Survival data acquisition
The GC patients’ survival data was downloaded from the GEPIA website (http://gepia.cancer-pku.cn). The dataset is an interactive web that includes 9,736 tumors and 8,587 normal samples from TCGA and the GTEx projects, which analyze the RNA sequencing expression. Another survival data was downloaded from TIMER (https://cistrome.shinyapps.io/timer/). Our centers’ survival data was obtained by our database that contained follow-up data. The detailed clinicopathological parameters were showed in Table 1 and 2.
Cell culture and treatment
MKN45, SNU216 and THP-1 monocytes were purchased from ATCC and maintained as described. Cells were routinely cultured and maintained in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco) and antibiotics (Gibco) according to the ATCC protocols. The plasmid or small interfering RNAs (siRNAs,100pmol) against human SLC2A3 were transfected into the MKN45 cells or SNU216 cells using Lipofectamine 3000 reagent (Thermo Scientific Dharmacon Inc., USA), while non-specific plasmid or siRNA was used as negative controls. Three siRNA were designed to knock down SLC2A3 expression and the most effective one - SLC2A3#siRNA#2 -was used for further examination (Supplementary Figure 3a). The oligonucleotide sequences used in this study were displayed in Supplementary Table 1. Lentiviral vectors plasmids were constructed by GENECHEM Biotech at Shanghai, China (http://genechem.bioon.com.cn/). Flag-tagged SLC2A3-overexpressed vectors (SLC2A3-LV) and control vectors (Control-LV) were transfected into SNU216 cells to generate cells with stable overexpression of SLC2A3.
Western blotting analysis
Protein from cells was separated by SDS-PAGE and transferred to PVDF membranes as described. The following primary antibodies were used for western blots: the rabbit β-Actin antibody, SLC2A3 antibody, GPI, HK2, LDHA, and SLC16A1 were used with the concentration of 1:500, Affinity. Cell signal pathway antibodies were purchased from CST group with a concentration of 1:1000, including STAT3, phosphorylated-STAT3.
Total proteins were extracted with cell lysis buffer supplemented with protease inhibitor and phosphatase inhibitor. Lysate (100 μg protein) was incubated with anti-SLC2A3 (1:50, Abcam), anti-p-STAT3(Tyr705) (1:50, Affinity), or IgG (as a negative control, 1:1000, Cell Signaling Technology) at 4°C overnight. Then the protein–antibody complex was incubated with protein A/G magnetic beads for 4-6 hr at 4°C. Immunoprecipitation was then collected by centrifugation at 1500 RPM for 15 min at 4 °C and washed the beads complex four times with PBS. After the final wash, protein A/G magnetic beads eluted by boiling 5min in 100°C with 2X protein loading buffer before western blot.
RNA isolation and real-time quantitative PCR analysis
Total RNA from cells was extracted using the Trizol reagent, following by reverse transcription for purified cDNA templates. A SYBR Premix Ex Taq II Kit (Takara) was used to perform real-time RT-PCR in the presence of oligo dT primers (Sangon) according to the manufacturer’s recommendation. The mRNA expression was normalized to β-Actin.
As previously described, immunohistochemistry (IHC) was performed to investigate protein expression in tissue. Tumor samples were obtained from the Department of General Surgery, NanFang Hospital, Southern Medical University. The sections of IHC were then incubated with antibodies against SLC2A3 (1:50, Affinity). Intensity of staining of cancer cells was scored as follows: 0 (no staining), 1 (weakly staining, light yellow), 2 (moderately staining, yellow brown), and 3 (strongly staining, brown). An intensity score of >=2 was considered as overexpression, whereas <2 in the intensity score was regarded as low expression. The discrepancies (<5%) were resolved by simultaneous reevaluation. All evaluation was analyzed by three independent observers using the same light microscope.
GC cells were transfected with SLC2A3 siRNA and negative control siRNA, and the CCK8 (Dojindo) proliferation assay was performed according to the manufacturer’s instructions for the indicated time.
Cell migration assay
Transwell chamber migration assay was measured using a transwell chamber with 8 μm filter inserts (Corning). 5 × 104 cells were mixed with 0.2 ml of serum-free medium and seeded to the upper chambers of transwell plates (Corning) as described. In the lower chamber, 0.6 ml of medium with 10% FBS was added to promote cell movement through the pores of the membrane. The inside of the inserts was cleaned thoroughly with a cotton swab, and cells which had migrated through the porous membrane were fixed with a methanol solution for 15 min, and Giemsa staining was performed. The numbers of cells in 4 randomly selected microscope fields were determined.
Extracellular acidification rate and basal oxygen consumption rate
Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using an XF24 extracellular analyzer (Seahorse Bioscience). A 24-well cell culture microplate was coated with Corning® Cell-Tak™ Cell and Tissue Adhesive (Corning Incorporated) to allow adhesion of suspended cells. After calibration of the analyzer, sequential compound injections, including oligomycin A, carbonyl-cyanide p-trifluoromethoxyphenylhydrazone (FCCP), antimycin A and rotenone, were applied on the microplate to test mitochondrial respiration. Sequential compound injections, including glucose, oligomycin A and 2-DG, were applied to test glycolytic activity.
Lactate production and glucose assay
To measure lactate production or mount of glucose, 1 × 105 cells per well were seeded in 24-well plates in triplicate for 24 h, then the medium was refreshed with RPMI1640 containing 1mM glucose overnight. The next day, culture medium was collected for measurement of lactate and glucose concentrations as determined by glucose (GO) assay kit (Sigma) and lactate assay kit (Biovision). Lactate production and mount of glucose were normalized by cell numbers.
Glucose Uptake Assay
2-NBDG (Life Technologies) was used as a glucose tracer. Briefly, 1 × 105 cells per well were seeded in 6-well plates in quadruplicate and incubated overnight at 37 °C with 5% CO2. The next day, cells were starved for glucose for 4 h, then one well was incubated with RPMI1640 medium with 25 μM glucose. as a negative control, and the remaining three wells were cultivated with 25 μM 2-NBDG for 2 h. After incubation, cells were digested and washed twice with PBS. The mean fluorescence intensity of cells was measured by flow cytometry with excitation light at 488 nm.
Chromatin immunoprecipitation (ChIP) assay
ChIP experiments were performed according to the protocol of Chromatin Immunoprecipitation kit (BersinBio). Immunoprecipitation reactions were performed with antibodies against p-STAT3 or with IgG used as a negative control. Purified DNA was then suspended for following qRT-PCR analysis. PCR products were then run on 2.5% agarose gels and visualized with ethidium bromide. Relative chromatin enrichment was calculated as the amount of amplified DNA normalized to input and relative to values obtained after normal IgG immunoprecipitation.
Tumor growth assay
All animal experiments were approved by the Institutional Review Board of Nanfang Hospital of Southern Medical University. MKN45 cells or SNU216 cells were suspended in 100 μl PBS at a final concentration of 1 × 106 cells and implanted subcutaneously into the flanks of 4- to 6-week-old male BALB/c nude mice (Laboratory Animal Unit, Southern Medical University, China). The sizes of the resulting tumors were measured weekly. Tumor volumes were calculated as follows: total tumor volume (mm3) =（Length×Width2）/2, where Length is the longest length.
AAV-GFP construction and intratumor injection
SLC2A3 knockdown and control recombinant Adeno-associated virus-green fluorescence protein vectors (AAV-GFP) were constructed (GENECHEM Biotech). The administration procedures were performed according to previous studies. Briefly, 1×109 physical particles of AAV in 100 μl of PBS were injected into the tumor of nude mice.
Data were analyzed using the student’s t-test or one-way analysis of variance, followed by the Student-Newman-Keuls test using SPSS v20.0 statistical software (SPSS, Inc. Chicago, IL, USA) and the results are expressed as the mean ± standard deviation (SD). A two-tailed probability (ρ)- value of <0.05 was considered statistically significant.