Patients enrollments and samples

Thirty cases of CRC tissues, adjacent paired noncancerous tissues, and matched liver metastasis tissues, were collected from the Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University. All the CRC patients underwent surgery at the Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University between January 2014 and January 2019. Basic information of patients was shown in Table S1. Inclusion and exclusion criteria were showed in supplementary materials and methods.

Sample collection

CRC tissues and noncancerous tissues in this study were quickly obtain from the specimen by surgical excision when the surgery was over. CRC tissue was obtained from the tumor area without apparent necrosis and noncancerous tissues was obtained 5 cm away from the tumor margin. All the tissue were separately loaded into the EP tubes and frozen in liquid nitrogen.

Cell experimentsReagents and inhibitors

Cholesterol was purchased from Sigma-Aldrich (St. Louis, MO, USA) (C3045) and dissolved in anhydrous ethanol for cell experiments. NVP-BHG712 (50 mg) was purchased from Selleck (Selleckchem, Houston, TX, USA) and dissolved in DMSO. A concentration of 25 nM was used in cell experiments, and mice were orally treated with 3 mg/kg. SH-4-54 was purchased from Selleck and dissolved in DMSO, and 150 nM solutions were used in cell experiments.

Cell culture

CRC cell lines used in this article, including SW620, LoVo, SW480, HT29, RKO, and HCT-116 (human CRC cell lines) cells and NCM460 cells were obtained and identified by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. Cell culture condition was 37 °C with a 5% CO2-humidified atmosphere.

Small-interfering RNA (siRNA) transfection

The siRNAs for EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, and EPHA4 were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Their sequences are shown in Table S2, and experimental methods were performed as previously described [39]. Experimental procedures are shown in Supplementary materials and methods.

Lentivirus transfection

Full length human EFNB2 cDNA was transfected into CRC cell lines using a lentivirus to generate Lentivirus-EFNB2 (EFNB2-OE). A plasmid with ectopic expression of a C-terminal truncated form of EFNB2 (ΔC EFNB2 and ΔC + H EFNB2) and an N-terminal truncated form of EFNB2 (ΔE EFNB2) was established. Lentivirus-NC was used as the negative control (vector). One short-hairpin RNA (shRNA) sequence against EFNB2 was transfected into CRC cell lines to generate sh-EFNB2, while sh-NC was used as the negative control. The sequences are shown in Table S2. All the lentivirus-EFNB2 cDNAs were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China).

Real-time quantitative polymerase chain reaction (RT-qPCR)

Trizol was used to extract RNA, and total RNA was reverse transcribed to cDNA using PrimeScriptTM Kits (Takara Bio Inc., Shiga, Japan). 18S RNA was used as an internal control. The sequences of the primers used are shown in Table S2. The relative expression of the target gene was calculated by the −△Ct or −△△Ct method.

Western blotting

Total protein was extracted and protein concentration was detected by BCA assay. Western blot analysis was performed as previously described [39]. EFNB2 (ab69858, Abcam, Cambridge, UK), EPHB4 (ab150545, ab98933, Abcam), LDLR (ab52818, Abcam), β-catenin (ab32572, Abcam), VLDLR (ab203271, Abcam), SCARB1(ab52629, Abcam), STAT3(ab68153, Abcam), p-STAT3(ab267373, Abcam), JAK2(ab108596, Abcam), p-JAK2(ab108596, Abcam), SREBP2(ab30682, Abcam), proliferating cell nuclear antigen (PCNA; Proteintech Group, Inc., Sankt Leon-Rot, Germany) primary antibodies were used. Horseradish peroxidase (HRP)-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00001-2) and HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (SA00001-1) were obtained from Proteintech Group, Inc (Chicago, US).

Immunohistochemistry

All tissues were paraffin-embedded and cut into 4 μm thick sections. All sections were dewaxed with xylene and hydrated with alcohol. Sodium citrate was used for antigen retrieval, and 0.3% hydrogen peroxide (H2O2) was used to block endogenous peroxidase. After blocking non-specific sites with bovine serum albumin, all the sections were incubated with an appropriate primary antibody and secondary antibody. We used 3,3′-diaminobenzidine (DAB) kits (ab64238, Abcam) for visualization, and hematoxylin was used to stain the nuclei. All sections were dehydrated with alcohol and sealed with neutral resin. The immunohistochemistry (IHC) staining score was calculated based on pixel intensity, as follows: no staining, 1; weak staining, 2; moderate staining, 3; and strong staining, 4.

Cholesterol detection

The cholesterol levels in the CRC cells and liver metastatic tissue of CRC were detected using Cholesterol/Cholesteryl Ester Quantitation Assay kits (ab65359, Abcam). An amount of 106 CRC cells or 10 mg liver metastatic tissue of CRC were used to detect the cholesterol level.

Luciferase reporter assay

The STAT3 overexpressed plasmids were transfected into CRC cells using Roche X-tremeGENE HP DNA Transfection Reagent (Roche Diagnostics, Basel, Switzerland). The luciferase plasmid of each group was added separately. CRC cells were co-cultured with Dual-Glo Luciferase Reagent. The fluorescence values were detected using Dual-Glo Luciferase Assays of SpectraMax M5.

ChIP PCR assay

ChIP PCR assays were conducted using Pierce™ Agarose ChIP Kits (26156, Thermo Fisher Scientific, Waltham, MA, USA). CRC cells were fixed with 1% formaldehyde and glycine. The cell chromosomes were fragmented using MNase Digestion Buffer Working Solution and Micrococcal Nuclease. A chromosomal solution of each group was incubated with STAT3 antibody, IgG4 (negative control) and RNA polymerase II antibody (positive control). The immune complex was precipitated and washed, and the DNA samples were recovered. The ChIP results were validated using PCR assays.

In vivo modeling

All mice were randomly divided into groups. Blindness is used in animal experiments. All animal experiments were approved by the Research Ethics Committee of Renji Hospital and adhered to the local or national requirements for the care and use of laboratory animals. Experimental procedures were showed in supplementary materials and methods.

LM redigestion and LM cell reculture

LM tissues were obtained from a liver metastasis model established by splenic injection and cut into 2 mm slices. All the LM tissues were treated with Collagenase/hyaluronidase and DNase I. After incubation at 37 °C for 30 min, the incubating mixture was filtered using cell strainers, and the LM cells collected. The LM cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin.

Statistical analysis

Measurement data are presented as the mean ± standard deviation (SD). SPSS 20.0 (Chicago, IL, USA) and GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA, www.graphpad.com) were used to conduct the statistical analyses. The correlation of EFNB2 expression with the values of categorical clinical variables in patients with CRC was evaluated using chi-square analysis or Student’s t-tests. Measurement data, such as age and tumor size, were evaluated using Student’s t-tests, while categorical variables and ranked data, such as gender, T stage, lymph node invasion, and distant metastasis, were analyzed using chi-square tests. Spearman’s rank correlation was used for the analysis of two-way ordered categorical data. Survival curves were generated using the Kaplan–Meier method, and analyzed using log-rank tests. Statistical significance was accepted at p < 0.05.