Patient and tissue samples
We collected 70 HNSCC tissue samples and samples from 18 healthy subjects from January 2007 to December 2008 to be assessed by immunohistochemical staining. Informed consent was obtained for all participants. All enrolled patients were pathologically diagnosed with squamous cell carcinoma and had complete clinical data. This study was approved by the Ethics Committee of the Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine and that it conformed to the provisions of the Declaration of Helsinki.
The HNSCC cell lines Cal27, HN4, HN6, HN30, SCC4 and SCC25, along with primary normal tongue epithelia cells, were used in this study. SCC4, SCC25 and Cal27 were purchased from the American Type Culture Collection (Manassas, VA). HN4, HN6 and HN30 were kindly provided by the University of Maryland Dental School, USA. All cell lines except SCC4 and SCC25 were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, Grand Island, NY, USA) supplemented with 1% glutamine, 1% penicillin-streptomycin and 10% fetal bovine serum. SCC4 and SCC25 were cultured in Dulbecco’s Modified Eagle’s Medium/F12 medium. Cells were cultured in a standard humidified atmosphere of 5% CO2 at 37 °C. Lactate was purchased from Sigma-Aldrich (St. Louis, MO, USA), and Compound 3K (a PKM2 inhibitor) and AZD3965 (a MCT1 inhibitor) were from Selleck (Houston, TX, USA).
Small interfering RNA (siRNA) for PKM2 (RiboBio, Guangzhou, China) and the scramble sequence were transiently transfected into HN4 and Cal27 using Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and plasmids encoding PKM2 (BioLink, Shanghai, China) and empty vector were transfected into HN4 and HN30. Treatments were administered 24 h after transfection. The sequences of PKM2 siRNA were: #1, 5’-CCAACACCATGCGTGTTGT-3’ and #2, 5’-GGATGTTGATATGGTGTTT-3’ and # 3,5’-GTGGTGATCTAGGCATTGA-3’. The sequence of #2 was chosen for the lentivirus package since it has perfect silencing efficiency. The siRNA sequences of galectin-9 were 5’-GAGGGUACGUGGUGUGCAA-3’.
Immunohistochemical (IHC) staining was performed to detect PKM2 expression in HNSCC patient tissue samples. Briefly, deparaffinized tissue was rehydrated with a graded series of ethanol, followed by antigen retrieval with citrate buffer (pH 6.0) in a water bath at 100 °C for 20 min, and then cooled at room temperature. Rabbit anti-human PKM2 (1:400 dilution, CST, Danvers, MA, USA), galectin-9 (1:800 dilution, CST, Danvers, MA, USA) and ki-67 (Proteintech, Rocky Hill, NJ, USA) antibodies were incubated at 4 °C in a humidified chamber overnight and then visualized using a 3,3'-diaminobenzidine (DAB) detection kit (Dako Cytomation, Denmark) containing secondary antibody. The intensity of PKM2 and galectin-9 immunoreaction was scored as follows: 0 = negative, absence of stained cells; 1 = weak; 2 = moderate; 3 = strong. The immunoreaction score (IRS) was calculated by multiplying the percentage of positive cells and the staining intensity as described in the literature .
Cellular proliferation capacity assay
Cell Counting Kit-8 (CCK8, Dojindo, Kumamoto, Japan), colony formation and EdU assays were used to analyze cell growth capacity, as described in our previous study . First, 500 ~ 1000 cells were plated in 6-well plates for 2 weeks until macroscopically visible colonies formed. Then, the colonies were fixed and stained using crystal violet. The EdU assay was performed with the Cell-Light EdU Apollo in vitro Imaging Kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions.
Cell migration and invasion assay
Migration ability was examined by both a wound healing assay and a transwell assay (uncoated insert). After yielding 90% confluent monolayers, the cells were scratched using a 10 µl pipette tip under FBS-free medium. Photographs were taken at 0 h and 24 h with a Nikon DMCI microscope (Nikon, Tokyo, Japan). For the transwell assay, 1 ~ 6 × 104 cells were seeded in the upper chambers with coated and uncoated inserts with serum-free medium. The lower chambers held 600 µl medium containing 20% FBS in 24-well plates. After 24 ~ 36 h, the migrating and invading cells were stained using 5% crystal violet and then counted and analyzed. All the experiments were performed in triplicate.
Real-time PCR assay
TRIzol Reagent (Takara, Japan) was used to extract total RNA from the indicated cell lines. Then, cDNA was synthesized from RNA using the PrimeScript RT reagent Kit (Takara, Japan). PKM2 mRNA expression was quantified using the SYBR Premix Ex Taq Reagent Kit (Takara, Japan) on an ABI StepOne real-time PCR system (Applied Biosystems, USA) as described in our previous study . The PKM2 primer sequences were 5’-TGACGAGAACATCCTGTGGC-3’ (forward), 5’-TTTTCCACCTCCGTCACCAG-3’ (reverse), and the GAPDH sequences were 5’-CCTCTGACTTCAACAGCGAC-3’ (forward), 5’-TCCTCTTGTGCTCTTGCTGG-3’ (reverse).
Western blot analysis
The cell protein was extracted in SDS lysis buffer (Beyotime, Shanghai, China). The protein samples were electrophoresed on 4–20% polyacrylamide gels (Genshare Biological, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, USA). The PVDF membranes were blocked with 5% skim milk for 1 h at room temperature and then incubated with specific primary antibodies overnight at 4 °C. The antibodies for PKM2, ERK, AKT, galectin-9, E-cadherin, MMP2 and MMP9 were purchased from Cell Signaling Technology (Danvers, MA, USA). β-actin, α-tubulin and GAPDH antibodies (Proteintech, Rocky Hill, NJ, USA) were used as internal controls. After incubation with an HRP-conjugated secondary anti-mouse or anti-rabbit antibody (Sangon, Shanghai, China), the immunoreactive bands were visualized with ECL Ultra (New Cell and Molecular Biotech, Suzhou, China).
Co-Immunoprecipitation (Co-IP) analysis
Cells were lysed with RIPA lysis buffer on ice. The lysis buffer was incubated with specific antibodies and normal IgG overnight at 4 °C, followed by incubation with Protein A/G Magnetic Beads (Bimake, Houston, TX, USA) for an additional 30 min at room temperature. After washing according to the manufacture’s instruction, the proteins were separated from the beads by using immunoblotting loading buffer for 5 min at 105 °C. Then collect supernatants for subsequent experiments.
Chromatin immunoprecipitation (ChIP)
As described in our previous study, the chromatin immunoprecipitation (ChIP) assay was performed according to the manufacturer’s instructions of a SimpleChIP Enzymatic Chromatin IP kit (CST, Danvers, MA, USA). NF-κB p65 antibody was used to pull down the promoter regions of galectin-9. The DNA fragments were purified analyzed by qPCR after the treated cells were fixed, lysed and sonicated.
Dual-luciferase reporter assay
293T cells were transfected with promoter-luciferase construct, and cotransfected with pRL-TK Renilla luciferase construct as an internal control (30:1). After 36 hours, the cells were harvest in lysis buffer and luciferase activity was detected using a dual luciferase reporter assay system (Beyotime, Shanghai, China).
The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking in 3% BSA for 30 min, cells were incubated with primary antibodies against NF-κB p65 (1:100 dilution) at 4ºC overnight and with secondary antibodies for 1 h at room temperature. The cells were costained with DAPI (Beyotime) for 5 min to detection of nuclei and then visualized using a fluorescence microscope (Zeiss, Jena, Germany) and a laser scanning confocal microscope (Zeiss, Jena, Germany).
Enzyme-linked Immunosorbent assay (ELISA)
Galectin-9 levels in medium supernatant were quantified using an enzyme-linked immunosorbent assay (ELISA) kit (R&D, Minneapolis, MN). The cell supernatant was collected and measured according to the manufacturer’s instructions. The concentration was determined spectrophotometrically at 450 nm and 540 nm. The supernatant lactate was measured according by a lactate assay kit (Sigma-Aldrich, St. Louis, MO, USA).
A FITC Annexin V Apoptosis Detection Kit (BD Biosciences, USA) was used for detecting cell apoptosis. Briefly, the cell suspension was incubated with 5 µl of annexin V and propidium iodide at room temperature for 30 min in the dark and then analyzed with a BD Beckman cytometer (BD Biosciences, Franklin Lakes, NJ, USA). PI/RNase staining buffer (BD Biosciences, USA) was used for detecting cell cycle distribution. A single-cell suspension was permeabilized with 75% ethanol at 4 °C overnight. Then, cells were suspended in 500 µL PI/RNase staining buffer and analyzed using a BD Beckman cytometer.
Actin-Tracker Green assay
Cell morphology was detected by Actin-Tracker Green staining (Beyotime, Shanghai, China). HN4, HN30 and Cal27 were washed twice with phosphate-buffered saline solution (PBS) and then fixed with 3.7% formaldehyde solution. Then, cells were incubated with Actin-Tracker Green working buffer (1:200 dilution) for 1 h. Cell morphology was observed under an AxioVert A1 fluorescence microscope.
Natural killer (NK) cell lysis assay
The transfected cells were cocultured with NK cells at different effector-to-target (E:T) cell ratios as indicated for 4 h. The cytotoxicity of NK cells was assessed using a Lactate Dehydrogenase (LDH) Cytotoxicity Assay Kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. Briefly, after working solution was added, the absorbance of each well was read at 490 nm on a Spectra Max i3 (Molecular Devices, Bedford, MA, USA).
Six-week-old male BALB/c nude mice purchased from the Shanghai Laboratory Animal Center (Shanghai, China) were used for our studies. Lentivirus stably transfected cells were established with Cal27 and HN30 cells. For the xenograft model, a total of 1 × 106 Cal27 cells were injected into the left or right flank of the mouse. Then, the tumor volume was measured once a week. For the lung metastasis model, a total of 1 × 106 HN30 cells were intravenously injected into the lateral tail vein. At the end point, the nude mice were sacrificed, and the xenograft tumors and lungs were excised. The lungs were fixed in Bouin’s fixative diluted 1:5 with neutral buffered formalin. Both lung and tumor tissues were fixed and stained for microscopic analysis. The animal studies were approved by the Animal Care and Use Committee of Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
The TUNEL assay was performed following the manufacturer’s instructions (Beyotime, Shanghai, China). Briefly, tissue sections were incubated with fluorescein-labeled dUTP. After staining the nucleus with DAPI, the apoptotic cells (green) were observed and analyzed using an AxioVert A1 fluorescence microscope.
Statistical analyses were performed with SPSS 13.0 for Windows (SPSS Inc., Chicago, IL). Excel and GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA) were used to process the raw data and plot the results. We performed student’s t-test to assess the statistical significance of differences. P values < 0.05 were considered significant (* P < 0.05, and ** P < 0.01). The data are presented as the means ± SD.