Cell culture
The MCF-10A cell line (ATCC, CRL-10317) was cultured with MEGM™ Mammary Epithelial Cell Growth Medium and incubated at 37°C in a humidified 5% CO2 atmosphere. The MDA-MB-231 cell line (ATCC, CRM-HTB-26) was cultured in DMEM (Gibco, 12491015) supplemented with 10% FBS (Gibco, 10091148) and 100 U/ml penicillin/streptomycin (Gibco, 15140122) and incubated at 37°C in a humidified 5% CO2 atmosphere.
Immunohistochemical assay
Tissue sections were obtained, dewaxed, rehydrated, and incubated with 3% hydrogen peroxide to eliminate endogenous peroxidase activity for 15 min at room temperature. The tissue antigen was retrieved with 0.01 M sodium citrate buffer (pH 6.0). The slides were incubated with the primary antibody at 4°C overnight. Then, the slides were washed three times with PBS. The slides were incubated with the secondary antibody at room temperature for 2 h. Then, the slides were washed three times with PBS. These tissue sections were stained with DAB and imaged by microscopy. The primary antibodies used were anti-CXCL10 (Abcam, ab306587), anti-CXCL12 (Invitrogen, MA5-23759), anti-CD4 (Abcam, ab133616) and anti-CD8 (Abcam, ab4055). The secondary antibody used was goat anti-rabbit IgG H&L (HRP) (Abcam, ab97080).
Western blot analysis
Western blot analysis
The cells were collected and split to extract plasma and membrane proteins. Proteins were denatured in boiling water for 10 min. Proteins were separated by SDS‒PAGE and transferred to PVDF membranes. The PVDF membranes were blocked in 5% skim milk for 1 hour. Then, the PVDF membranes were incubated with primary antibodies overnight at 4°C. The PVDF membranes were washed three times with TBST and incubated with HRP-labeled secondary antibodies for 1 h at room temperature. Then, the PVDF membranes were washed three times with TBST and imaged with a chemiluminescence instrument.
ELISA
MDA-MB-231 cells (1×104 cells per well) were seeded into 96-well plates. After reaching the indicated time points, the culture supernatants were analyzed with an ELISA kit, including a human CXCL10 ELISA kit (R&D Systems, QK266). The experiment was performed according to the manufacturer’s instructions. The OD values at 450 nm were read with a Multiskan™ FC microplate photometer (Thermo Scientific, 1410101).
Q-PCR assay
Total RNA was extracted with TRIzol solution (Invitrogen, 15596026). Reverse transcription of cDNA was performed with HiScript QRT SuperMix for qPCR (+ gDNA wiper) according to the manufacturer’s instructions (Vazyme, R123-01). qPCR was performed in triplicate with AceQ qPCR SYBR Green Mastermix on an ABI VII7 ABI/viia7 real-time fluorescence quantitative PCR instrument. The following qPCR primers were used: CXCL10, 5′-AACCTCCAGTCTCAGCACCATGAA-3′ (forward) and 5′-AGGTACAGCGTAA GGTTCTAGAGAG-3′ (reverse); CXCL12, 5′-TGCCAGAGCCAACGTCAAG-3′ (forward) and 5′-CAGCCGGGCTACAATCTGAA-3′ (reverse); and GAPDH, 5’-GAGTCAA CGGATTTGGTCGT-3’ (forward) and 5’-TTGATTTTGGAGGGATCTCG-3’ (reverse).
Woundhealing assay
MDA-MB-231 cells were seeded in a 24-well plate and grown to 90% confluence to form a confluent monolayer. A wound was formed by dragging the tip of the pipette. Cell migration images were captured at 0 and 24 h by microscopy, and the cell pixel areas were calculated at 0 and 24 h.
Migration area = 24 h-cell pixel area − 0 h-cell pixel area.
Transwell assay
MDA-MB-231 cells were seeded into the transwell upper chamber at 5000 cells/well and cultured for 24 h. The upper chamber was washed with PBS. The migrating cells were fixed with methanol for 30 min, and 0.1% crystal violet was added for 20 min. Finally, the cells were viewed under a microscope, photographed, and counted.
Statistical analysis
R 3.4.3 and GraphPad Prism 8.01 (GraphPad Software, La Jolla, CA, USA) were used for our statistical analyses. Categorical variables were evaluated using Fisher's exact test or the χ2 test. For continuous variables, paired-samples Student's t test was utilized, and for multiple groups of continuous variables, one-way ANOVA was used. Genes linked to overall survival were identified by calculating hazard ratios (HRs) with 95% confidence intervals (CIs) based on univariate and multivariate Cox regression models, which were used to measure survival. For statistical significance, a cutoff of P < 0.05 was utilized.