of 100 µg/mL (named STING agonist group). STING agonist (final concentration 100 µg/mL) and anti-IFNART (final concentration 10 ng/mL) were both used to treat 4T1 cells (named STING agonist + anti-IFNART group). 4T1 cells treated by PBS (final concentration 100 µg/mL) were used as Vehicle group. Cells were cultured for 48 h at 37°C, 5% CO2, followed by being collected to subjected to Western blot.
Enzyme-linked immunosorbent assay (ELISA)
The xenograft tumor mass was homogenized at 15000 r/min for 40 s, followed by being centrifuged at 1000 r/min for 5 min. The supernatant was collected. Using an ELISA kit, the level of TNF-α, IFN-β and IL-10 in peripheral blood samples and the above supernatant was detected in line with the instructions.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Clinical samples and xenograft tumor mass was prepared into powder in liquid nitrogen. Total RNA in tissue powder samples was extracted using TRIzol reagent (Beyotime, Shanghai, China). The extraction procedures were strictly based on the instructions. Thereafter, cDNA was synthesized using 2 µg of total RNA samples according to the instruction of the Roche Transcriptor First Strand cDNA Synthesis kit (Roche, Mannheim, Germany). The cDNA was subjected to PCR using the ABI7500 instrument (Applied Biosystems, Waltham, MA, USA) in line with the following procedure: 95˚C for 5 min, 38 cycles of (95˚C for 45 s, 50˚C for 45 s and 72˚C for 40 s), and 72˚C for 3 min. Genes and corresponding primers were: STING, forward 5'-CAAGGACCAACTACAACC-3', reverse 5'-TGCCTCTTCTTTAATTG-3'. PD-1, forward 5′-GGTGTGAGGCCATCCACAA-3′, reverse 5′-CCATTCTGTCGGAGCCTCTG-3′. PD-L1, forward 5′-TATGGTGGTGCCGACTACAA-3′, reverse 5′-TGGCTCCCAGAATTACCAAG-3′. TNF-α, forward 5′-CCCGCATCCCAGGACCTCTCT-3′, reverse 5′-CGGGGGACTGGCGA-3′. IFN-β, forward 5′-TCCGAGCAGAGATCTTCAGGAA-3′, reverse 5′-TGCAACCA-CCACTCATTCTGAG-3′. IL-10, forward 5'-CTTCGAGATCTCCGAGATGCCTTC-3', reverse 5'-ATTCTTCACCTGCTCCACGGCCTT-3'. Actin, forward 5′-TCCTGTGGCATCCACGAAAC-3′, reverse 5′-GAAGCATTTGCGGACGAT-3′. Actin was used as control to normalize the expression of other mRNA expression by 2−ΔΔCT method.
Immunohistochemistry (IHC)
Clinical samples and xenograft tumor mass were treated by 10% formaldehyde solution for 24 h. Paraffin was used to embed tissues and then tissues were cut into sections (4 µm). Sections were sequentially treated by xylene and gradient alcohol for dewaxing and rehydration. Antigen retrieval was carried out by immersing sections into citrate buffer (10 mM, pH = 6.0). After 10 min treating with 3% H2O2, sections were treated for 1 h with 5% goat serum. Primary antibodies (1:100) were added to treat section for 12 at 4˚C. Primary antibodies were: rabbit anti-STING (ab252560, Abcam, Shanghai, China), rabbit anti-PD-L1 (ab233482, Abcam, Shanghai, China), mouse anti-PD-1 (ab52587, Abcam, Shanghai, China), rabbit anti-CD8+ (ab138727, Abcam, Shanghai, China) and rabbit anti-FOXP3+ (ab4728, Abcam, Shanghai, China). Goat anti-rabbit (ab150080, Abcam, Shanghai, China) or anti-mouse (ab6789, Abcam, Shanghai, China) secondary antibodies (1:200) was then applied for 2 h treating of sections. Diaminobenzidine (DAB) (Solarbio, Beijing, China)was used to stain sections, followed by hematoxylin staining (Solarbio, Beijing, China). Dehydration of sections was performed using gradient alcohol. Transparency of sections was carried out by xylene. After sealed in neutral resin, sections were observed using a microscope.
Hematoxylin-eosin (HE) staining
As described above, mice lung, liver, brain-cortex and kidney were treated by 10% formaldehyde solution for 24 h. Tissues embedded into paraffin were prepared into sections (4 µm), followed by treated with xylene and gradient alcohol for dewaxing and rehydration. According to instructions, hematoxylin and eosin (Solarbio, Beijing, China) were sequentially applied for sections staining. Dehydration and transparency was conducted by using gradient alcohol and xylene. After sealed in neutral resin, sections were observed using a microscope.
Western blot
Xenograft tumor mass was prepared into powder in liquid nitrogen to extract total proteins using lysis buffer (Beyotime, Shanghai, China) on ice. Additionally, 4T1 cells were lysed by lysis buffer. Total proteins in the supernatant were collected after centrifuged at 5000 × g, 20 min. The concentration of total proteins was determined using a BCA kit (Beyotime, Shanghai, China) according to the instructions. After that, 20 µg of protein sample was experienced separation using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS‑PAGE). After being separated, proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. For blocking, 5% skimmed milk was used to incubate the PVDF membrane for 1 h. TBS containing 0.1% Tween-20 (TBST) was used to wash the PVDF membrane. Afterwards, the PVDF membrane was probed by rabbit anti-mouse primary antibodies for 12 h at 4˚C. Rabbit anti-mouse primary antibodies were: p-STING (1:500, AF7416, Fushen Biotechnology, Shanghai, China), p-TBK1 (1:500, NY-1725R-Phospho, Anyan Biotechnology, Nanjing, China), TBK1 (1:1000, ab227182, Abcam, Shanghai, China), p-IRF3 (1:500, ab192796, Abcam, Shanghai, China), IRF3 (1:1000, ab25950, Abcam, Shanghai, China), p-STAT1 (1:500, ab30645, Abcam, Shanghai, China), STAT1 (1:1000, ab47425, Abcam, Shanghai, China) and Actin (1:1000, ab8227, Abcam, Shanghai, China). TBST was used to wash the PVDF membrane again. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2000, ab6271, Abcam, Shanghai, China) was added to incubate the PVDF membrane for 2 h at room temperature. Enhanced chemiluminescent (ECL) reagent (Beyotime, Shanghai, China) was applied for protein blots visualization. The density of protein bands were quantified by Image J software (National Institutes of Health, Bethesda, MD, USA). Actin was served as control to normalize other proteins expression.
Statistical analysis
SPSS Software 19.0 was applied for statistical analysis. Data were displayed as mean ± standard deviation. Paired Student's t-test and analysis of variance (followed by Tukey's post hoc test) were utilized respectively for the comparison between two groups and in multiple groups. P < 0.05 meant a statistically significant difference. All data were obtained from 3 independent repeated experiments.