The use of human samples was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University. Human tissue samples were collected from Department of Pathology, Nanfang Hospital, Southern Medical University (Guangzhou, China). All patients given informed consents. None of these patients received chemotherapy or radiotherapy before operation.
Human CRC cell lines (HCT116, HT29, SW480, SW620) and murine CRC cell lines (MC38, CT26) were purchased from Cell Resource Center, Shanghai Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China) and maintained at the Department of Pathology, Southern Medical University (Guangzhou, China). Human cell lines were cultured in RPMI-1640 medium, and murine cell lines were cultured in DMEM medium containing 10% fetal bovine serum (Gibco), at 37 °C, in the atmosphere of 5% CO2.
Immunohistochemistry and immunofluorescent staining
For immunohistochemistry (IHC) staining, tissue slides were deparaffinized and hydrated, incubated with antibody anti-STOML2 (1:1000, Proteintech, Wuhan, China) and anti-Ki-67 (1:200, BD, CA, US) overnight at 4 °C. For negative controls, the antibodies were replaced with normal non-immune serum. Tissue slides were reviewed and scored by two independent observers, based on percentage of positive cells and the degree of positive staining. Positive cells at each intensity of staining were recorded on a scale of 0-3 (0, no staining; 1, weak staining = light yellow; 2, moderate staining = yellowish brown; 3, strong staining = brown). A score of ≥2 with at least 50% of malignant cells with positive STOML2 staining was classified as tumors with high expression of STOML2, and <50% of malignant cells with nuclear staining or <2 intensity score was classified as tumors with low expression of STOML2. For cell immunofluorescent (IF) staining, a multiplexed tyramide signal amplification method (TSA; PerkinElmer, Inc., US) was performed on 4-μm sections for detection of the co-localization of STOML2 and PHB proteins. Prior to each immunofluorescence labeling, antigens were retrieved with a single microwave step. Each labeling cycle consists of application of a primary antibody, a secondary antibody conjugated to horse radish peroxidase (HRP), and TSA conjugated to a fluorophore. Tissue slides were incubated with antibody against STOML2 and PHB for 30 min respectively. TSA conjugated Fluorescein was used for STOML2 and CY5 for PHB. Images were captured using inverted confocal microscope (Olympus, Japan) and suite software. Images were processed using Image J and Photoshop CS5 software (Adobe Systems Inc., San Jose, CA).
Establishment of stably transfected cell lines
SW620 cells were infected with STOML2-knockdown (shSTOML2) or scramble shRNA (Scr; GENECHEM, Shanghai, China). SW480 cells were infected with STOML2-overexpressed (3×Flag-STOML2) or control lentivirus (mock; GENECHEM, Shanghai, China). MC38 and CT26 cells were infected with STOML2-knockdown (shSTOML2) or control lentivirus (Scr; GENECHEM, Shanghai, China). Cells were seeded in 6-well plates at a density of 2×10^5 cells per well, 24 h before transfection. 2×10^6 TU of corresponding lentivirus and 5 μg of polybrene (Merck, Darmstadt, Germany) were mixed to 1 mL serum-free medium to transfect cells. After transfection for 48 h, 1 μg/mL of puromycin (Merck, Germany) was added to each well to screen out the stably transfected cells, and the cells were then transferred to conventional medium. Transfection efficiency was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunoblot assay.
Total RNA extraction and real-time quantitative PCR
Total RNA was extracted with Trizol reagent (TaKaRa, Dalian China) following manufacturer’s protocol. cDNA synthesis was performed with PrimeScript™ RT reagent Kit (TaKaRa, Dalian China). qRT-PCR was carried out using SYBR Premix Ex Taq™ II (TaKaRa, Dalian China) on ABI-7500 instrument (Applied BioSystems). Data were normalized to the mean Ct values of housekeeping gene GAPDH and calculated using –ΔΔCt method to compare variation in gene expression.
Cell proliferation, colony formation assay and transwell assay
For Cell Counting Kit-8 (CCK8) assay, cells were seeded in 96-well plates at a density of 800 or 5000 cells per well, respectively for murine or human CRC cell lines, one day before proliferation assay. Where indicated, SW480 cells were pre-treated with 10μM sorafenib or equal volume of DMSO before CCK8 assay. Cell medium of each well was discarded and replaced by CCK-8 reagent 2 h before testing. Absorbance value was detected at 450nm wavelength by Microplate Reader (Perkin Elmer, MA, US), continuously for 4 days. For colony formation assay, cells were sufficiently distributed in 6-well plates with 3 ml complete medium. After 14-day incubation at 37°C in an atmosphere of 5% CO2, the colonies formed by single cells were fixed in 75% ethanol and stained with Giemsa for quantification. For transwell assay, 2×10^5 cells were seeded with 500 μl serum-free medium into transwell chamber (Corning, NY, US), which was insert to 24 well plate. Medium containing 30% FBS was added to the bottom chamber. After 24 h incubation, cells invaded into lower chamber were fixed in methanol, stained in crystal violet (Sigma, MO, US) and counted under microscope. All experiments were replicated for three times.
Immunoblot and co-immunoprecipitation assay
Total protein of cultured cells were extracted using lysis buffer (KeyGEN, Jiangsu, China), with PMSF, protease and phosphatase inhibitor reagents added according to the manufacturer’s instruction. Equal mass of protein extract was separated in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes (Merck Millipore, MA, US). After BSA blocking for 1 hour, the protein-loading membranes were incubated with primary antibody overnight at 4℃. These membranes were then incubated with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (zsbio, Beijing, China) for 1 hour at room temperature. Blotting images were captured and analyzed using Image Lab Software (Bio-Rad, CA, US). GAPDH was set as endogenous reference.
For immunoprecipitation assay, total protein of cultured cells were lysed and incubated with 50 μl protein-A Sepharose beads (Santa Cruz Biotechnology, TX, US), and anti-His, anti-PHB or anti-SLP2 antibody as where indicated, at 4 °C overnight with gentle mixing and anti-IgG was set as a control. Then the samples were washed and denaturized for western blotting.
Referred antibodies were purchased from commercial sources as follows: anti-STOML2 (1:1000, Proteintech, Wuhan, China), anti-GAPDH (1:1000, Proteintech, Wuhan, China), anti-PHB (1:500, Genetex, CA, US), anti-His (1:800, Proteintech, Wuhan, China), anti-IgG (Santa Cruz Biotechnology, TX, US), anti-RAF1 (1:100, Cell Signaling Technology, MA, US), anti-p-RAF1 (1:100, Cell Signaling Technology, MA, US), anti-MEK1/2 (1:200, Cell Signaling Technology, MA, US), anti-p-MEK1/2 (1:100, Cell Signaling Technology, MA, US), anti-ERK1/2 (1:500, Cell Signaling Technology, MA, US), anti-p-ERK1/2 (1:200, Cell Signaling Technology, MA, US).
Isolation, culture, transfection and immunofluorescent staining ofprimary murine colon tumors
Colon tumors of 16-week ApcMin/+ mice were isolated, collected and embedded onto Matrigel (BD Biosciences) at 50 μl per well in 24-well plates, following previously published methods . The culture medium was DMEM/F12, supplemented with 1 unit/ml of penicillin, 1 μg/ml of streptomycin, 2.5 ng/ml of amphotericin B, 10 mmol/L HEPES, 2mM Glutamax, 1× N2 supplement, 1× B27 supplement, and 50 ng/ml murine EGF. Culture medium was changed every 2 days and organoids were passaged by mechanical disruption once a week. Organoid transfection was conducted as described protocol . Briefly, organoids were trypsinized for 10 min at 37 ℃, to obtain single cell suspension. 2×10^6 TU of corresponding lentivirus and 5 μg of polybrene (Merck, Germany) were mixed to 1 mL serum-free medium to transfect cells. After incubated for 48 h, 1 μg/mL of puromycin (Merck, Germany) was added to each well to screen out the stably transfected cells, which were re-embedded to Matrigel afterwards. For IF staining , organoids were grown in matrigel plated onto an 8-well chamber slide (Lab-Tek II, 154534). After culture medium was discarded, organoids were fixed in 4% PFA-PME (50 mM PIPES, 2.5 mM MgCl2, 5 mM EDTA) for 20 minutes, then permeabilized in 0.5% Triton for 20 minutes and blocked in IF Buffer (PBS, 0.2% Triton, 0.05% Tween, 1% BSA) for 1 h. Organoids immunofluorescent (IF) staining was conducted as previously described. Chamber slide were incubated with antibody against E-cadherin and Ki67 for 30 min. Images were captured using inverted confocal microscope (Olympus, Japan) and suite software. Images were processed using Image J and Photoshop CS5 software (Adobe Systems Inc., San Jose, CA).
Animals and tumor growth assay
All mice experiments were approved by Animal Research Ethics Committee of Southern Medical University and proceeded in accordance with the guidelines on the care and use of animals for scientific purposes. Stable-transfected murine CRC cells (2×106) of MC38-Scr, MC38-shSTOML2 or CT26-Scr, CT26-shSTOML2 suspended in 200 µl PBS were injected subcutaneously into the left or right hind limb of 6-week-old male wild type (WT) C57BL/6 or BALB/c mice (n = 7/group), accordingly. Tumor size was measured every three days with a vernier caliper, and tumor volume was calculated as 0.52×L×W2 (cm3; L stands for length and W for width of the tumor). Mice were anesthetized and sacrificed at indicated time, around 4 weeks after injection. Tumors were dissected, measured and photographed, then fixed with formalin and embedded in paraffin for further immnuohistological assessment.
Orthotopic model and murine endoscopy monitoring
Murine CRC cell lines MC38 and CT26 were suspended at density of 2×10^6 in 50μL PBS for each injection. Mice were anesthetized by inhaling 1.5 to 2% isoflurane (RWD Life Science Co., Ltd, Shenzhen, China). Optical colonoscopy was performed using a Karl Storz (Tuttlingen, Germany) Image 1 HD Camera System, Image 1 HUB CCU, 175 Watt Xenon Light Source, and Richard Wolf 1.9mm/9.5 Fr Integrated Telescope (part number 8626.431). Indicated cells were injected into mouse colonic lamina propria under colonoscopy, at around 1cm from anal orifice, using a custom injection needle (Hamilton Inc., 33-gauge, small Hub RN NDL, 6 inches long, point 4, 45 degree bevel, like part number 7803-05), syringe (Hamilton Inc. part number 7656-01), and transfer needle (Hamilton Inc. part number 7770-02). Tumor growth was assessed by serial endoscopy monitoring and recording carried out every 6 days after injection, and tumor size was scored by the diameter of the colonic lumen occupied by tumor . Mice were anesthetized and sacrificed at indicated time, around 30 days after injection. Total colons were dissected, measured and photographed, then fixed in formalin and embedded in paraffin for further immnuohistological assessment.
Yeast two-hybrid screening assay
Human CRC cell line SW620 was selected to construct CRC cDNA library. Following total RNA extraction, cDNA single/double strand synthesis and purification, SW620 cDNA library was co-transformed with pGADT7-Rec (Clontech) into Saccharomyces cerevisiae strain Y187. Transformation efficiency and titer of CRC cDNA library was verified. The full-length of STOML2 coding sequence was cloned to inframe with the Gal4 DNA binding domain of the bait plasmid pGBKT7 (Clontech) by PCR. pGBKT7-STOML2 plasmid was transformed into Saccharomyces cerevisiae strain Y2H to establish bait strain, and double strand library cDNA synthesized from SW620 was co-transfected with pGADT7-Rec plasmid into Y187 to establish library host strain. Bait strain was first tested to be non-toxic to Y2H, and pGBKT7-STOML2 could not activate reporter gene by itself. Bait and library host strain were then co-cultured at 30C with 30-50 rpm swirling, and after mating for 20 hours, zygotes in typical three-lobed shape were present in the mating culture. Mating mixture was spread on three 100mm media (SD/-Trp, SD/-Leu, SD/-Leu/-Trp) at dilution of 1:10, 1:100, 1:10^3,1:10^4. After 3 to 5 days, the number of colonies screened and mating efficiency were calculated, and remaining mixture was spread on forty-four 150mm QDO/A media (SD/-Ade/-His/-Leu/-Trp/Aba) for 3-to-5-days culture. On QDO/A media, clones over 2 mm at diameter were selected and transferred to QDO/X/A media, to underwent higher selective pressure. Blue-stained colonies on QDO/X/A media were randomly selected to verify multiple prey genes by amplification with polymerase chain reaction. These plasmids with prey genes (pGADT7-Prey) were extracted from each blue-stained monoclone, and respectively co-transfected with pGBKT7-STOML2 and pGBKT7 into competent yeast strain pGBKT7-S+pGADT7-Prey and pGBKT7+pGADT7-Prey. To retest the interaction and exclude false positive in yeast, these two Y2H strain were spread on DDO/X and QDO/X/A conditioned media, with pGBKT7-53+pGADT7-T set as positive control and pGBKT7-lam+pGADT7-T as negative control.
Data were all presented as mean ± standard error of mean (SEM) unless otherwise annotated. Statistical analysis was performed by two-tail unpaired Student’s t test for experiments where two means were compared. Two-way analysis of variance (ANOVA) was used to compare means of three or more experimental groups. Factorial design ANOVA was used to analyze experiments with two independent variables. Gene set enrichment analyses (GSEA) were conducted using GSEA 4.0.3 (Broad Institute, MA, US) [29, 30]. Statistical analyses were performed using GraphPad Prism software 5.0 (GraphPad Software, CA, US) and SPSS software (Version 22.0, IL, US).