Mice experiments
All animal experiment protocols were approved by the Animal Ethics Committee of the Affiliated Hospital of Jining Medical University (No. 2022B021).
Subcutaneous tumour model
For the subcutaneous tumour model, three million indicated cells were injected subcutaneously and bilaterally into BALB/c nude, NVSG, or C57BL/6J mice. Once tumour sizes reached 0.1-0.15 cm3, the mice were randomly assigned into different groups and treated with vehicle, OX40L recombinant protein (200 mg/mouse, biweekly, I.P., SinoBiological, #53582-M04H), anti-OX40 antibody (1 mg/mouse, once weekly, I.P., BioXCell, #BE0031, ). Drug treatment lasted for 3 weeks. The tumour volume was measured at the indicated time points and calculated as length × width2/2.
Colon orthotopic tumour model
In the orthotopic mouse colon cancer model, mouse colon cancer MC38 cells were stably transfected with the firefly D-luciferin-GFP expression vector. Severe immunodeficient NVSG mice were anesthetised using chloral hydrate. A midline incision was made in the abdominal cavity of each mouse. By using a sterilised micro syringe, a 30-μL cell suspension containing 500,000 indicated cells (pre-mixed with matrix) was gently injected into the distal colon. The incisions were closed using 6-0 nylon sutures. After 2 weeks, tumour imaging was performed using the FluorVivo imaging system (INDEC Biosystems, Santa Clara, CA, USA). Thereafter, the mice were the randomly assigned to different groups and treated with vehicle, OX40L recombinant protein, or anti-OX40 antibody, as described above. Drug treatment lasted for 3 weeks. Survival analysis was performed based on the mouse survival status and time. Finally, the mice were sacrificed, and liver metastases were evaluated based on GFP signals using an OV100 microscope (Olympus).
Tail-vein injection metastasis model
In the tail-vein injection mouse model, mouse colon cancer MC38 cells, glioma GL261 cells, and ovarian cancer ID8 cells were stably transfected with the firefly D-luciferin-GFP expression vector. Two million cells were injected into the tail vein of athymic BALB/c nude mice. Drugs were administered as described above. Survival analysis was performed based on the mouse survival status and time. Finally, the mice were sacrificed, and liver metastases were evaluated based on GFP signals using an OV100 microscope (Olympus).
Splenic injection for liver metastasis models
Splenic injection of LUC-GFP-MC38 cells was performed to establish a pre-clinical murine model of hepatic metastasis. Briefly, trypsinised LUC-GFP-MC38 cells were resuspended in cold phosphate-buffered saline (PBS) at a final concentration of 106/mL. The mice were anesthetised with isoflurane, and laparotomy was performed on the left abdomen. Thereafter, 100-μL MC38 cells were injected slowly into the spleen. The mice were euthanised at the study endpoint for analysis and sampling. Finally, the mice were sacrificed, and liver metastases were evaluated based on GFP signals using an OV100 microscope (Olympus).
PDX model
Mouse PDX experiments were conducted following protocols and ethical guidelines approved by the Institutional Animal Care and Use Committee. Primary human CRC tumour fragments (2-3 mm in diameter) were subcutaneously injected into 6-week-old female immunodeficient BALB/c nude mice. CRC patients had not received treatments or procedures. Anti-OX40 antibody was administered as earlier described. Anti-PD-1 antibody (200 µg/mouse, once weekly, I.P., BioXCell, #BE0146), Verteporfin (100 mg/kg, biweekly, I.P., Selleck, #S1786), and Gefitinib (80 mg/kg, biweekly, oral gavage, Selleck, #S1025) were administered. Drug treatment lasted for 3 weeks. The tumour volume was measured at the indicated time points and calculated as length × width2/2.
Generation of transgenic OX40fl/fl;Cd31cre/- mice
Single-stranded RNAs (sgRNAs) suitable for the CRISPR/Cas9 system were designed to target exons 3-7 of Tnfrsf4. These sgRNAs can accurately guide Cas9 to the target site. To achieve finger site cleavage, we synthesised a DNA template containing the desired mutation. These templates were obtained through chemical synthesis of DNA fragments or PCR amplification, ensuring the correct loci and containing repair template sequences compatible with the CRISPR/Cas9 system.
The designed sgRNA sequence and repair template were inserted into the appropriate CRISPR/Cas9 vector to construct a donor for editing. We injected the screened CRISPR/Cas9 vector into the fertilised egg and implanted the fertilised egg into the uterus of a surrogate mother mouse. After pregnancy, some mice carried the required sequence. Genotyping of newborn mice was conducted via PCR amplification and sequencing to confirm whether they carry the target mutation. Tnfrsf4 flox mice were hybridised with endothelial Cre (Tek-Cre) mice to obtain flox-homozygous and Cre-positive mice, achieving conditional Tnfrsf4 knockout in ECs.
Generation of transgenic OX40ki/ki;Cd31cre/- mice
The gRNA to Tnfrsf4-ROSA26 gene, the donor vector containing the ‘CAG promoter-loxP-PGK-Neo-6*SV40 pA-loxP-Kozak-mouse Tnfrsf4 CDS-rBG pA’ cassette and Cas9 mRNA were co-injected into fertilised mouse eggs to generate targeted conditional knockin offspring. F0 founder animals were identified using PCR and sequencing and were bred with wild-type mice to test germline transmission and F1 animal generation. We bred F1-targeted mice with mice carrying a tissue-specific Tek-Cre deletion to generate mice that were heterozygous for a targeted allele and hemizygous/heterozygous for the Cre transgene. Mice genotyping primers and probs are listed in Table S5.
Human participants
Human primary CRC specimens for the PDX model were obtained from the Affiliated Hospital of Jining Medical University. None of the patients underwent preoperative radiation or chemotherapy. This study was approved by the Research Ethics Committee of the Affiliated Hospital of Jining Medical University and was conducted following the Declaration of Helsinki (No. 2022B021). Before inclusion in the study, the participants provided written informed consent. The clinical characteristics and pathological information of the participants with the primary CRC specimens are listed in Table S6.
Cell culture and transfection
The mouse CRC cell line MC38, mouse glioma GL261 cells, mouse ovarian cancer ID8 cells, and human CRC cell lines HCT116, SW480, LS174T, HT29, and LOVO were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS). Human CRC cell lines, DLD1 and RKO, and the human colon epithelial cell line, NCM460, were cultured in RPMI 1640 medium supplemented with 10% FBS. HUVECs were cultured in DMEM supplemented with 10% FBS and 1% NEAA. Cells were incubated at 37℃ in a humidified atmosphere with 5% CO2. Regular testing for mycoplasma contamination was also performed. Cell lines were authenticated by Genetica DNA Laboratories using STR profiling. Transfection was performed using Lipofectamine 3000 reagent following the manufacturer’s instructions.
Gene silencing
The siRNAs targeting the indicated genes and scramble siRNA controls were purchased from Biotend (Shanghai, China). siRNAs were transfected into cells using Lipofectamine 3000. Cells were trypsinised at 48-72 h post-transfection for various assays. The sequences of shRNAs and siRNAs are listed in Table S5.
Laser capture microdissection (LCM) of CD31+ ECs
Human CRC tissues were frozen in isopentane. Serial 10-μm sections of the CRC tissues were cut longitudinally on a Jung Frigocut 2800E cryostat at -20℃ and mounted onto Superfrost Plus glass slides at room temperature. Sections were immediately fixed in 70% ethanol for 30 s, washed with distilled water, rinsed in 95% ethanol, immersed in filtered Eosin-Y for 10 s, dehydrated in 100% ethanol, and washed consecutively for 5, 10, and 15 min with fresh xylene. The slides were air-dried for 1 h and transferred to a desiccator at room temperature. An Arcturus PixCell II LCM system equipped with an Olympus microscope (Arcturus Engineering, Mountain View, CA, USA) was used to capture principal cells from the sections. One LCM cap (Capture Transfer Film TF100, Arcturus) was used per tissue section, and optimal conditions for LCM included a laser power of 40 mW, duration of 1.5-2.5 ms, and laser spot size of 7.5 m for epithelia. Captured cells were mixed with TRIzol lysis buffer in an Eppendorf tube, microcentrifuged, and stored at 80 ℃. RNA was extracted within 72 h. The cell capture process, from tissue sectioning to lysis, was completed rapidly to limit RNA degradation.
RNA extraction and quantitative reverse transcription-PCR (qRT-PCR)
Total RNA was extracted and purified using the RNeasy Mini Kit (Qiagen,# 74104) following the manufacturer's instructions. Thereafter, 1 μg of total RNA was reverse transcribed using the PrimeScript RT kit (TakaRa, #RR047R). qRT-PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen,# 204141). GAPDH was used as the reference gene for normalisation. The average of three independent analyses was calculated for each gene. Fold changes were determined via relative quantification (2ΔΔCt). The qRT-PCR primer sequences are listed in Table S5.
Coimmunoprecipitation (Co-IP) and western blotting
The cells were lysed in WB&IP buffer supplemented with protease inhibitors. The cell lysates were immunoprecipitated with the indicated primary antibodies overnight at 4°C, followed by protein A/G precipitation for 2 h or direct incubation with magnetic beads conjugated with tagged antibodies. The beads were washed thrice with lysis buffer and eluted in SDS sample buffer. The eluted immune complexes were separated via SDS-PAGE, followed by western blotting. Equal amounts of total protein were separated on 7.5/10/12.5% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were incubated overnight with primary antibodies against the target proteins. The membranes were washed thrice with 1× TBST buffer and incubated with secondary antibodies for 1 h at room temperature. The signals were visualised using Luminata Crescendo Western horseradish peroxidase substrate. Antibobies in this study include anti-phospho-YAP Ser127 (CST, #13008, 1:1000 dilution), anti-phospho-YAP Ser397 (CST, #13619, 1:1000 dilution), anti-YAP (CST, #14074, 1:2000 dilution), anti-phospho-IκBα Ser32/36 (CST, #9246, 1:5000 dilution), anti-IκBα (Proteintech, #10268-1-AP, 1:2000 dilution), anti-phospho-Akt Ser473 (Proteintech, #66444-1-Ig, 1:500 dilution), anti-Akt (Proteintech, #60203-2-Ig, 1:2000 dilution), anti-α-SMA (CST, #19245, 1:1000 dilution), anti-Vimentin (CST, #5741, 1:2000 dilution), anti-VE-Cadherin (CST, #2158, 1:1000 dilution), anti-β-tubulin (ABclonal, AC015, 1:4000 dilution), anti-phospho-MOB1 Thr35 (CST, #8699, 1:2000 dilution), anti-MOB1 (CST, #13730, 1:1000 dilution), anti-phospho-MST1 Thr183 (CST, #49332, 1:2000 dilution), anti-MST1 (Proteintech, #22245-1-AP, 1:500 dilution), anti-phospho-LATS1 Thr1079 (CST, #8654, 1:1000 dilution), anti-LATS1 (CST, #3477, 1:1000 dilution), anti-β-TrCP (ABclonal, A21951, 1:1000 dilution), anti-OX40 (CST, #61637, 1:2000 dilution), anti-GAPDH (ABclonal, AC001, 1:4000 dilution), anti-Histone H3 (CST, #4499, 1:4000 dilution), anti-Spns2 (Abcam, ab59972, 1:500 dilution), and anti-TEAD4 (ABclonal, A23774, 1:500 dilution).
Immunofluorescence staining
A total of 1×105 cells were seeded in a 24-well culture plate (slides were placed in advance), and the culture medium was discarded after 48 h of culture. The cells were fixed in 4% paraformaldehyde and permeabilised with 0.5% Triton X-100. The cells were blocked with 5% bovine serum albumin. Primary antibodies were added and incubated overnight at 4°C on a shaker. The fluorescent secondary antibody was diluted with the blocking solution and incubated in the dark for 1 h at room temperature. Nuclei were stained with DAPI for 5 min. Slides were mounted using a fluorescent mounting medium. An upright fluorescence microscope was used to observe and assess the staining index of positive cells.
For multiple IHC of tissues slices, experiments were performed following the instructions from the Panovue TSA kit (Panovue, #10079100020). Briefly, the sections were treated with 0.5% Triton X-100 for 30 min and blocked with goat serum for 1 h at room temperature, washing with PBS 3× between each step. The sections were incubated with antibodies including anti-CD3 (Abcam, ab231775, 1:200 dilution), anti-CD31 (Abcam, ab182981, 1:200 dilution), anti-Ki67 (Abcam, ab15580, 1:500 dilution), anti-YAP (CST, #14074, 1:100 dilution), anti-OX40 (CST, #61637, 1:200 dilution) overnight at 4 ℃ in a humidified chamber, followed by incubation with secondary antibodies and DAPI. Anti-fade mounting medium was used to seal the sections. Immunofluorescent images were obtained using a confocal microscope.
HUVEC migration assay
HUVECs suspended in serum-free medium were placed in the upper chamber of a 24-well Transwell system (Corning) with polycarbonate filters (8-μm pores, Corning). Then, 500 μL of conditional medium was added to the lower chamber. After 12 h of incubation, the cells that migrated to the bottom of the membranes were stained with 0.25% crystal violet for 20 min, followed by imaging and counting.
HUVEC migration assay
HUVECs suspended in serum-free medium were placed in the upper chamber of a 24-well Transwell system with polycarbonate filters (8-μm pores, Corning). Thereafter, 500 μL of the conditional medium was added to the lower chamber. After 12 h of incubation, the cells that had migrated to the bottom of the membranes were stained with 0.25% crystal violet for 20 min, followed by imaging and counting.
HUVEC tube formation assay
We pre-coated 48-well plates with 150 μL precooled Matrigel (Corning, #354234) per well and polymerised at 37 °C for 30 min. HUVECs (1×104 cells) suspended in 200 μL of the conditional medium were seeded into each well and further cultured for 3 h. Thereafter, the fields of tube structure were randomly chosen and photographed for quantification.
Tumour transendothelial migration assay
HUVEC monolayers on Transwell inserts were cultured in DMEM medium containing 10% FBS for 4 h at 37℃. The inserts were placed over 24-well plates and coated with a thin layer of Matrigel. GFP-labeled HCT116 cells were added to the top chamber with HUVECs and allowed to migrate through HUVECs. After 48 h of migration, the top chamber was removed, and the cells in the bottom chamber were fixed and stained. Transwell inserts were counterstained with DAPI and observed via fluorescence microscopy. The results were quantified by counting the number of HCT116 cells passing through the endothelium in the same field (20×) and were expressed as standardised values for at least three independent experiments. The assay was quantified in at least three independent experiments, with each Transwell counting five fields.
Nuclear/cytoplasmic fractionation
Cell pellets were resuspended in 1 ml fractionation buffer (0.1% NP-40, complete Protease Inhibitor, PhosSTOP, and PMSF in PBS), gently pipetted 15 times, and immediately centrifuged at 13,500×g for 30 s. The supernatants were labelled as cytoplasmic fractions. The pellets were washed twice with fractionation buffer and dissolved in 160 μL of fractionation buffer as the nuclear fraction. Each fraction was sonicated for 10 s at a 60% output.
Ubiquitination assay
The ubiquitination assay was performed following established protocols. Briefly, the indicated cells were treated with the proteasome inhibitor MG132 (50 mM, Selleck, #S2619) for 6 h before harvesting. Cell extracts were subjected to immunoprecipitation and western blotting with antibodies against ubiquitin.
Untargeted metabolomics analysis
Metabolite extraction
For cultured cells, approximately 107 cells were harvested, and 800 μL of cold methanol/acetonitrile (1:1, v/v) was added to remove proteins and extract metabolites. The mixture was transferred to a new centrifuge tube and centrifuged at 14,000×g for 5 min at 4°C. The supernatant was collected, and the remaining solution was dried using a vacuum centrifuge at 4°C. For liquid chromatography-MS (LC-MS) analysis, the dried samples were re-dissolved in 100 μL of acetonitrile/water (1:1, v/v) solvent and transferred to LC vials.
LC-MS analysis
To assess polar metabolites in untargeted metabolomics, the extracts were analysed using a Sciex TripleTOF 6600 quadrupole time-of-flight mass spectrometer. The mass spectrometer was coupled with hydrophilic interaction chromatography via electrospray ionisation (ESI). LC separation was performed on an ACQUIY UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 µm particle size, Waters, Ireland) using a gradient of solvent A (25 mM ammonium acetate and 25 mM ammonium hydroxide in water) and solvent B (acetonitrile). The mass spectrometer was operated in the negative and positive ionisation modes. The product ion scan was obtained using information-dependent acquisition in the high-sensitivity mode. The following parameters were used: fixed collision energy, 35 V±15 eV; declustering potential, 60 V (+) and -60 V (-); exclusion of isotopes within 4 Da; and monitoring of 10 candidate ions per cycle.
Quantitative measurement of S1P
The homogenate was sonicated on ice for 30 min, and the mixture was centrifuged for 10 min at 14,000×g and 4°C. Subsequently, 500 μL of the supernatant was used to extract metabolites using a hydrophilic-lipophilic balance elution system. The system was pre-activated with 200 μL of methanol and equilibrated with 200 μL of water. The loaded system was washed successively with 200 μL of water and 200 μL of 10% methanol aqueous solution and eluted with 50 μL of acetonitrile. The analyses were performed using a UHPLC system (I-Class LC, Waters) coupled to a QTRAP mass spectrometer (AB Sciex 5500). The mobile phase comprised solvents A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). The samples were kept in the automatic sampler at 4°C, with a column temperature of 45°C. The gradient was run at a flow rate of 400 μL/min, and a 4 μL aliquot of each sample was injected. The gradient started at 30% B from 0 to 1 min, linearly increased to 80% B from 1 to 7 min, further increased to 90% B from 7 to 9 min, and was sustained at 90% B from 9 to 11 min. Quality control samples were used to test and evaluate the system’s stability and repeatability. In the ESI negative mode, the following conditions were set: source temperature at 450°C, Ion Source Gas1 at 55, Ion Source Gas2 at 60, Curtain gas at 30, and Ion Spray Voltage Floating at 4,500 V. The multiple reaction monitoring (MRM) mode was used to detect ion pairs. The Multiquant software was used to extract the chromatographic peak areas and retention times. The relative quantitative analysis of each metabolite was based on the peak area.
Single-cell transcriptome sequencing
Sample collection, library preparation and sequencing of scRNA
Five patients diagnosed with primary CRC were recruited for this study. The scRNA-sequencing was conducted on the Illumina sequencing platform by Genedenovo Biotechnology Co., Ltd (Guangzhou, China). Single cells were isolated and sorted from freshly dissected tumors using standard protocols. In brief, tumors were cut into 1 mm3 pieces and enzymatically digested using the MACS Tumor Dissociation Kit (Miltenyi, #130-095-929) according to the manufacturer's instructions. The dissociated cells were then passed through a 70-μm Cell-Strainer and centrifuged. After removing the supernatant and lysing the red blood cells, we performed single-cell transcriptome amplification using the SMART-seq2 protocol. Libraries were prepared using the chromium controller and the 10× Chromium Next GEM Single Cell 3' v3.1 protocol. The cell suspension was mixed with the master mix and loaded onto a chromium Next GEM chip G along with Single Cell 3' v3.1 Gel Beads and Partitioning Oil. Within the droplets, RNA transcripts from single cells were uniquely barcoded and reverse-transcribed. The resulting cDNA molecules were pooled and subjected to end repair, addition of a single 'A' base, and ligation of adapters. Subsequently, the products were purified and enriched using PCR to generate the final cDNA library. Finally, the libraries were sequenced on the Illumina HiSeq platform, following the read length specifications provided in the user guide.
scRNA-seq data processing
The raw sequencing data from scRNA-seq were processed using Cell Ranger 3.0.2 (10× Genomics). For scRNA-seq data, filtering was applied, and the gene expression matrix was generated using DNBelab C Series scRNA analysis software (MGI). The reference genome (Ensemble assembly: Sscrofal1.1) was downloaded for the analysis. Cells were retained based on criteria including the detection of more than 200 and less than 5000 genes and a mitochondrial gene detection percentage (MT%) of less than 30%. Following the generation of UMI count profiles, Seurat 3.0 was utilized for quality control and subsequent analysis. The gene expression measurements for each cell were normalized using the 'LogNormalize' method, which scales the total expression by a default scale factor (10,000) and applies a logarithmic transformation to each dataset. Data alignment involved the selection of 1,000 highly variable genes from each data matrix, followed by the implementation of the 'FindIntegrationAnchors' and 'IntegrateData' functions in Seurat 3.0. Subsequently, clustering was performed using the 'FindClusters' function in Seurat to identify sub-cell type clusters. UMAP visualization was used to represent each dataset's clusters if they were derived from both donors. To identify differentially expressed marker genes in each cluster, the 'FindAllMarkers' function in Seurat, based on the Wilcoxon rank-sum test, was utilized. The top 10 differentially expressed genes (DEGs) in each cluster were visualized using a heatmap generated by Seurat. Cell cycle scoring was conducted using the 'CellCycleScoring' function in Seurat, with cell cycle phase marker genes.
RNA-Sequencing
RNA was isolated from HUVECs treated with PBS or OX40L using TRIzol. Each sample was purified using an RNeasy Mini Column (Qiagen, Limburg, Netherlands), treated with DNase, and assessed for quality using an Agilent 2100 Bioanalyzer. The samples were subjected to paired-end sequencing (2×100 bp) using the Illumina HiSeq 2000 platform. Read mapping to the human genome (hg19) was performed using TopHat v2.0.11 (http://tophat.cbcb.umd.edu) with default options and a TopHat transcript index generated from Ensembl_GRCh37. The RNA-sequencing was performed by Genesky Biotechnologies Inc. (Shanghai, China). To identify differentially expressed genes (DEGs) between the two samples, the expression level of each transcript was calculated as fragments per kilobase of exon per million mapped reads (FPKM). Fold change ≥2.5 or ≤−2.5 and adjusted p-value <0.01 were the criterion to obtain DEGs.
Trace-level quantitation of S1P binding with YAP
Protein immunocomplex preparation
To test the direct binding of S1P with YAP, we expressed the recombinant YAP WT construct in Escherichia coli cells and purified both proteins to homogeneity. For in vitro S1P-YAP binding detection, 1.0 μg of purified 6× His-YAP WT protein was incubated with S1P in 1 mL of adjusted immunoprecipitation lysis buffer at 37℃ for 6 h. The immunoprecipitation assay was performed using standard protocols, and the protein-bound beads were washed thrice. Methanol was used to extract metabolites.
Trace-level quantitation of metabolites
The immunocomplexes were directly sorted into 100 μL of 100% acetonitrile. This resulted in a final concentration of approximately 80% acetonitrile due to flow-PBS contamination. The samples were vacuum-concentrated using an EZ2 elite system (Genevac) and stored at -80℃ until further processing. Targeted quantification of these metabolites via LC-MS was conducted using Agilent 1290 Infinity II UHPLC coupled with a ProLab Zirconium Ultra microLC pump, with an Agilent 6495 QQQ-MS operating in the MRM mode. ESI coupling was achieved using a prototype microLC ESI source (ProLab). MRM settings were optimised for S1P using pure standards, and the optimised settings were applied to detect their respective isomers. LC separation was performed on a 100×0.3 mm column with 1.8 μm Zorbax Eclipse Plus C18 (Agilent). The solvent gradient ranged from 100% buffer A (10 mM ammonium formate in 90:10 water:methanol) to 100% buffer B (10 mM ammonium formate in 90:10 propanol: acetonitrile). The flow rate was set at 5 μL/min. The autosampler temperature was maintained at 5℃, and the injection volume was 5 μL. Data processing was performed using Agilent MassHunter Software. For each experiment, at least two negative controls were used to assess background metabolite levels. Metabolites were included only if they were detected above background levels and had a retention time similar to that of the standard qualifier peak. The area under the curve was calculated to evaluate the metabolic differences.
Molecular docking analysis of S1P with YAP
The structure of YAP1 was not fully analysed using nuclear magnetic resonance spectroscopy and X-ray crystallography; therefore, we selected the full-length YAP1 structure predicted using AlphaFold for the next docking step. The 3D structure of S1P was downloaded from PubChem (PubChem, CID 5283560). Subsequently, the YAP and S1P structures were uploaded to Webina, an AutoDock-based web server (https://durrantlab.pitt.edu/webina/). Among the top 10 docking complex models generated, S1P bound within the central cavity formed at the interface of TEAD-binding, WW structural, SH3 domain-binding, and PDZ domain-binding domains of YAP. The model with the highest binding affinity was chosen for further analysis.
MST binding assay
MST measurements were conducted using the Monolith NT.115 system (NanoTemper). Full-length YAP WT protein was expressed and purified in E. coli cells. The purified YAP WT protein was labelled with Atto 488 fluorescent dye following the manufacturer's instructions. The labelling efficiency was 1:2 (protein:dye) by measuring the absorbance at 280 and 488 nm. A solution of S1P or S1P-acetate in 0.01 M HEPES (pH=7.4), 0.15 M NaCl, and 0.005% v/v Surfactant P20 was serially diluted, ranging from 1,000 μM to 30 nM, in the presence of 200 nM labelled YAP. After incubation at room temperature for 15 min, samples were loaded into silica capillaries (Polymicro Technologies). Measurements were taken at 22°C with 20% LED and 40% IR-laser power. Additional measurements were taken at 20% and 60% IR laser powers for comparison. Data analysis was performed using the Nano Temper Analysis software, utilising the Kd curve fitting function.
Enzyme-linked immunosorbent assay (ELISA)
Serum samples from CRC patients and healthy controls or cell culture media were collected and stored at -80℃ until testing. EGF levels in the serum or cell culture media were measured using a protein-specific ELISA kit (Solarbio, #SEKH-0052), following the manufacturer's instructions. All measurements were performed in duplicate, and the average value was calculated from standard curve analyses.
Luciferase reporter assay
Three kb promoter of OX40 was amplified and cloned into pGL3 basic luciferase reporter vectors. To test whether STAT3 regulates OX40 transcription, the pGL3-OX40 promoter and internal control Renilla LUC were co-transfected into 293T cells. Forty-eight hours post-transfection, a luciferase reporter assay was conducted using a Dual-Luciferase Reporter Assay System (Promega, #E1910) following the manufacturer’s instructions. Luminescence was measured using a Gen5 microplate reader (BIOTEK, USA).
Reverse-ChIP
An Reverse-ChIP kit (BersinBio, #Bes5005) was used for this experiment. Cells (3×108) were cross-linked with 3% formaldehyde for 10 min at room temperature, and 0.125 M glycine was added to terminate the reaction. Cells were scraped and harvested, and chromatin DNA was sonicated to obtain approximately 500-bp fragments. Biotin-labelled probes for OX40/TNFRSF4 were provided by Bersin Bio, and the probe sequences are listed in Table S5. The mixed probes at a final concentration of 1 nM per probe (pre-denatured at 85℃ for 3 min) were added to the chromatin supernatant followed by hybridisation (85℃ for 10 min, 37℃ for 30 min, 70℃ for 5 min, 37℃ for 30 min, 55℃ for 2.5 min, and 37℃ for 60 min). The supernatant was incubated for 2 h at 37℃ with streptavidin magnetic beads. After rinsing five times, elution buffer was added to resuspend the beads, and the proteins were eluted by heating with shaking. Next, the protein samples were subjected to polyacrylamide gel electrophoresis, and binding between the OX40/TNFRSF4 promoter and TFs was verified via western blotting.
ChIP
ChIP assays were performed in HUVECs using a ChIP kit (Abcam, #ab117137) following the manufacturer's instructions. Rabbit IgG was used as a control, and an anti-STAT3 antibody was used to pull down the promoter regions of OX40 genes with the STAT3 regulatory element. The DNA fragments were purified and used for real-time PCR with primers targeting the OX40 promoter. The results are presented as fold changes and were calculated by dividing the ChIP signals obtained with the anti-STAT3 antibody by those obtained with the IgG control. The primers used for this analysis are listed in Table S5.
Mining the TCGA datasets
The dataset files for COAD were downloaded from the TCGA website. The files included RNA-seq files that provided normalised FPKM values, survival status, and time for each patient. For gene expression analysis, the FPKM values of the indicated genes from tumour samples and corresponding normal tissue samples, if available, were plotted. The statistically significant difference between tumours and NTs was calculated using Student’s t-test. Gene expression correlations between OX40/TNFRSF4 and CD31, CD34, and VWF levels were assessed using Spearman’s correlation tests.
Statistical analysis
Statistical analyses were conducted using the SPSS version 19.0, GraphPad Prism 5, and ImageJ software. Two-tailed and unpaired Student's t-tests were used to compare the two groups. The data obtained from at least three independent experiments performed in triplicates are presented as means ± standard error or deviation. Spearman's correlation test was used to analyse the correlation between two genes. For comparisons between three or more groups with comparable variations, a one-way analysis of variance was used. If the results showed a significant difference, the Student-Newman-Keuls analysis was used to determine the difference between the two groups. Survival curves were estimated using the Kaplan-Meier method and compared using the log-rank test. All statistical tests and p-values were two-sided. Statistical significance was set at P<0.05.