Gene enrichment analysis
Gene enrichment analysis of CD274 (PD-L1) and SLC16A3 (MCT4) was performed using the Metascape online database (http://www.metascape.org/) based on the dataset retrieved from the String database (https://string-db.org/). We utilized UALCN-TCGA (The Cancer Genome Atlas) online database (http://ualcan.path.uab.edu/cgi-bin/ualcan-res.pl.) to search the expression level of MCT4 and PD-L1 in TNBC.
The human BC cell lines MDA-MB-231, MDA-MB-468 and BT-549 were cultured in RPMI 1640 medium (BI) with 10% fetal bovine serum. For lactate treatment, MDA-MB-231, MDA-MB-468 and BT-549 were treated with lactate for 12h in high glucose DMEM (Dulbecco's Modified Eagle Medium) after starved. MCT4 inhibitor 7ACC1 (MCE, Shanghai, China) (20μM, 30μM, 40μM, 50μM, 0.1mM) was added to cell medium on a time gradient.
Western blot (WB) analysis
The total-cell protein was extracted by lysis buffer. The extraction of membranous and cytoplasmic protein was performed according to the kit (KeyGEN Biotech, Jiangsu, China) instructions. Protein extracted from cells was electrophoresed and transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, America). After blocked, membranes were incubated with the primary antibodies anti-MCT4 (G-9) (IgG2b κ, Santa Cruz Biotechnology, santa cruz, USA, 1:1500), anti-MCT4 (G-7) (IgG3 κ, Santa Cruz, Santa Cruz Biotechnology, santa cruz, USA, 1:1500), anti-PD-L1 (Proteintech Group, Chicago, USA, 1:1000), anti-GAPDH (ZEN-BIOSCIENCE, 1:1000) overnight at 4℃, and then incubated with secondary antibodies. Membranes were scanned by Tanon-5200 apparatus (Tanon, Shanghai, China) with ECL luminescence kit (Millipore, Massachusetts, USA).
Wound closure assay and lactate concentration measurement
Cells were seeded into 6-well plate (1*10 6 cells per well) and incubated for overnight at 37℃. Different processing monolayer cells were scraped using 10 ml pipette tips for 0 h and 24 h, and image analyses of the wound closure area were conducted. The supernatant of cell culture was detected utilizing Human Lactate Elisa Kit (J&L, Shanghai, China) for detecting lactate concentration according to the manual.
Quantitative real time PCR (qRT-PCR) analysis
Total RNA was extracted using lysis reagent Trizol kit (Vazyme, Nanjing, China), and cDNA was obtained by Hifair® Ⅱ 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China). qRT-PCR was carried out using UltraSYBR Mixture (Cwbio, Beijing, China), and the data obtained was analysed by the ΔΔCt method. The primers used for qRT-PCR was listed in Table 1.
CRISPR-Cas9-mediated gene editing
MCT4 small-guide RNA (sgRNA) oligo sequences (http://crispr.mit.edu:8079/) were acquired (Table 2). The recombinant plasmid of Lenti-CRISPR V2 was inserted with MCT4 sgRNA, and it was transfected into MDA-MB-231 cells via Lipofectamine® 2000 reagent (Invitrogen, Carlsbad, California, USA) for generating MCT4 knock-out (KO) MDA-MB-231 cell line, which was selected by puromycin (3μg/mL).
Establishing of the stable MDA-MB-231 line expressing MCT4
The human complementary DNA fragment encoding full-length MCT4 was cloned into pEGFP-N1 (Shanghai Gene chem, Shanghai, China). The plasmid pEGFP-N1-MCT4-wildtype(wt) was transfected into HEK-293T cell lines via Lipofectamine® 2000 reagent. The virus supernatant was subsequently transfected to MDA-MB-231 cells for 48h to overexpress MCT4, the infected cells were selected with Kanamycin (50ug/mL).
Cells grown in round glass slides were fixed by 75% alcohol and then were incubated with anti-MCT4 (G-9) (Santa Cruz, 1:100) and anti-PDL1 (Proteintech, 1:100) primary antibody overnight following goat serum blocking, and then incubated with HRP-conjugated fluorescent secondary antibodies for 1h and DAPI for 10min at room temperature, and images were photographed by confocal microscope (Olympus, FV1000, Japan).
Flow cytometry analysis
The cells were stained by primary antibodies PDL1 (Proteintech, 1:100) and MCT4 (G-7) (Santa Cruz, 1:100) for 30 min and fluorescent secondary antibody for 30min respectively. The stained cells were analysed by a flow cytometer, and data obtained were presented with FlowJo software.
Hematoxylin-Eosin (HE) staining and multiple immunohistochemical staining (mIHC)
HE staining and PD-L1, MCT4 and EpCAM staining were performed on formalin-fixed, paraffin-embedded human TNBC tumor tissue sections provided by Tianjin cancer hospital with the permission of Nankai University medical ethics committee. We trusted WiSee Biotechnology Company to perform HE and mIHC staining and assay (protocal seen as supplemental material 1), which provided simultaneous detection and quantitation of PD-L1, MCT4 and EpCAM, and we provided MCT4 (G-7) (Santa Cruz) and MCT4 (G-9) (Santa Cruz) antibody.
Statistical analyses were performed with analysis of variance (one-way ANOVA) and Student’s t test using SPSS 13.0 Statistical Software (SPSS Inc., Chicago, IL, USA) and are presented as mean±s.d. from triplicated independent experiments. A significant difference was considered when the P-value was <0.05.