After insufficient radiofrequency ablation, tumor-associated endothelial cells exhibit enhanced angiogenesis and promote invasiveness of residual hepatocellular carcinoma

Fresh HCC tissues were obtained from seven patients who had positive α-fetoprotein (AFP) with curative liver resection at the Liver Cancer Institute and Zhongshan Hospital, Fudan University (Shanghai, China). The study design was approved by the Ethics Committee of Fudan University, and all the study participants provided informed consent.

Stable green fluorescent protein (GFP)-expressing HepG2 and HCCLM3 cells, two human HCC cell lines established at the Liver Cancer Institute and Zhongshan Hospital[20, 21], were maintained in high-glucose DMEM (Gibco, Los Angeles, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Los Angeles, USA), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. TAECs were isolated as described before[22]. Briefly, TAECs were obtained from surgical HCC specimens immediately after removal from patients. Specimens were minced and digested by incubation for 1 h at 37°C in 1640 medium (Gibco, Los Angeles, USA) containing 0.1% collagenase IV (Sigma-Aldrich, St. Louis, MO, USA). After washing in PBS (Gibco, Los Angeles, USA), the cell suspension was forced through a graded series of meshes to separate the cell components from stroma and aggregates. TAECs were isolated from cell suspension using anti-CD31 monoclonal antibodies (mAbs) coupled to magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) and magnetic cell-sorting using the MACS system (Miltenyi Biotech, Bergisch Gladbach, Germany). To increase the purity of isolated TAECs after positive magnetic bead isolation, the cell pellets underwent a second isolation with anti-CD31 mAbs. Cells were grown in complete EGM-2 medium (Lonza, Basel, Switzerland) supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. TAECs were used at passages 1–6.

TAECs were grown on 24-well plates to 40-50% confluence, then fixed and blocked. Cells were then incubated with primary monoclonal or polyclonal antibodies against CD31, CD34, vascular endothelial growth factor receptor-1 (VEGFR1), vascular endothelial growth factor receptor-2 (VEGFR2), von Willebrand factor (vWF), CD68 and αSMA (Santa Cruz, California, USA) overnight at 4°C. The next day, plates were washed and incubated with anti-mouse or anti-rabbit fluorescein isothiocyanate- and/or tetramethyl rhodamine isothiocyanate-conjugated secondary antibody (Invitrogen, Carlsbad, USA). Cells were counterstained with 4'-6-diamidino-2-phenylindole (DAPI; KeyGen Biotech, Nanjing, China) to visualize cell nuclei and observed using an inverted fluorescence microscopy (Olympus IX51) equipped with an Olympus Qcolor 3 digital camera (Olympus).

TAECs were incubated in serum-free EBM-2 medium containing 10 μg/ml rhodamine-labeled 1,1V-dioctadecyl-3,3,3V,3V-tetramethylindocarbocyanine acetylated low-density lipoprotein (Dil-Ac-LDL; Sigma-Aldrich, St. Louis, MO, USA) for 4 h at 37°C. The cells were fixed in 4% paraformaldehyde for 20 min at room temperature. Cells were counterstained with DAPI to visualize cell nuclei and analyzed using an inverted fluorescence microscope (Olympus IX51) equipped with an Olympus Qcolor 3 digital camera (Olympus).

TAEC proliferation was measured using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. Briefly, TAECs were cultured in 96-well plates at a concentration of 3 × 10³/well. After 24-h incubation, the plates were heat treated for 10 min at 37, 42 or 47°C. After incubation for 24, 48 or 72 h, 5 μL of CCK-8 reagent was added to each well. The absorbance was measured at 450 nm after 2.5-h incubation at 37°C.

TAECs were plated into the 6-well plates, and after 24-h incubation the plates were sealed and submerged for 10 min in a water bath set to 37, 42 or 47°C. At 24, 48 or 72 h after heat treatment, TAECs were trypsinized and resuspended for further experiments involving migration and tube formation.

Quantitative cell migration assays were performed using a modified Boyden chamber (Costar-Corning, New York, USA) with 8.0-μm pore polycarbonate filter inserts in 24-well plates as described previously[9]. Briefly, the lower chamber was filled with EGM-2 with 10% FBS, and TAECs (5 × 10⁴ cells/well) in serum-free medium were added into the upper chamber. The cells were allowed to migrate for 5 h at 37°C. The non-migrated cells were removed from the upper surface of the membrane by scraping with a cotton swab, and the migrating cells were fixed with methanol, stained with crystal violet (Beyotime, Nantong, China) and photographed under an inverted fluorescence microscope (Olympus IX51) equipped with an Olympus Qcolor 3 digital camera (Olympus). Migration was assessed by counting the number of stained cells from 10 random fields at ×100 magnification.

TAECs tube formation was studied on growth factor-reduced Matrigel (Becton Dickinson, San Jose, USA) diluted 1:1 in ice with cold EBM-2 in a 96-well plate. Cells (1 × 10⁴ cells/well) were added to Matrigel-coated 96-well plates. TAECs were photographed under an inverted fluorescence microscope (Olympus IX51) equipped with an Olympus Qcolor 3 digital camera (Olympus). Tube formation was assessed by counting the number of loops from 10 random fields at ×50 magnification.

TAECs were cultured in a T25 culture bottle at a concentration of 5 × 10⁵ cells in EGM-2 containing 10% FBS. After 24-h incubation, 1 ml of fresh EBM-2 was added to the culture bottle with replacement of EGM-2. The culture bottles were submerged in a water bath for 10 min at 37, 42 or 47°C. Immediately after heat treatment, 2 ml of fresh EBM-2 was added to each culture bottle and cells were cultured in the incubator at 37°C. After 24-h incubation, the medium was collected and spun down at 3000 rpm for 20 min, and the supernatant was collected and stored at −80°C. Cells were lysed using cell lysis buffer (150 mM NaCl; 50 mM Tris–HCl; pH 8.0; 0.1% SDS; and 1% Triton X-100) containing protease, phosphatase inhibitors, and protein was used for further experiments.

The invasiveness of hepatoma cells was assessed by measuring their ability to invade through Matrigel-coated transwell inserts with 8.0-μm pores (Costar-Corning, New York, USA). HepG2-GFP or HCCLM3-GFP cells were trypsinized and resuspended with conditioned medium. 200 μl of cell suspension (2 × 10⁴ or 5 × 10⁴ cells) was added to each upper well, and the lower chamber was filled with DMEM with 10% FBS. Hepatoma cells were allowed to invade for 36 h at 37°C. The non-invaded cells were removed from the upper surface of the membrane by scraping with a cotton swab, and the invaded cells were fixed with methanol, stained with crystal violet, and photographed under inverted fluorescence microscopy (Olympus IX51) equipped with an Olympus Qcolor 3 digital camera (Olympus). Invasion was assessed by counting the number of stained cells from 10 random fields at ×100 magnification.

Cytokines secreted into the conditioned medium were quantified using an enzyme-linked immunosorbent assay (ELISA) kits (for IL-6, MCP-1, IL-8 and GRO-α; Boster, Wuhan, China) according to the manufacturer’s instructions. The concentration of cytokines was normalized to the total cellular protein.

Equivalent amounts of whole cell extracts were subjected to SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 2 h and then incubated with respective primary antibody overnight at 4°C followed by the incubation with the appropriate HRP-conjugated secondary antibody for 1.5 h at room temperature. Blots were visualized with an ECL detection kit (Pierce, USA) and analyzed using Quantity One 1-D Analysis Software (Bio-Rad, Hercules, USA).

TAECs were plated onto 96-well plates and allowed to grow until complete confluence was achieved. The plates were submerged in a water bath for 10 min at 37, 42 or 47°C. After 24-, 48- or 72-h incubation, cells were rinsed with PBS twice, and HepG2-GFP or HCCLM3-GFP cells (1 × 10⁴ cells) in fresh DMEM were added to the wells. The plates were incubated at 37°C in 5% CO₂ for 20 min. Non attached cells were removed by washing thrice with PBS and the attached hepatoma cells in each well were visualized under a microscope and counted using an inverted fluorescence microscope (Olympus IX51) equipped with an Olympus Qcolor 3 digital camera (Olympus).

TAECs were plated onto 96-well plates and allowed to grow until 80% confluence. The plates were submerged in a water bath for 10 min at 37, 42 or 47°C. After 24-, 48- or 72-h incubation, cells were washed with PBS twice and fixed with PBS containing 4% paraformaldehyde at room temperature. The plates were blocked with 2% BSA at 37°C for 2 h. Cell surface expressions of adhesion molecules were determined by means of primary binding with specific antibody for VCAM-1, ICAM-1 or E-selectin (Santa Cruz, California, USA), followed by secondary binding with an HRP-conjugated goat anti-rabbit IgG antibody. Quantification was performed by determination of colorimetric conversion at OD at 450 nm of 3,3’,5,5’-tetramethylbenzidine using a TMB peroxidase EIA substrate kit (Bio-Rad, Hercules, USA).

Student’s t test or the ANOVA test was used for comparison of two groups or three groups using GraphPad Prism (GraphPad Software Inc., La Jolla, CA). A P value of <0.05 was set as the level of statistical significance.

Seven strains of CD31+ TAECs were obtained from seven HCC patients. In addition to CD31, TAECs virtually expressed the EC-specific markers vWF, VEGFR1, VEGFR2 and CD34 (Figure1A). Negative expression of CD68 and α-SMA in isolated TAECs excluded the contamination of macrophages and fibroblasts (Figure1A). Western blot confirmed the immunofluorescence results and that AFP expression could be detected in tumor cells but not in TAECs (Figure1B). TAECs also took up complexes of Dil-Ac-LDL (Figure1C).

In order to simulate the growth pattern of TAECs that were not killed after the heat stress generated by RFA, TAECs were exposed to a 10-min heat treatment at temperatures ranging from 37 to 55°C, and after 36 h, morphological changes in TAECs were observed. It was found that TAECs could not be continuously cultured once the temperature exceeded 47°C (Figure2A). To monitor the potential effect of insufficient RFA on the function of TAECs in vitro, we initially observed the cell proliferation, migration and tube formation of TAECs at 24, 48 and 72 h after heat treatment. It was found that the proliferation of TAECs after insufficient RFA was significantly decreased as compared with the control treatment (Figure2B). However, it was interesting that the insufficient RFA treatment promoted the migration and tube formation of TAECs; this was not observed after the control treatment (Figure2C and D).

HepG2-GFP or HCCLM3-GFP cells were used to investigate the adhesion ability of TAECs after insufficient RFA. TAECs were treated with insufficient RFA or the control treatment. After a 24-, 48- or 72-h interval, they were incubated at 37°C and hepatoma cells were added. The results showed that after insufficient RFA TACEs adhered to HepG2-GFP cells at a significantly higher level than the case after the control treatment (all P <0.001; Figure3A and B). Similar results were observed in HCCLM3-GFP cells (all P <0.001; Additional file1: Figure S1). In order to explore the mechanism involved in the process, we measured the surface expressions of the TAEC adhesion molecules after insufficient RFA using cell ELISA. The results showed that the expression of E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were significantly up-regulated on the surface of TAECs at 24, 48 and 72 h after insufficient RFA (Figure3B). Western blot also confirmed the cell ELISA results (Figure3C).

Using the conditioned media from TAECs with or without insufficient RFA treatment, we further explored the effect of TAECs on the invasiveness of hepatoma cells. Conditioned medium from TAECs after insufficient RFA significantly enhanced the invasiveness of HepG2-GFP cells relative to the control (Figure4A). Similar results were observed in HCCLM3-GFP cells (Additional file2: Figure S2). To test the possible mechanism involved in the promotion of the invasiveness of hepatoma cells by TAECs after insufficient RFA, we measured the levels of cytokine secreted by TAECs in the conditioned medium. We found that insufficient RFA significantly increased the secreted levels of IL-8, IL-6, MCP-1 and GRO-α by TAECs (all P<0.05; Figure4B).

To further determined the associated signal pathways involved in the process as described above, we investigated the expression levels of total and phosphorylated ERK1/2, NF-κB, Akt and STAT3 protein in TAECs at 24 h after insufficient RFA. It was found that total protein levels of ERK1/2, NF-κB, Akt and STAT3 were not changed after insufficient RFA, whereas phosphorylated ERK1/2 (p-ERK1/2), NF-κB and p-Akt were up-regulated and p-STAT3 was substantially down-regulated in TAECs after insufficient RFA (Figure5).