Patients and specimens
The specimens of 12 patients were collected after obtaining the informed consent from the Biomedical Ethics Committee of Ruijin Hospital (Table 1). All these patients were diagnosed as CRC pathologically and accepted laparoscopic surgery in Minimally Invasive Surgery Centre, Ruijin Hospital, Shanghai Jiaotong University.
Specimens processing and tumor cell preparation
Surgical specimens were washed with PBS containing penicillin-streptomycin solution and minced into meat emulsion using tweezers and disposable surgical blades. Tissue fragments were transferred to a 50ml centrifuge tube, and digested with 0.5mg/ml type IV collagenase in DMEM at 37°C for 1 hour until fully digested. The digested tissue suspension was filtered through 500 μm and 100 μm cell strainers in sequence to remove residual tissue. Then the filtrate was re-filtered through a 40μm cell strainer, and the cell clusters retained in the strainer were collected and washed twice with PBS. Cell clusters were transferred to the stem cell culture medium (DMEM/F12 medium with glutamine, non-essential amino acids, 8 ng/mL bFGF, 2-mercaptoethanol, serum substitute and penicillin-streptomycin solution) and cultured at 37°C, 5% CO2 and 20% O2. After 24 hours of cultivation, organoids with obvious spherical structures and smooth surfaces are formed.
The cultivation and expansion of PDOs
Once PDOs were formed, added the growth factors required for CRC organoid culture in the stem cell medium, including 50 ng/mL EGF, 1μg/ml R-Spondin1, 500 nM A8301, 50 ng/mL Noggin, 3 μM SB202190, 10 nM prostaglandin E2, 1 x B27, 10 mM nicotinamide. A 23-gauge needle was used to tear the PDO for expansion. For 3D culture, PDOs were embedded in Cellmatrix type I-A (Nitta gelatin) and overlaid with organoid culture medium. 0.2 mg/mL type 4 collagenase was used to digest Cellmatrix to release PDOs.
Flow cytometry analysis
PDOs were collected and washed with PBS, and digested into single cell suspension using 0.25% trypsin/EDTA. The cells were washed with PBS, and then incubated with fluorescent-conjugated primary antibodies in PBS containing 0.5% BSA at 4°C in the dark for 30 min. Antibodies used including EpCAM, CEA CAM1, CD31 and CD45. More information about the antibody was listed in Table S1. Samples were detected using flow cytometry (BD Biosciences) and analyzed by FlowJo software (Tree Star).
Tissues and PDOs were fixed in 4% neutral buffered formalin and embedded in paraffin. H&E staining and immunohistochemical staining were performed on the slices. Antibodies of IHC analysis included Ki-67, EpCAM, MUC2, α-SMA, CD68. More information about the antibody was listed in Table S2. TUNEL apoptosis detection kit was used according to the manufacturer's instructions. Positive cells were scored irrespective of the intensity of staining. The percentage of positive cells was scored semi-quantitatively by two independent individuals.
Immunofluorescence assays and confocal microscopy
Immunofluorescence assays were performed as previously described. PDOs were collected and fixed with 4% paraformaldehyde for 15 min. After washing with PBS, PDOs were permeabilized with 0.1% Triton X-100 at room temperature for 5 min. Then PDOs were washed with PBS and blocked with 5% BSA for 1 hour at room temperature. PDOs were incubated with primary antibodies overnight at 4°C, including E-Cadherin, β-catenin, Ki-67 and EpCAM. Details of the antibody were listed in Table S3. After that, PDOs were washed and incubated with Alexa Fluor 488 or Alexa Fluor 555 Secondary Antibody (Abcam) at 37°C for 2 hours. After washing with PBS, diamidino phenylindole (DAPI, Santa Cruz, USA) was used to counterstain the nucleus. The results were visualized using a laser scanning confocal microscope (Zeiss, LSM510).
Flow cytometric assays of apoptosis were performed as previously described. Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen) was used to detect cell surface Annexin V expression and propidium iodide (PI) uptake by flow cytometry. Cells were collected, washed twice with ice-cold PBS, and suspended in 100 μl binding buffer. Using 3 µl Annexin V-FITC and 5 µl PI to stain cells in the dark at room temperature for 15 minutes. Apoptosis was analyzed by flow cytometry using the FACSCalibur system (BD Biosciences, USA). According to the manufacturer's instructions, using TUNEL Apoptosis Detection Kit (Beyotime) to stain PDOs, and detecting apoptosis by fluorescence microscope. GreenNuc™ Caspase-3 Assay Kit (Beyotime) was used to detect Caspase-3/7 activity in PDOs according to the manufacturer's instructions. Using Ac-DEVD-CHO, a reversible inhibitor of Caspase-3/7, as a negative control. The results were visualized by fluorescence microscope.
Four-week-old male BALB/c nude mice were purchased from the Shanghai Institute of Zoology, Chinese Academy of Sciences. All experiments were performed in accordance with the official recommendations of the Chinese Zoological Society, and animals were cared for humanity in accordance with the standards outlined in the "Guidelines for the Care and Use of Laboratory Animals". PDOs were injected subcutaneously in the flanks of nude mice. Mice were observed for up to 3 months and killed when tumor diameter reaches 10 mm. For drug treatment, when tumors reached about 5mm in diameter, all mice were randomly divided into four groups (3 in each group, Group 1: Vehicle-only solution; Group 2: ML264 25mg/kg; Group 3: oxaliplatin 5 mg/kg; Group 4: oxaliplatin 5 mg/kg +ML264 25mg/kg. These compounds are dissolved in NS (physiological saline solution) and administered intraperitoneally (ip). The dosing regimen is: oxaliplatin once a week, ML264 twice a week, each treatment regimen lasting for a duration of 14 days. Use a vernier caliper to measure the tumor size and record the data twice a week. Three days after the last injection, the animals were sacrificed by cervical decapitation, and tumors were excised and retained for further analyses. Samples were prepared for Western blot analysis and histological staining.
Evaluation of PDOs growth
The evaluation of PDOs growth was performed as previously described. PDOs were evaluated after cultured in Cellmatrix type I-A for 1 week. The PDOs growth ratio was calculated as follows: (major axis length) × (minor axis length) after cultivation / (major axis length) × (minor axis length) before cultivation.
Drug sensitivity analysis
Stable growing CRC PDOs were used for drug sensitivity test. ~20 PDOs were embedded in Cellmatrix type I-A and plated in 96-well plates, and overlaid with organoid culture medium. PDOs were treated with oxaliplatin at 0, 0.25, 0.5, 1, 2 and 4μM respectively, and DMSO was added to the culture as a solvent control. The plate was incubated at 37°C, 5% CO2 for 7 days, and images were captured on days 1, 3, 5, and 7 to evaluate PDOs growth and calculate growth ratio. Curve fitting of drug sensitivity data was conducted as mentioned, and the data was expressed as a percentage of growth starting from 0μM.
The human CRC cell lines used in our study were purchased from the American Type Culture Collection (ATCC, USA). All these cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS), penicillin (107 U/l), and streptomycin (10 mg/l) and incubated at 37 °C and 5% CO2.
RNA extraction and quantitative real-time PCR (qRT-PCR)
RNA extraction and qRT-PCR were performed as previously described. Total RNA of PDOs was extracted using RNAprep pure Micro Kit (Tiangen, China) according to the manufacturer's instructions. Total RNA was reversed to cDNA using HiScript III RT SuperMix (Vazyme, China) and qPCR was performed using SYBR Green (Vazyme, China) according to the manufacturer's instructions. The primers of qPCR were purchased from Genewiz, China and the sequences were listed in Table S4. GAPDH was used as control. The relative expression ratio of mRNAs in each group was calculated by the 2−ΔΔCT method.
Protein extraction and Western blotting
Protein extraction and western blot analysis was performed as previously described. Cells were collected at a 70-80% confluence, and total protein was extracted by RIPA (Solarbio, China) in the presence of Protease Inhibitor and Protein Phosphatase Inhibitor Cocktail (APExBIO, USA). The concentration of total protein lysate was quantified using BCA Protein Assay Kit (ThermoFisher, USA). Proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Tanon, China). Antibodies of western blotting included KLF5, Cleaved Caspase-3, Caspase-3, Bcl-2, Bax and GAPDH. Details of the antibody were listed in Table S5. Goat anti-rabbit or goat anti-mouse HRP-conjugated IgG was used as the secondary antibody (proteintech, USA), and samples were incubated at room temperature for 2 h. The ECL chemiluminescence agent (Millipore, USA) was used to visualize the membrane. The image was captured by Tanon Chemiluminescence Imaging System (Tanon, China). GAPDH was used as the internal control.
CRC cells were transiently transfected with BCL2 or Bax siRNA (TSINGKE, China) using Lipo3000 (Invitrogen) according to the manufacturer’s instructions. Western blot was used to test the downregulation efficiency of siRNA. The targeting site sequences were listed in Table S6.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed as previously described. SimpleChIP® Plus Enzymatic Chromatin IP Kit (CST, USA) was used according to the manufacturer’s instructions. The presence of predicted transcription factor binding regions pulled by antibodies was examined by qPCR. The primers of qPCR were purchased from Genewiz, China and the sequences were listed in Table S4.
Luciferase reporter assay
Luciferase reporter assay was performed as previously described. The BCL2 promoter was cloned into the pGL3-Basic luciferase plasmid (Promega, Madison, USA) to construct WT BCL2 reporter. BCL2 truncat plasmid and mutant plasmid were also constructed（Fig. 5E, H）. For Renilla luciferase reporter assay, each reporter construct was co-transfected into HEK293T cells together with KLF5 plasmid or Control plasmid. After 48 h of incubation, luciferase activities were measured using the Dual luciferase Reporter Assay System (Promega).
All of the statistical analysis were performed by SPSS 20.0 software or R software 3.1.2 (R Core Team). Quantitative variables were analyzed by Student's t-test. Data were shown in mean ± SD. All experiments were performed in triplicate. A two-tailed value of P < 0.05 was considered statistically significant.