Cell culture
HT-29, HCT-116 and Caco-2 cells were purchased from American tissue culture collection (ATCC, Manassas, VA, USA). Dulbecco’s Modified Eagle Medium (DMEM; Biological Industries, Israel) was used as the culture medium for HT-29 and Caco-2 cells while McCoy’s 5A Modified Medium (Biological Industries, Israel) was used for HCT-116, each supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco, USA). All cells were maintained at 37 °C in a humidified atmosphere with 5% CO2.
Gene editing
KO- (sh-CBS) was performed using lentiviruses with short hairpin RNA (shRNA) targeting CBS mRNA sequence (Sigma, USA). Lentiviruses with shRNA of scrambled sequence served as negative control (Sigma, USA). Caco-2 cells were infected at a MOI of 30 with 10 μg/ml of polybrene, and were then selected with 6 μg /ml puromycin for 7 days. Similarly, overexpression of CBS for HT-29 cell line was achieved through transfection with lentiviruses containing h-CBS sequence (Hanbio Biotechnology Co., Ltd., China) at a MOI of 30 with 10 μg/ml of polybrene followed by screening with 4 μg /ml puromycin for 7 days.
Stable knockout of CBS (KO-CBS) for HT-29 cell line was conducted using CRISPR/Cas9 system. Gene-specific sgRNA was designed to target CBS coding regions as shown below: CTGATGAGATCCTGCAGCAG. We first phosphorylated, annealed, and cloned the guide oligonucleotide into the BsmBI site of the pHBLV-U6-gRNA-EF1-CAS9-PURO vector (Hanbio Biotechnology Co., Ltd., China), and then verified the constructed vector by sequencing. Next, we transformed the transfer plasmid with the oligonucleotide into Escherichia coli strain DH5α bacteria, and used Plasmid DNA purification kit (Macherey–Nagel, Germany) to isolate the amplified plasmid from the bacteria. Transferable lenti-CAS-puro plasmid (Hanbio Biotechnology Co., Ltd., China), packaging plasmids psPAX2 (Hanbio Biotechnology Co., Ltd., China) and pMD2G (Hanbio Biotechnology Co., Ltd., China) were transfected into 293T cells to produce the lentivirus. Virus-containing supernatant was collected at 48 and 72 h post transfection and was used to infect Caco-2 cells. Sixteen hours after the infection, fresh medium containing 10 μg/ml puromycin was utilized to replace the lentivirus-containing medium. We collected the puromycin-resistant cells after 7 days of screening.
Finally, western blotting was used for all gene-edited cell lines to assess the silencing or overexpression efficiency.
Cell counting kit-8 (CCK-8) assay
CCK-8 assay was performed to measure the capacity of cell proliferation using CCK-8 kit (Sigma-Aldrich, USA). Cells were seeded in 96-well plates at a density of 4000 cells per well with or without GYY4137 supplement. After incubation, 10 μl CCK8 were added to the wells at different time. The absorbance was measured at 450 nm by microplate reader (Bio-Rad, Hercules, CA, USA).
Colony formation assay
Colony formation assay was used for cell proliferation analysis. Cells were seeded into 6-well plates at a density of 1000 cells per well for 14 days. Colonies were then fixed with 10% formaldehyde for 20 min and stained with 0.1% crystal violet for 10 min at room temperature. Colonies containing more than 50 cells were counted and the mean colony numbers were calculated. Each clone was plated in triplicate in each experiment.
Sphere formation assay
Sphere formation assay was used to detect cell-renewal ability. Five hundred cells were seeded into 24-well non-treated cell culture plates (Nest, China). Serum- free F12/DMEM (Gibco, USA) supplemented with 2% B27 (Gibco, USA), 20 ng/ml human recombinant epidermal growth factor (PeproTech, USA), 20 ng/ml human recombinant fibroblast growth factor-10 (PeproTech, USA), 10 ng/ml human recombinant hepatocyte growth factor (PeproTech, USA), 100 units/ml antibiotics penicillin and 100 μg/ml streptomycin (Gibco, USA) were used for cell culture. After 10 days, spheres larger than 50 μm were counted and photographed under a light microscope. Each clone was plated in triplicate in each experiment.
Transwell migration assay
For migration assay, 2.5 × 105 cells suspended in 200 μl serum-free medium were added into the upper transwell chamber (8 mm, Corning Costar, USA), and 700 μl medium supplemented with 20% serum was placed in the lower chamber. Each transwell chamber contains a 6.5 mm diameter membrane with 8.0 μm pore size. After incubation, cells on the upper surface of membrane were removed gently with a cotton swab. Cells invading to the lower surface were fixed with methanol before staining with 0.1% crystal violet for 20 min at room temperature. The stained cells were counted in 5 randomly selected fields under a light microscope. Each clone was plated in triplicate in each experiment.
Real-time quantitative PCR (RT-qPCR)
Total RNA from xenografts and cell lines were isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Total RNA (1 mg) was eluted with RNase-free water and stored at – 80 °C. RT-qPCR was performed using SYBR-green PCR Master Mix in a Fast Real-time PCR 7500 System (Applied Biosystems). The primers for CD44 were as follows: 5′-CCTTTGATGGACCAATTACCATAAC-3′ (forward); 5′-TCAGGATTCGTTCTGTATTCTCCTT-3′ (reverse). GAPDH was used as the internal control. Fold change of CD44 was calculated by the 2−ΔΔCt method.
Western blotting
Total protein was extracted using RIPA buffer and the extracts containing equal quantities of protein (30 μg) were electrophoresed in 10% polyacrylamide gel, transferred with PVDF membranes and blocked for 1 h (5% BSA in TBS-Tween 20 buffer) at room temperature. Incubations with primary antibodies to detect CD44, CBS, and β-actin (CST, USA) were followed by incubations with secondary antibodies conjugated with horseradish peroxidase (CST, USA). Blots were developed with ECL detection reagents (Millipore, USA). Images were collected utilizing Syngene GeneGenius gel imaging system (Syngene, UK) according to the manufacturer's instructions.
Immunohistochemistry (IHC) analysis
Xenografts from subcutaneous injection and liver samples were immediately fixed in 10% neutral buffered paraformaldehyde. After fixation, the tissue was dehydrated in a graded ethanol series and then embedded in paraffin. Each section (3-μm) of the paraffin-embedded tissue was mounted on a glass slide and either stained with hematoxylin and eosin (H&E) or processed for IHC. For the latter, each slide was completely deparaffinized by immersion in xylene twice for 10 min and rehydrated with water following incubation in graded ethanol (100, 90, 80, and 70%). The antigen retrieval procedure was carried out by microwaving the slides for 10 min in citrate buffer (pH 6.0; Biogenex, San Ramon, CA) followed by incubation in 3% H2O2 in methanol to block endogenous tissue peroxidase activity. The sections were blocked with 1.5% goat serum for 1 h and incubated with CD44 or Ki-67 antibody overnight at 4 °C. Mouse anti-CD44 (CST, USA) and mouse anti-Ki-67 (CST, USA) antibody was used. After washing with PBS, slides were then incubated with a biotinylated secondary antibody for 30 min at room temperature. The antigen signal was amplified using the ABC method (Vectastain ABC kit, catalogue no. PK-6105, Vector Laboratories, Burlingame, CA). The antigen–antibody–avidin complex was detected using the chromogenic substrate 3,3-diaminobenzidine, which produced a dark brown color. Immunostained sections were counterstained with hematoxylin and examined by light microscopy. H-score was used for the semi-quantitive analysis of IHC results. The calculation of H-score was based on the intensity of staining (3, strong; 2, moderate; 1, weak; 0, none) and the proportions of positively stained tumour cells as previously described (H-score = % strong staining × 3 + % moderate staining × 2 + % weak staining × 1 + % no staining × 0) [22]. For each sample, five fields were randomly selected and the average H-score was calculated.
Immunofluorescence assay
For the immunofluorescence assay, after fixation with 4% paraformaldehyde for 10 min, PBS was used to gently wash the cells thrice. Then, the cells were immunostained with primary antibodies targeting SP-1 (CST, USA) at 4 °C overnight, and the secondary antibodies used were Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific, USA). Lastly, the cells were again washed with PBS and then mounted with ProLong Gold mounting medium with DAPI (Molecular Probes, USA). The sections were observed by confocal laser scanning microscopy (Zeiss LSM780, Carl Zeiss, Germany). Pearson’s correlation coefficient was used for the semi-quantitive analysis of the colocalization values as previously described [23].
Fluorescent detection of intracellular H2S
To visualize intracellular H2S level, 2 × 104 cells were seeded in a glass-bottom 35 mm well (Corning, USA) and cultured overnight. After adding 10 µmol/L of H2S-specific near-infrared fluorescence probe to the culture medium and incubated for 30 min, the living cells were immediately sent for fluorescence imaging [24].
Flow cytometry
For surface marker detection, the cells were collected and resuspended at a density of 1 × 104 per test. After incubation for 30 min at room temperature with CD44-APC antibody (eBioscience, USA), the cells were washed twice with PBS and resuspended in 400 µL of PBS for flow cytometry using Calibur 2 (Beckman Coulter, USA). The results were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). The mean fluorescence intensity (MFI) ratio (sample ΔMFI (specific marker MFI − isotype control MFI)/control sample ΔMFI × 100) was calculated for all samples.
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed according to the instructions of Agarose ChIP Kit (Thermo Fisher Scientific, USA). Antibody against SP-1 (CST, USA) was used to precipitate the DNA–protein complex and subsequently elute the DNA from the antibody. Primers specific for the CD44 promoter were 5′- CTCTTTCCACTTGGAAGATTCACCA-3′ (forward) and 5′- TGGATATCCTGGGAGAGGAGCT-3′ (reverse). The immunoprecipitated DNA was amplified by real-time PCR using SYBR-green PCR Master Mix in a Fast Real-time PCR 7500 System (Applied Biosystems).
Xenograft model
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University First Hospital. Twelve male BALB/c nude mice at age of 6 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.. Each mouse was subcutaneously inoculated with 1 × 106 of HT-29 cells in a volume of 200 μl PBS with or without CBS overexpression. After a palpable tumor was developed, the tumor length A and width B were measured twice a week using a caliper. The formula used for calculating the tumor volume was A × B2/2. Mice were humanely sacrificed by exposure to a fixed flow rate of CO2 (30% chamber replacement rate) 34 days after inoculation. Subcutaneous tumor grafts were harvested and analyzed by western blotting and IHC analysis.
Intrasplenic injection model
Twelve 6-week-old male Balb/c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.. To investigate the tumor metastasis in vivo, 1 × 106 of HT-29 cells containing stably-expressed human CBS sequence or empty vector in a volume of 100 μl in PBS were injected into spleen subcapsular followed by spleen-resection after 5 min. After 8 weeks, the livers of nude mice were surgically removed after euthanasia with CO2 (30% chamber replacement rate), fixed in 10% neutral buffered formalin, embedded in paraffin, and prepared into 3-μm sections for H&E staining and IHC analysis.
Statistical analysis
Data are expressed as means ± SD. All statistical analyses were undertaken using Prism for macOS software, version 8.4.1 (GraphPad Software, La Jolla, CA, USA). Student’s t-test or the Mann–Whitney U-test were used for comparisons between two experimental groups with or without normal distribution, respectively. p-values < 0.05 were considered statistically significant.