The LOXL1 expression levels were assessed using IHC on the paired paraffin-preserved tissue sections of 30 CRC patients and 15 CRC patients with liver metastasis. Immunohistochemistry was performed on 2 μm sections using the BenchMark ULTRA automated stainer (Ventana Medical Systems, Inc., Tucson, Arizona, USA) in accordance with the manufacturer’s protocols. Primary LOXL1 antibody was obtained from Sigma (HPA042111, anti-LOXL1 diluted 1:50). Each specimen was scored according to the proportion of positive cancer cells as follows: 1, 0-25%; 2, 25-50%; 3, 50-75%; and 4, > 75%. Specimens were also scored according to the staining intensity of cancer cells as follows: 0, negative; 1, light yellow; 2, dark yellow; 3, brown. The IHC staining score was calculated by multiplying the proportion of positive cancer cells by the staining intensity of cancer cells. The staining results were evaluated by two independent pathologists who had at least five years working experience.All samples were obtained with approval from the Institutional Ethics Committee of The First Affiliated Hospital of Soochow University (authorisation number ECSU-2019000212).
Human colorectal cancer cell lines, such as DLD1, HCT116, HCT8, HT29, LoVo, SW480, SW620, and RKO, were purchased from American Type Culture Collection (ATCC). All CRC cell lines were maintained in RPMI 1640 supplemented with 10% foetal bovine serum and 1% antibiotic (penicillin and streptomycin) at 37°C in an atmosphere of 5% CO2 and 95% air.
Lentiviral vector construction and packaging
Lentiviral constructs of LOXL1 encoding pLenti-EF1a-FH-CMV-GFP-P2A-puromycin were prepared as described previously . Lentivirus expressing LOXL1 was produced in HEK293T cells and then packaged using pMD2.G and psPAX2. The HCT8 and SW480 cell lines were infected with the viral supernatant using 8 μg/mL polybrene (TR-1003-G, Sigma), and the infected cells were incubated for 48 h. Single colonies were obtained through puromycin selection (8 μg/mL), which were detected using western blotting.
Wound healing assay
First, 1×106 cells were cultured in six-well plates and incubated for 24 h. The cultured cells were rinsed thrice using phosphate buffered saline (PBS), and three wounds (scratches) were created in parallel using a sterile 200-μL pipette tip. The wells were washed thrice with PBS to discard any floating cells. Representative images of their migration were captured immediately using a microscope (Nikon, Eclipse Ti-S) 24 h and 48 h after scratching.
In vitro Transwell migration and invasion
Cell migration and invasion experiments were performed using 24-well plates with 8 μm-polycarbonate filter inserts (#3422, Corning). HCT8-N/HCT8-LOXL1, SW480-N/SW480-LOXL1, HT29-N/HT29-LOXL1 (knockdown), and RKO-N/RKO-LOXL1 (knockdown) cells were seeded at densities of 2×105 cells/200 μL and 1×105 cells/200 μL per well, respectively, in serum-free RPMI 1640. All cells were either uncoated or Matrigel-coated (#354234, Biocoat) and incubated in chambers containing 600 μL of RPMI 1640 with 10% foetal serum as a chemoattractant. The cells were imaged, and their migration and invasion were captured using a microscope (Nikon, Eclipse Ti-S). The migrating and invading cells were eluted using acetic acid and quantified by measuring their absorbance at 570 nm. All experiments were performed thrice independently.
Plate colony formation assay
HCT8-N/HCT8-LOXL1, SW480-N/SW480-LOXL1, RKO-N/RKO-LOXL1 (knockdown), and HT29-N/HT29-LOXL1 (knockdown) cells were independently seeded in six-well plates at densities of 5000 cells/well at 37°C. The medium, RPMI 1640 containing 10% foetal bovine serum, was changed every alternate day. After 10 days, the cells forming colonies were immersed in 4% paraformaldehyde for 20-30 min, stained using crystal violet for 2 h, and rinsed thrice with PBS to remove the excess crystal violet. Finally, images were captured using a microscope, and the number of colony-forming units was counted.
Luciferase reporter assay
The 3× GTIIC promoter was subcloned into the XhoI/HindIII site of the pGL4.2 vector (Promega). HEK293T cells were transiently co-transfected with the pGL4.2-3× GTIIC, pcDNA3.1-YAP, and LOXL1/LOXL1 ∆SP/LOXL1 mutants. The pRL-TK vector was co-transfected in each experimental well as an internal control. After 24 h of transfection, the cells were collected and analysed using the Dual-Luciferase Reporter Assay Kit (E1910, Promega).
Immunoprecipitation and western blotting
HEK293T cells were transfected with FL LOXL1 and its mutants. After 24 h, the medium was collected and centrifuged at 1000 g for 5 min. The supernatant was subjected to immunoprecipitation using M2-conjugated magnetic beads (M8823, Sigma) by rotating for 4 h at 4°C. The immunoprecipitates were washed three times using PBS and subjected to western blot analysis. Additionally, the cells were lysed using a lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 10% glycerol, 1 mM DTT, and the complete protease inhibitor cocktail) for 10 min on ice and centrifuged at 20,000 g for 30 min. The cell lysates were analysed by subjecting them to SDS-PAGE and immunoblotting with antibodies as indicated in the figures.
Total RNA isolation and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Expression of the genes CYR61, CDC20, CDX2A, and CTGF was detected using qRT-PCR and normalized to that of GAPDH. Total RNA was extracted using TRIzol (DP424, TIANGEN), according to the manufacturer’s protocol. Total RNA (1 μg) was reverse transcribed using the PrimeScript RT reagent Kit (RR037A, TaKaRa). SYBR green (B21202, Bimake) and an ABI Step One Plus real-time PCR system (Applied Biosystems) were used to conduct qRT-PCR. The primers used in this study are listed in Table 1.
Cell apoptosis analysis
To analyse the fraction of apoptotic cells, HCT8-N/HCT8-LOXL1 and SW480-N/SW480-LOXL1 cells were assessed with an annexin V-APC/7-AAD apoptosis detection kit (KA3808, Abnoya). Briefly, each sample containing 1×105 cells was washed twice with cold phosphate-buffered saline (PBS) and then resuspended cells in 100 µL 1×binding buffer. Then, 5 µl of 7-AAD and 5 µl of APC annexin V were added to each sample. The cells were incubated in the dark for 15 minutes at room temperature. Approximately 10,000 cells/sample were analysed by flow cytometry (Becton, Dickinson and Company, FACS Canto II).
To analyse the apoptotic cells in xenografted tumours, the samples were fixed in 10% formalin and were paraffin-embedded in the Pathology Facility of First Affiliated Hospital of Soochow University. TUNEL analysis was conducted by a commercial company (Wuhan Servicebio Technology CO., Ltd).
HCT8 cells grown on slides were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 15 min, and permeabilized in 0.1% Triton X-100 in PBS. Fixed cells were incubated with anti-YAP antibody (#1407S, CST) overnight at 4°C. Alexa fluorescence 546-labelled secondary antibody was applied for 60 min at room temperature (Invitrogen Life Technologies). All the samples were then stained with 4’,6-diamidino-2-phenylindole (DAPI). All images were collected using a Nikon A1 confocal microscope.
To carry out the xenograft tumorigenesis assays, 1×107 HCT8-N/HCT8-LOXL1 cells were subcutaneously injected into 4-week-old male nude BALB/c mice. The tumour sizes were monitored every 3 d, and their volumes were determined using the following formula: volume (mm3) = (length×width2)/2. Subsequently, they were subjected to H-E staining and immunohistochemistry (IHC).
To carry out the tail vein metastasis assay, cells were injected into the lateral tail veins of 4 week-old male nude BALB/c mice. Eight weeks later, the mice were anesthetized using nembutal (pentobarbital, TRC). The mice were sacrificed and examined at necropsy for the presence of metastases. Their lungs, livers, and bones were fixed in formalin. Subsequently, the samples were subjected to H-E staining and IHC.
To conduct the liver metastasis assay, cells were harvested using 0.25% trypsin, washed thrice with PBS, and suspended in PBS at a final concentration of 1.5×107 cells/mL. The 6-week-old BALB/c nude mice were anesthetized through an intraperitoneal injection of nembutal at a dose of 75 mg/kg. Then, a small incision, approximately 10 mm in length, was made through the skin over the spleen. Using a 27 gauge needle, 100 μL of the tumour cell suspension was slowly injected into the spleen, after which it was placed back in the abdominal cavity. The incision was closed through simple continuous suturing. The mice were sacrificed after 20 d and liver metastasis was confirmed pathologically .
All animal experiments were approved by the Animal Care and Use Committee as well as the Ethical Committee of Soochow University (SYXK2017–0043). All surgeries were performed under sodium pentobarbital anaesthesia with minimum fear, anxiety and pain.
The data obtained were statistically analysed using SPSS (version 20.0; IBM, New York) and represented as the mean ± SD. A t-test (for two groups) was used to determine differences between the groups, which were considered statistically significant at P < 0.05.