Isolation of fibroblasts from cancer tissues and paracancerous tissues in HCC samples

Human HCC tissues were obtained in post-surgical samples, from the department of hepatobiliary surgery, at the Fifth Affiliated Hospital of Sun Yat-sen University. All subjects signed an informed consent form, and the study was approved by the Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University.

Cancer-associated fibroblasts (CAFs) and paracancerous normal fibroblasts (NFs) were isolated from HCC tissues and non-tumor tissues adjacent to the HCC, respectively. Fresh HCC tissues and paracancerous tissues were washed with phosphate buffer (PBS; GenDEPOT, Barker, TX, USA) and mince into small pieces (< 1 mm3). Five minuted small tissues were attached to the cell culture dishes and treated with Dulbecco's modified eagle medium (DMEM medium, Gibco, USA), containing 10% fetal bovine serum (FBS; Invitrogen, Waltham, MA, USA), 50 U/mol penicillin (Sigma-Aldrich, USA) and 50 mg/ml streptomycin (Sigma-Aldrich, USA). The DMEM medium was changed every two days. The fibroblasts extending from the HCC tissue were then trypsinised and transferred to the dish, followed by incubation in fresh medium to facilitate attachment of the isolated fibroblasts to the dish. Cells were maintained in complete medium at 37° C in a humidified incubator with 5% CO2 and 21% O2.

Cell lines

HCC cell lines (HepG2, Huh7) were obtained from the Cancer Center, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China. Cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) were cultured from postoperative HCC tissues in our center. All cells were cultured in Dulbecco’s modified eagle medium (DMEM medium, Gibco, USA), which was supplemented with 10% fetal bovine serum (FBS), 50 U/mol penicillin (Sigma-Aldrich, USA) and 50 mg/ml streptomycin (Sigma-Aldrich, USA). AMD3100, an inhibitor of CXCL12, was purchased from Selleck (S8030). CXCL12 protein was purchased from MedChemExpress (HY-P7287). The anti-CXCR4 was purchased from Proteintech (60042–1-Ig). All cultured cells were kept in a humidified incubator at the temperature of 37°C, under the concentration of 5% CO2, and 21% O2.

Cell transfection

For the transfection of human hepatic carcinoma cells (Huh7, HepG2), lentivirus with scramble (control) and sh-CXCR4 was used in the experiments, which was constructed and purchased from Genechem (Shanghai, China). Fourty-eight hours after transfection, stable cell lines were screened by DMEM (10% FBS) containing 2ug/ml puromycin. Western blot analyses were performed to verify the success of transfection.

HCC cells were cocultured with CAFs and NFs

CAFs (1 × 106 cells/ml) or NFs (1 × 106 cells /ml) were cultured in Dulbecco’s modified eagle medium (DMEM medium, Gibco, USA), which were supplemented with 10% fetal bovine serum (FBS) for 48 h. Then, their supernatant was collected to treat HCC cells (HepG2 and Huh7) for 48 h.

Western blotting analyses

After all sample proteins were separated by SDS-PAGE gel, they were transferred to the PVDF membrane and then blocked with 5% skimmed milk. Then, they were subsequently incubated with primary and secondary antibodies. The primary antibodies used to detect the target protein were β-actin (abclonal, AC026, 1:1000), FOLR1 (Proteintech, 23355–1-AP, 1:1000), CXCR4 (Proteintech, 60042–1-Ig, 1:1000), Cleaved Caspase-3 (Cell Signaling Technology, 5A1E, 1:500). The targeted bands were analyzed by ImageJ software (v1.8.0; National Institutes of Health, USA). The β-actin was used as the internal control. The relative protein levels were quantified through comparison to β-actin.

Cell viability assays

Cell proliferation was analyzed by the cell counting kit-8 (CCK-8, MedChemExpress, Cat. No. HY-K0301). Cells were seeded at a density of 1 × 104 /well into 96-well microplates. Then, the cells were treated with various concentrations of Sorafenib (0.25, 0.5, 1, 2, 4, 8, 16, and 32 μM). The CCK-8 assay was performed after 48 h of treatment. Treated cells were incubated for 4 h with a culture medium containing the CCK-8 reagent, and absorbance was recorded at 450 nm using the iMark™ Microplate Absorbance Reader (Bio-Rad, iMark, United States). All experiments were repeated three times. The inhibition of cell proliferation was expressed by the absorbance.

Flow cytometry apoptosis assay

Cell apoptosis analysis: cells were implanted into a 6-well plate for apoptosis analysis. Then, the medium was replaced with a fresh medium supplemented with 3 μM Sorafenib. After treatment of 48 h, the cell apoptosis was detected by Apoptosis Detection Kit (eBioscience™ Annexin V Apoptosis Detection Kits, Thermo Fisher Scientific, Lot No. 2106736). All cells were detected by Polychromatic analytical flow cytometry (Beckman, Cytoflex LX, United States).

Colony formation assay

HCC cells (800 cells/well) were implanted into a 6-well plate. Then, they were treated with the supernatant of CAFs or NFs, CXCL12 protein, AMD3100 (20 μM), and sorafenib (3 μM). After treatment of 15 days, cells were fixed with 4% paraformaldehyde and stained with crystal violet (5%). Each experiment was done thrice. Cells colonies formation rate = Number of colonies formed in each treatment group / Number of implanted cells (800 cells) × 100%.

Enzyme-linked immunosorbent assay (Elisa)

The supernatants of CAFs and NFs were carried out with ELISA analyses. The OD value was detected with the enzyme plate analyzer. Meanwhile, the amount of target protein secreted per 100,000 cells was calculated. The Elisa kit was a Human stromal cell-derived factor 1β (CXCL12β/SDF1B) ELISA kit (Cusabio, B04011121).

Immunohistochemistry staining (IHC)

Human HCC tissues and the xenograft tumors of mice were sectioned into 5 μm slices. Hematoxylin and eosin staining was applied to confirm the status of cancer or cancer-free. All tissue sections were baked, dehydrated, hydrated, and antigen-retrieved. Then, they were incubated with primary and secondary antibodies. An N-ACHROPLAN microscope (ZEISS, Germany) was used to photograph the representative areas. Image-Pro Plus v6.0 software (Media Cybernetics Inc., Bethesda, MD, USA) was used to analyze the Information Object Definition (IOD) values of all images. We calculated the relative IOD values based on three parameters (sum of area, average density, and IOD), which were used for further analysis.

IHC was performed according to the kits (Boster, SA1028, SA1027). Primary antibodies were used for IHC staining: α-SMA (Abcam, ab119952, 1:100), Cleaved Caspase-3 (Cell Signaling Technology, 5A1E, 1:100).

Immunohistofluorescence staining (IF)

Human HCC tissue samples were sectioned into 5 μm slices. Hematoxylin and eosin staining was applied to confirm the status of cancer or cancer-free. IF was performed according to the kits (Boster, SA1028, SA1027). Primary antibodies were used for IHC staining: CXCL12 (Boster, BA1389, 1:100), and α-SMA (Abcam, ab119952, 1:100).

Animal xenograft models

The growth of the tumor was observed in vivo. A total of 9 BALB/c mice (4 weeks old; 9 males) were purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China) and housed with a 12 h light/dark cycle and fed standard laboratory food and water. We randomly divided the 9 mice into the NC group, CAFs group, and CAFs + AMD3100 group equally, with 3 mice in each group.

1 × 106 HepG2 cells (50 μL) and 1 × 106 NFs cells (50 μL) were mixed and then injected subcutaneously into the left and right flanks of nude mice in the NC group. Meanwhile, 1 × 106 HepG2 cells (50 μL) and 1 × 106 CAFs (50 μL) were mixed and then injected subcutaneously into the left and right flanks of nude mice in the CAFs group, and CAFs + AMD3100 group. Therefore, we would get 6 tumors in each group. The tumor volume (mm3) = (length of tumor × width of tumor2)/2. When tumor volume reached 100 – 150 mm3, the mouse of the NC group and CAFs group were began to receive tail vein injections of an equal volume of normal saline + Sorafenib (30 mg/kg) for 3 weeks. Meanwhile, the mouse of the CAFs + AMD3100 group was began to receive tail vein injections of an equal volume of AMD3100 (2.5 mg/kg) + Sorafenib (30 mg/kg) for 3 weeks.

Statistical analysis

Statistical analysis was performed using GraphPad Prism V.8 software. All data were repeated at least three times. Data were presented as mean ± SEM and analyzed by Student’s t-test and Pearson correlation. One-way analysis of variance (ANOVA) and Brown-Forsythe tests were carried out for multiple group comparisons. Kaplan-Miere analysis and Log-rank test as used to analyze the survival of HCC patients. For each test, values of p < 0.05 were considered statistically significant. *p < 0.01; **p < 0.001; ***p < 0.0001; ****p < 0.00001. N.S, not significance.