Cell lines and culture
CAL27 and SCC9 cell lines were purchased from American Type Culture Collection. CAL27 cells were cultivated in DMEM. SCC9 cells were cultivated in DMEM/F-12. Both culture mediums were added with penicillin/streptomycin (ST488, Beyotime, Shanghai, China) and 10% fetal bovine serum (10099141, Gibco, Australia). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.
Wound healing assay
The HNSCC cells were incubated in 6-well plates to become a confluent layer. A scratch was made in each well using a pipette tip. The plates were washed gently with PBS to remove exfoliated cells. Then, we changed the medium to glucose-free DMEM containing 1% FBS with or without 3 mM lactate and captured the images with a light microscope (Leica, Wetzlar, German). The next day, the images at the same position of the scratch were acquired again. The closing area of the wound was analyzed with ImageJ to assess the migration capacity of cancer cells.
Matrigel invasion assay
Basement Matrigel (356237, Corning, NY, USA) was dissolved on ice and diluted 10 times with serum-free DMEM. The diluent was spread evenly on the membrane of the 6.5 mm Transwell chambers with 8 µm pores (3422, Corning) and placed in a 37°C incubator to solidify for at least 4 h. 2 × 105 cells were suspended in an FBS-free medium and seeded on the upper of the membrane. In the lower chamber existed a medium containing 10% FBS with or without 3 mM lactate. 48 h later, the chambers were fixed (4% paraformaldehyde for 20 minutes) and dyed (Crystal violet for 10 minutes). The cells on the lower membrane were counted under a light microscope (Olympus, Tokyo, Japan) after wiping away the cells on the upper membrane of the chambers.
CCK-8 assay
3 000 cells were seeded in 96-well culture plates. 3 mM lactate was added to the experimental group after 4 ~ 8 h. Cell proliferation was measured using a Cell Counting Kit-8 (BS350, Biosharp, Hefei, China) each day.
EdU assay
EdU assay was used to measure the proportion of proliferating cells. HNSCCs were uniformly seeded on 24-well plates and mixed with 3 mM lactate for 48 h. An equal volume of pre-heated Edu working solution (2X, 20 µM) was added to the previous medium in the plates which were continued to place in the incubator. The plates were fixed in 2 h and stained using the EdU Cell Proliferation Kit (C0078, Beyotime) as instructed. Cells were observed and photographed under a fluorescence microscope (Biozero BZ-8000, Keyence, Osaka, Japan).
Western Blot
The membranes were handled with the primary antibodies for 4°C overnight incubation after being blocked with confining liquid (P0252, Beyotime) for 20 min. The dilution of corresponding secondary antibodies were used to treat protein membranes for 1 h. Then ECL kit was used to display the results. Protein membranes were washed by TBST three times between every step. The antibodies were as follows: P4HA1, LDHB, MCT1, CD133, ALDH1A1, BMI1, Cyclin D1 and E1 were purchased from proteintech (Chicago, IL, USA). OCT4 and HIF1-α were purchased from Cell Signaling Technology (Danvers, MA, USA). Phospho-RB was purchased from ABclonal (Wuhan, China).
Lactate uptake and PA detection
10 000 cells were inoculated in ultra-low attached plates (3741, Corning) with standard CSC medium with or without 3 mM lactate to measure lactate and PA metabolism of HNSCC cells. The supernatant and cells were collected every 12 h. At the same time, the cells' densities were measured to correct the final results. The lactate concentration in the supernatants and the PA content of the cells were evaluated according to the manufacturer’s instructions of the Lactate Acid Test Kit (A019-2-1, Jiancheng, Nanjing, China) and the PA Content Assay Kit (BC2205, Solarbio, Beijing, China).
Sphere formation assay
5 000 cells were inoculated in ultra-low attached plates with standard CSC medium with or without 3 mM lactate. 1% exogenous Col Ⅰ (356236, Corning) was added as a recovery experiment. The DMEM/F12 medium was supplemented with 2% B27 (17504, Gibco, CA, USA), 20 ng/ml human EGF (AF-100-15, PeproTech, Rocky Hill, NJ, USA), 20 ng/ml human bFGF (100-18B, PeproTech), and penicillin/streptomycin. After 10 days, photographed and counted under a microscope, then collected the spheres.
Colony formation assay
200 cells were implanted in each well of the 12-well plate and incubated for 10 days with or without 3 mM lactate. 4% paraformaldehyde fix solution was used to deal with the colonies for 20 minutes, and then the plates were stained with crystal violet for 10 minutes. Wash and dry the cell mass before observing it under the microscope.
Immunofluorescence
Cells were seeded on 24-well plates containing medium with or without 3 mM lactate. Spheres were embedded in frozen cutting compound (SAKURA, Torrance, CA, USA) and sliced at 8 µm. The plates and sections were fixed with paraformaldehyde and blocked with BSA for 1 h. The PBS was used to wash between the two steps twice. The samples were incubated with primary antibodies at 4°C overnight and washed 3 times afterward. Corresponding secondary fluorescent antibodies were used to incubate for 1 h. Antifade Mounting Medium with DAPI was added for nuclear staining after 3 times sample washing. Fluorescence microscopy (Biozero BZ-8000; Keyence, Osaka, Japan) and confocal microscopy (Andor Revolution XD, Andor Belfast, UK) were utilized to acquire images of stained samples.
Chromatin immunoprecipitation (ChIP)
For ChIP assay at least 4×106 cells were collected. Chromatin was cross-linked by 1% formaldehyde and the reaction was terminated by glycine solution. Membrane extraction buffer, MNase, and S220 focused-ultrasonicator (Covaris, Woburn, MA, USA) were exerted to fragment the crosslinked chromatin. The resulting chromatin fragments were incubated with a diluted HIF1-α primary antibody or IgG in IP dilution buffer at 4°C overnight. ChIP Grade Protein A/G Magnetic Beads were exploited to attain chromatin fragments with specific binding sites. The chromatin fragments were eluted, reversed, and the specific DNA was identified by qPCR assay after recovery and purification. The detailed steps of these processes were instructed by the Pierce Magnetic ChIP Kit (26157, Thermo Fisher Scientific, Rockford, IL, USA). The primers for 4 regions of the P4HA1 promoter were listed in Table S2.
Real-time PCR
Cells were dealt with TRIzol (Takara, Tokyo, Japan) to procure total RNA that was reverse-transcribed to cDNA by HiScript III RT SuperMix (R323-01, Vazyme, Nanjing, China). CDNA, ChamQ SYBR (VQ311-02, Vazyme), and specific primers were included in the loading mixture. RT-PCR was operated in the Quant-Studio 6 Flex qPCR System. Changes of the mRNA expression were evaluated according to the Ct results.
Cell cycle analysis
Normal and Col1A1 knockdown cells of CAL27 and SCC9 were seeded on 6-well dishes with or without 3 mM lactate. Pre-measured cells were fixed in 70% ice ethanol for at least 2 h and stained with Propidium and RNase A. More details were operated according to the instruction of the Cell Cycle Analysis Kit (C1052, Beyotime), and the results were measured with Flow cytometry (BD Biosciences, San Jose, CA, USA).
SiRNAs transfection and lentivirus infection
The experiments proceeded as previously described (21). The siRNAs targeting HIF-1α, P4HA1, MCT1, and LDHB were designed and purchased from Hanbio (Shanghai, China). Non-influential sequences were assembled as negative control (siNC). The recombinant lentivirus Col1A1i and HIF1-α/LDHB were constructed by GeneChem (Shanghai, China). The details of the interference sequences were described in Table S1.
Transcriptome sequencing
The data of NC and COL1A1i group with 3 replicates of CAL27 cells were provided by Novogene Co., Ltd (Beijing, China). Experiments were performed and results were reported in 1 month.
Mouse xenografts
All animal experiments were approved by the Ethics Committee of the Hospital of Stomatology at Wuhan University (Approval number: S07922040J). 20 female BALB/c nude mice (Vital River, Beijing, China) were divided into group negative control (NC), lactate treatment (LAC), HIF1-αi, and LDHBi with lactate treatment (LAC + HIF1-αi + LDHBi) and Col I recovery (LAC + HIF1-αi + LDHBi + Col I) (n = 5). 3×106 pre-infected CAL27 cells were injected subcutaneously. The above 4 groups were sequentially injected with PBS (50 µL), 3 mM lactate (50 µL), 3 mM lactate (50 µL) or 3 mM lactate with 1% exogenous Col Ⅰ (50 µL) once every 4 days. 1 month later, Euthanasia was used for mice (automated CO2 delivery systems) and the tumors were collected and measured.
Statistical analysis
Quantification analysis of experimental data was performed using GraphPad Prism 9. Student's t-test was employed when analyzing data with only two groups, whereas One-Way ANOVA was utilized for data with three or more groups. Differences with P-values < 0.05 were considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).