The human CRC cell lines were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). HCT-116 were cultured in McCoy’s 5A medium (Biological Industries, Beit HaEmek, Israel) and SW620 cells were cultured in RPMI 1640 medium (Biological Industries) supplemented with 10% fetal bovine serum (FBS) (Biological Industries). Cells were cultured in 37℃ with 5% of CO2 concentrations in incubators.
Infection with lentiviral con℃structs
Lentiviral constructs expressing Sestrin2 (LV-Sestrin2) and a non-targeted green fluorescence protein (GFP) virus (LV-GFP) were purchased from GeneChem (Shanghai, China). Cells (1.5´104) were plated in 24-well plates on the day before transfection. Lentiviral constructs were transduced at multiplicity of infection 10 in HCT-116 and SW620 cells using HiTransG P transfection reagent (Genechem). The infected cells were maintained in fresh medium without FBS for 12 h and then washed with phosphate-buffered saline (PBS), after which the medium was replaced with fresh medium supplemented with 10% FBS. Puromycin (2 mg/mL; Beyotime, Beijing, China) was used to generate stable expression cell lines in HCT116 and SW620 cells.
The RNAsimple Total RNA Kit (TIANGEN, Beijing, China) was used to extract total RNA according to the manufacturer’s instructions. All-in-One cDNA Synthesis SuperMix (Bimake, USA) was used to generate the first-strand cDNA. The expression level of cDNAs was normalized to that of β-actin by the comparative CT method. The primer sequences used in this study are provided in Table 1.
Cell proliferation assay
A total of 500 cells were plated in the wells of a 96-well plate. Cell viability was measured using Cell Counting Kit-8 (CCK-8) (Bimake) and the Thermo Scientific™ Varioskan™ LUX Multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm once a day for 7 days.
Sphere cells were cultured in stem cell medium (SCM), which is a DMEM/F-12 medium supplemented with epidermal growth factor (20 ng/mL, MedChemExpress, MCE), basic fibroblast growth factor (20 ng/mL, MCE), and B-27 (2%; Invitrogen, Carlsbad, CA, USA). A total of 200 cells were plated into each 96-well with 200 µL SCM. The number of spheres was counted after 2 weeks. For second sphere assay, the sphere cells were separated using ACCUTASE (YEASEN). A total of 200 cells were plated into each 96-well with 200 µL SCM. The number of spheres was counted after 2 weeks.
Limiting dilution assay in vitro
The procedure is same as described before . Briefly, cells were seeded in U-bottom 96-well at final cell concentrations 1000, 100, 10, 1 per well with 200 µL SCM. After 2 weeks, the wells contain cell spheres were counted and empty wells were excluded. The frequency of CSCs was calculated on website (http://bioinf.wehi.edu.au/software/elda/) .
Total 1×106 suspended cells were added in the upper chamber (0.8 um in 6 well) with serum-free culture medium of transwell chamber (JET, China). The lower chamber contained medium supplemented with 10% FBS. After 24 h cultured, cells on the upper side of the filter membrane were removed. The rest of the cells were fixed with 5% paraformaldehyde for 20 min and dyed using Crystal Violet Staining Solution (Beyotime, China), then photographed at 200× and counted in five random fields.
Cells were detached into single cells and washed with cold PBS, pre-incubated with Human TruStain FcX™ (422301; BioLegend, San Diego, CA, USA), and then incubated with CD44-APC (B265921; BioLegend) on ice in the dark. Cells were assayed by flow cytometry.
Soft agar colony formation assay
A concentration of 1×104 cells was suspended in SCM containing 0.36% agar and poured on an agar bed (SCM with 0.75% agar). After 3 weeks, the sphere number was counted after 0.04% crystal violet staining.
Wnt/β-catenin pathway inhibition assay
HCT-116 cells in the LV-Sestrin2 and LV-GFP groups were treated with 0.5 µM BML-284 (Wnt signaling activator; MedChemExpress, Monmouth Junction, NJ, USA) in dimethyl sulfoxide for 24 h and then collected for Western blot analysis. In the sphere formation assay, 0.1 µM BML-284 was mixed with cells and then plated in 96 wells with SCM. The number of spheres was counted after 2 weeks.
Western blot analysis
Cells were washed with PBS and lysed in RIPA buffer with Protease Inhibitor Cocktail (Bimake) on ice. Cell lysates were centrifuged (12,000 rpm) at 4°C for 20 min and then quantified using the BCA Protein Assay Kit (Beyotime). The lysate was denatured with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Sample Loading Buffer (Beyotime), followed by SDS-PAGE and electrotransfer to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). The membranes were incubated overnight at 4°C with anti-Sestrin2, anti-CD44, anti-Sox2, anti-Oct4, anti-CXC chemokine receptor 4 (Cxcr4), and anti-β-actin, (Proteintech Group, Wuhan, China) at the appropriate dilution, and incubated with secondary antibody at room temperature for 2 h. The bands were visualized using BeyoECL Plus (Beyotime).
Xenograft mouse model
A xenograft mouse model was determined by subcutaneously injection. One million HCT-116 cells in the LV-Sestrin2 and LV-GFP groups injected into the hips of 6-week-old female BALB/c-nu mice (Animal Center of Chongqing Medical University, Chongqing, China). All animal studies were approved by the Ethics Committee of Chongqing Medical University. The tumour size was measured every 3 days. The tumour volume (V= l×w2/2) was calculated by measuring the length (l) and width (w). Mice were euthanized by increasing volume of CO2in a chamber, 21 days after cells injection.
The Colorectal cancer dataset used comprised mRNA-seq data from TCGA tumors ( https://tcga-data.nci.nih.gov/tcga/). The two-gene correlation map is realized by the R software package ggstatsplot, and the multi-gene correlation map is displayed by the R software package pheatmap. We used Spearman’s correlation analysis to describe the correlation between quantitative variables without a normal distribution. A p-value of less than 0.05 was considered statistically significant.
Except where otherwise noted, experiments were repeated at least three times. All statistical analyses were performed using GraphPad Prism Version 8. The t test was used to evaluate the significance of the differences between two groups of data. P <0.05 was considered statistically significant.