Immunohistochemistry (IHC) analysis of CRLM patients
LM and adjacent normal liver tissues from 18 clinical diagnosed patients with CRLM were collected from the Pathology Department (from 2013 to 2020), Sir Run Run Shaw Hospital, Hangzhou, China. The expression of BMI-1 was investigated with IHC staining. Anti-BMI-1 (1:100; Cell Signaling Technology, USA) was used as Primary antibody before secondary antibody incubation.
Cell culture and treatment
High metastatic human CRC cell line (HCT116), low metastatic human CRC cell line (DLD1), mouse CRC cell line (CT26) and human hepatic stellate cell line (LX2) were purchased from the American Type Culture Collection (Manassas, USA). Cells were cultured in DMEM medium (Gibco, USA) with 10% FBS and 1% penicillin/streptomycin in a 5% CO2 humidified incubator at 37°C. BMI-1 overexpressed lentiviral construct (pGC-FU-GFP-BMI-1) and negative control (pGC-FU-GFP) were obtained from Genechem (Shanghai, China). The control (LX2 NC) and stable BMI-1 overexpressed LX2 (LX2 BMI-1) were established using lentivirus transfection under the manufacturer’s instructions. Besides, to make conditioned medium (CM), we firstly cultured transfected LX2 in a 10cm plate with 1×106 cells. Then we used 6mL serum-free DMEM medium to culture cells for 24h. We collected the supernatants, centrifuged at 1000× rpm for 10 min and filtrated through a 0.22mm filter unit (Millex, USA). CM from NC LX2(NC CM) and BMI-1 LX2(BMI-1 CM) were made by mixing the different supernatant with complete medium (1:1). After that, we cultured CRC cells with CM for 24 h. All CM would be used in 2 d. To inhibit Smad2 phosphorylation, after SB-505124 (0.05 µM) was utilized to pretreat CRC cells for 1 hour, the cells were then incubated with BMI-1 CM for 24 hours.
Western blot
Whole protein from the tissue and cell samples were extract using RIPA buffer with a 1% protease/phosphatase inhibitor. After centrifugation of the extraction solutions, BCA Protein Concentration Assay Kit (Solarbio, China) was utilized to quantify the proteins. After SDS-PAGE, proteins were migrated to PVDF membranes. Antibodies against BMI-1, GAPDH, β-actin, Vimentin, Smad2/3, phosphorylated Smad3 (1:1000; Cell Signaling Technology, USA), α-SMA, TGF-β1, phosphorylated Smad2 (1:1000; Abcam, USA), Fibronectin (1:1000; BD Biosciences, USA), E-cadherin, ZEB1, Twist-1 (1:1000; Novus Biologicals, USA), Snail (1:1000; Proteintech Group, USA) were utilized as primary antibodies. After washing with TBST buffer three times every 10 minutes, goat anti-rabbit/mouse IgG (Abcam, USA) was utilized as secondary antibodies. A Bio-Rad CD Touch detection system (USA) with ECL detection reagents was utilized to detect protein bands.
qPCR
Whole RNA from the tissue and cell samples were extract using Trizol reagent (Cwbio, China). Afterward, HiScript II Q RT SuperMix (Vazyme Biotech, China) was utilized for reverse transcription. ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, China) was utilized quantitative polymerase chain reaction (qPCR). A Bio-Rad CFX-96 Real-Time PCR system (USA) was utilized to analyze the results. The sequences of all primers were listed in Table S1.
Immunofluorescent staining
Immunofluorescent staining was performed on 96-well plates. Cells were fixed in paraformaldehyde and permeabilized with Triton X-100. Before incubating with a secondary antidody, antibodies against Vimentin (1:100; Cell Signaling Technology, USA) and Snail (1:100; Proteintech Group, USA) were used as primary antibodies. A Zeiss AXIO Observer A1 inverted fluorescence microscope system (Germany) was utilized to obtain images.
Cell viability assay
Cells were cultured with CM separately for 24, 48, 72, and 96 h. After treatments, CCK-8 reagent (Yeasen Biotechnology, China) was added and incubated for 2 h. A spectrophotometer (Thermo Fisher, USA) was utilized to detect the optical density (OD) value (450nm).
Colony formation assay
CRC cells were cultured with CM for 2 weeks. Cells were fixed in paraformaldehyde and stained by crystal violet. Colonies were viewed with an Olympus CKK53 microscope, and photos were taken. Image J was utilized to measure number of colonies.
Wound healing assay
CRC cells were cultured to reach 90% confluence. After creation of a linear wound, cells were cultured with CM for 48 h. Wounds were viewed with an Olympus CKK53 microscope, and photos were taken at 0, 24, 48 h. Image J was utilized to measure the migration rate.
Transwell migration assay
Transwells chambers were purchased form Corning. CRC cells were plated in the upper chambers with serum-free DMEM, while CM was in the lower chambers. After 48 h, cells in the upper chambers were removed, and cells on the lower membrane surface were fixed and stained. The migrated cells were viewed with an Olympus CKK53 microscope, and photos were taken. Image J was utilized to measure number of cells.
IHC
Tissue samples were fixed, dehydrated, embedded and sectioned. After deparaffinization, sections were blocked with goat serum and incubated with primary antibodies for 24 h. Then, the sections were incubated with secondary antibody for 30 min, followed by counterstaining with Mayer’s hematoxylin. At last, an Olympus CKK53 microscope was utilized to view and photograph the staining of the sections.
Animal experiments
All animal experimental procedures were approved by the Committee on the Ethics of Animal Experiments of Zhejiang University.
Treatment protocol 1
5-week-old male C57 mice (19-20g) were randomly divided into five groups (n = 3). Isoflurane (inhal.) was utilized to anesthetized the mice. Then, the spleen was exteriorized through a left flank incision. CT26 cells (2×106) were intrasplenic injected to establish the tumor, and then injection site was pressed for 5 min. Surgical thread was used to close the peritoneum and skin. The mice were sacrificed at 7, 14, 21 and 28d after CT26 cells inoculation. Resected liver tissues were collected for qPCR, WB and IHC assay.
Treatment protocol 2
5-week-old male BALB/c nude mice (17–18 g) were randomly divided into four groups (n = 5): (1) HCT116 cells (5×106) mixed with LX2 NC cells (1×106); (2) HCT116 cells (5×106) mixed with LX2 BMI-1 cells (1×106); (3) DLD1 cells (5×106) mixed with LX2 NC cells (1×106); (4) DLD1 cells (5×106) mixed with LX2 BMI-1 cells (1×106). The mixed cells were subcutaneously injected into the flanks to establish the tumor. Tumor sizes were estimated every two days after the tumors were measurable. The mice were killed at 28d after cell inoculation. Resected tumor tissues were collected for qPCR, WB and IHC assay. Total tumor volume (mm3) = L× W2/2 (L = length and W = width).
Statistical analysis
In all experiments, the date is showed as the mean + SD based on three independent experiments. The difference between the groups was analyzed using the χ2 test, Fisher’s exact probability, Student’s t test or one-way Analysis of Variance (ANOVA) properly in GraphPad Prism 8 Software or SPSS 25.0. P < 0.05 was regarded as statistically significant.