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Clinical Cancer Research Vol. 11, 8557-8563, December 15, 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Overexpression of Platelet-Derived Growth Factor Receptor {alpha} in Endothelial Cells of Hepatocellular Carcinoma Associated with High Metastatic Potential

Ti Zhang, Hui-Chuan Sun, Yang Xu, Ke-Zhi Zhang, Lu Wang, Lun-Xiu Qin, Wei-Zhong Wu, Yin-Kun Liu, Sheng-Long Ye and Zhao-You Tang

Authors' Affiliation: Liver Cancer Institute and Zhong Shan Hospital of Fudan University, Shanghai, People's Republic of China

Requests for reprints: Zhao-You Tang, Liver Cancer Institute and Zhong Shan Hospital of Fudan University, 136 Yi Xue Yuan Road, Shanghai 200032, People's Republic of China. Phone: 86-21-6403-7181; Fax: 86-21-6403-7181; E-mail: zytang{at}scrap.stc.sh.cn.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Little information is available on the heterogeneity of the vascular endothelium in hepatocellular carcinoma. The aim of this study was to identify the differentially expressed genes in tumor endothelial cells from highly metastatic hepatocellular carcinoma.

Experimental Design: Magnetic beads conjugated with anti-CD31 antibody were used to isolate vascular endothelial cells from hepatocellular carcinoma xenografts with different metastatic potentials in nude mice. Gene expression profiles for different endothelial cells were compared by use of cDNA microarray. The up-regulated gene was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry.

Results: cDNA microarray analysis revealed differential expression patterns in seven genes consistently presented in endothelial cells isolated from hepatocellular carcinoma with different metastatic potentials. Overexpression of platelet-derived growth factor receptor {alpha} was found only in the endothelium of highly metastatic hepatocellular carcinoma, which was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry. Oral administration of STI571 (imatinib mesylate or Glivec), a protein tyrosine kinase inhibitor of platelet-derived growth factor receptor, combined with s.c. injection of IFN-{alpha} not only effectively reduced tumor weight (by 81.8%) and microvessel density (by 70.2%) but also inhibited lung metastasis (by 100%). Furthermore, immunohistochemical analysis of platelet-derived growth factor receptor {alpha} in human hepatocellular carcinoma tissues revealed its correlation with postoperative recurrence, especially in patients without microvessel invasion.

Conclusions: The gene expression of hepatocellular carcinoma vascular endothelium is different between tumors with different metastatic potential. Platelet-derived growth factor receptor {alpha}, which is overexpressed in endothelium of highly metastatic hepatocellular carcinoma, may serve as a biomarker for predicting metastasis and a therapeutic target for highly metastatic hepatocellular carcinoma.

Hepatocellular carcinoma is one of the most prevalent cancers in Asia and Africa (1), and even in the West, its incidence is steadily increasing. In Europe, mortality from primary liver cancer increased 4-fold for men and 2-fold for women between 1979 and 1994 (2), whereas in the United States there was a 41% increase in the mortality rate from primary liver cancer over the past two decades (3). Despite efforts to improve its prognosis, the overall survival of patients with hepatocellular carcinoma is still unsatisfactory (4, 5). More than 70% of patients were not candidates for surgical treatment when diagnosis of hepatocellular carcinoma was established, and systemic treatment is not effective in most cases; even in the group of patients receiving curative resection, metastasis and recurrence are still common and responsible for the poor prognosis (6).

Antiangiogenesis is an attractive treatment for hepatocellular carcinoma, a typical hypervascular cancer, because its growth, metastasis, and hepatocarcinogenesis depend on angiogenesis (7, 8). However, few studies have reported success with antiangiogenesis treatment for hepatocellular carcinoma. Different responses to the same treatment have been observed in patients undergoing chemotherapy, an outcome associated with heterogeneity of tumor cells. Likewise, the heterogeneity of tumor vessels, which has been widely accepted, may result in a heterogeneous response to antiangiogenesis treatment. As a major part of tumor vessels, endothelial cells are heterogeneous in different organs, tissues, and tumors (9, 10), and the distinct molecules on tumor endothelial cells (TEC) can serve either as diagnostic markers or as therapeutic targets (11, 12).

Several molecules have been identified as tumor-specific endothelial markers (13–15), and tumor type- and stage-specific vascular markers have been reported (16, 17). Unfortunately, TEC-specific molecules have not been well defined in hepatocellular carcinoma, and there is no such molecule reported that could discern different metastatic potential of patients with hepatocellular carcinoma. For instance, CD34 can be used as a TEC marker, and microvessel density (MVD) outlined by CD34 antibody was correlated with the prognosis of hepatocellular carcinoma patients (7, 8). However, CD34, like other reported endothelial markers, is expressed in both metastatic and nonmetastatic hepatocellular carcinoma and does not reflect the intrinsic characteristics of TEC itself or serve as a therapeutic target.

A human hepatocellular carcinoma metastasis model system has been established at the Liver Cancer Institute of Zhong Shan Hospital that consists of a highly metastatic model of human hepatocellular carcinoma in nude mice (18), metastatic human hepatocellular carcinoma cell line MHCC97 (19), and cell clones with high and low metastatic potentials (MHCC97H and MHCC97L; ref. 20). The rate of metastasis to lungs was 100% using orthotopic inoculation with highly metastatic cell line MHCC97H and 40% with MHCC97L. This model system has been used for in vitro and in vivo studies of hepatocellular carcinoma–related invasion, metastasis, and angiogenesis (21–23).

In the present study, we isolated TECs from hepatocellular carcinoma tumors with different metastatic potential by immunomagnetic methods and analyzed the angiogenesis-related gene expression profile by cDNA microarray. We found several differentially expressed genes in TEC and confirmed that the expression of platelet-derived growth factor receptor {alpha} (PDGFR{alpha}) was related to growth and metastasis of hepatocellular carcinoma.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals and tumor models. Male athymic BALB/c nu/nu mice (5 weeks old) were obtained from Shanghai Institute of Materia Medica, Chinese Academy of Science. All mice were bred in laminar-flow cabinets under specific pathogen-free conditions. The experimental protocol was approved by the Shanghai Medical Experimental Animal Care Committee.

Hepatocellular carcinoma tumor models produced by MHCC97H (human hepatocellular carcinoma cell line with high metastatic potential), MHCC97L (human hepatocellular carcinoma cell line with lower metastatic potential), and Hep3B (human hepatocellular carcinoma cell line with very low invasiveness; ref. 24) were established in nude mice by orthotopic inoculation as described previously (refs. 18–20; see Supplementary Data). Five weeks later, when tumors reached a diameter of 8 to 10 mm, mice were sacrificed by cervical dislocation, and the tumors were removed for TEC isolation.

Human hepatocellular carcinoma cell line Hep3B and murine fibroblast line NIH3T3 were obtained from The Shanghai Institute of Cell and Biology, Chinese Academy of Science, and cultured in DMEM or RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Life Technologies), respectively.

Immunomagnetic isolation of endothelial cells. Tumors or corresponding livers from six to eight mice were harvested and dissociated into cell suspension by incubation with 0.1% collagenase type IV (1 mg/mL in serum-free DMEM). Magnetic beads (Dynabeads M-450 sheep anti-rat IgG; Dynal, Oslo, Norway) were used for immunomagnetic purification of cells according to the manufacturer's instructions. The antibody of CD31/platelet-endothelial cell adhesion molecule-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), an endothelial marker widely used in this approach (25–28), was coupled with magnetic beads and added to unfractionated cells. Cells were incubated with beads for 25 minutes at 4°C and separated with a magnetic particle concentrator (Dynal; see Supplementary Data).

Cell yield was determined in a hemocytometer, and viability was assessed by trypan blue exclusion. Some of the fractionated cells, used for verification, were plated into wells or chamber slides (BD Falcon, Heidelberg, Germany) precoated with attachment factor (Cascade Biologics, Portland, OR) and cultured in M131-MVGS medium (Cascade Biologics). Other cells were pelleted, snap frozen in liquid nitrogen, and stored at –70°C for not more than 2 months before analysis.

Immunofluorescence analysis. Acetone-fixed, 6-µm-thick frozen sections of hepatocellular carcinoma tumors and normal livers were stained with rat anti-mouse CD31 (1 µg/mL) for 18 hours at 4°C. Normal rat IgG was used as a negative control. Bound antibodies were detected by incubation with rhodamine or FITC-conjugated goat anti-rat IgG (Santa Cruz Biotechnology) for 30 minutes at 37°C. The slides were cross-stained with 4',6-diamidino-2-phenylindole (1:1,000; Sigma, St. Louis, MO) for nucleus staining.

For immunocytochemistry, endothelial cells were incubated on chamber slides. When they reached subconfluence, endothelial cells were incubated with rat anti-mouse CD31 antibody (1 µg/mL), rabbit anti-mouse VE-cadherin antibody (2 µg/mL; Alexis Biochemical, San Diego, CA), or goat anti-mouse PDGFR{alpha} antibody (2 µg/mL; R&D Systems, Minneapolis, MN). The bound antibodies were detected with rhodamine, FITC-conjugated secondary antibodies, or biotinylated secondary antibodies (KPL, Gaithersburg, MD) followed by avidin-Alexa Fluor 488 (Molecular Probes, Eugene, OR), respectively. The murine fibroblast line NIH3T3 and human hepatocellular carcinoma cell line MHCC97H were used as negative controls. For additional negative controls, samples were exposed to secondary antibodies alone with primary antibodies replaced by PBS.

To localize the distribution of PDGFR{alpha} in hepatocellular carcinoma tumor and normal liver, sections were simultaneously stained with CD31 and PDGFR{alpha} antibody. Vascular pericyte staining with anti–{alpha}-smooth muscle actin (Sigma), coupled with CD31 or PDGFR{alpha} antibody, was also done to exclude exceptional PDGFR{alpha} distribution.

Antibody staining was observed using a Leica TCS SP2 Microscope Confocal System (Leica, Heidelberg, Germany) or Olympus BX40 fluorescence microscope (Olympus, Tokyo, Japan). (A detailed protocol is available in Supplementary Data.).

Internalization of acetylated low-density lipoprotein. The isolated endothelial cells were incubated in serum-free M131 medium containing 10 µg/mL rhodamine-labeled 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine acetylated low-density lipoprotein (Biomedical Technologies, Inc., Stroughton, MA) for 4 hours at 37°C. The cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature and stained with 4',6-diamidino-2-phenylindole. The NIH3T3 and unfractionated cells (cells after collagenase digestion but before immunomagnetic isolation) served as controls. Samples were analyzed under fluorescence microscopy and the Leica confocal imaging system.

Reverse transcription-PCR analysis. Total RNA from CD31-negative cells with endothelial cells depleted (NC), TEC (from MHCC97H, MHCC97L, and Hep3B tumors), and liver endothelial cells (LEC; CD31-positive cells from normal liver) was extracted and reverse transcribed into single-stranded cDNA using RevertAid Moloney Murine Leukemia Virus Reverse Transcriptase (MBI Fermentas, Vilnius, Lithuania) according to the manufacturer's instructions. To assess the purity of the isolated endothelial cells, PCR was done on cDNA generated from TEC, LEC, or NC. Hepatocellular carcinoma cell-specific transcript {alpha}-fetoprotein and pan-leukocyte marker CD45 were analyzed to exclude the contamination of tumor cells or leukocytes, whereas three endothelium-specific transcripts, CD31, VE-cadherin (9), and CD146 (14), were amplified to confirm the identity of endothelial cells. Gene expression level of PDGFR{alpha} in different TEC and NC was also determined by reverse transcription-PCR (RT-PCR) analysis. Glyceraldehyde-3-phosphate dehydrogenase mRNA was detected to serve as an internal reference. (Sequences of the primers used in this study are available in Supplementary Table S1.).

Gene expression profiling. GEArray Q Series Mouse Angiogenesis Gene Array kit (SuperArray, Bethesda, MD) was used to characterize the gene expression profiles of different TEC, LEC, and NC (from MHCC97H tumors). The chip includes 96 angiogenesis-related genes and 2 housekeeping genes, ß-actin and glyceraldehyde-3-phosphate dehydrogenase. Hybridization was done according to the manufacturer's instructions as described previously (29). Experiments were done in duplicate, and the reproduction rate was >98% between experiments.

Real-time PCR. QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA) and DNA Engine Opticon System (MJ Research, Reno, NV) were used in real-time PCR analysis of gene expression levels of PDGFR{alpha} in different TEC and NC (MHCC97H). Data were analyzed with Opticon Monitor software version 1.02. The thermal cycling conditions comprised an initial denaturation step at 95°C for 15 minutes and 45 cycles at 94°C for 15 seconds and 57°C for 1 minute. The glyceraldehyde-3-phosphate dehydrogenase mRNA was used as an internal standard. (Primer sequences are shown in Supplementary Table S1.).

In vitro cytotoxicity assay. MHCC97H cells (3 x 103) were seeded into 38-mm2 wells of flat-bottomed 96-well plates in quadruplicate and allowed to adhere overnight. The cultures were refed with new medium (negative control); medium containing recombinant IFN-{alpha}-1b (1,000 units/mL; Kexing Bioproduct, Shenzhen, People's Republic of China); medium containing different concentrations (0.1-10 µmol/L) of STI571 (imatinib mesylate or Glivec; Novartis Pharma, Basel, Switzerland), a protein tyrosine kinase inhibitor of PDGFR (30); or medium containing different concentrations of STI571 plus IFN-{alpha}. After 3 days of treatment with different agents, the number of metabolically active cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (31).

Treatment and assessment of nude mice bearing MHCC97H tumors. Seven days after the implantation of tumor into the liver, the mice were randomly assigned to receive one of the following four treatments (six mice in each treatment group): (a) a daily oral dose of vehicle solution (water) and a daily s.c. injection of distilled water (control group), (b) a daily oral dose of STI571 at 50 mg/kg (STI571 group), (c) a daily s.c. injection of IFN-{alpha} at 7.5 x 106 units/kg (IFN-{alpha} group), or (d) a daily oral dose of STI571 at 50 mg/kg plus a daily injection of IFN-{alpha} at 7.5 x 106 units/kg (STI571 plus IFN-{alpha} group).The mice were treated for 5 weeks. After the mice were sacrificed, tumor weight was measured and MVD of tumor was evaluated as described previously (32). The incidence of lung metastasis was evaluated independently by two pathologists.

Immunohistochemical double staining for CD34 and platelet-derived growth factor receptor {alpha} in clinical specimens of human hepatocellular carcinoma. For immunohistochemical analysis, formalin-fixed, paraffin-embedded surgical specimens from 77 hepatocellular carcinoma patients who had received curative resection were analyzed. Forty-five of them suffered from recurrence within 1 year, whereas 32 were disease-free for at least 3 years. Histologic diagnosis was made according to WHO criteria. (The main clinicopathologic features are available in Supplementary Table S2.).

To assess the distribution of PDGFR{alpha} in human hepatocellular carcinoma specimens, sections were simultaneously stained with mouse anti-CD34 antibody (10 µg/mL; Santa Cruz Biotechnology) and rabbit anti-PDGFR{alpha} antibody (10 µg/mL; NeoMarkers, Fremont, CA). Staining was evaluated by two independent observers without knowledge of the patient's survival. The positive or negative PDGFR{alpha} expression was based on whether it could be detected in the vessel of examined tissues. For negative controls, primary antibodies were replaced by PBS (see Supplementary Data).

Statistical analysis. Data were analyzed by the computer program SPSS 10.0 (SPSS, Inc., Chicago, IL) using ANOVA and Fisher's exact test. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Immunofluorescence detection and immunomagnetic isolation of endothelial cells in mice model. CD31 was detected in both tumor vessels and normal liver vessels. However, CD31-positive vessels were not evenly distributed in tumor tissue. Hotspots of microvessels were visible at the periphery of the tumor. Notably, CD31 staining was mainly observed in large vessels of normal liver tissue, although it was positive in almost all kinds of tumor vessels. Both immature and mature vessels were found in hepatocellular carcinoma tissues, and abnormal vessels were relatively common. No staining was observed in the negative control group.

The yield of endothelial cells ranged from 5 x 105 to 15 x 105 for six to eight tumor nodules and from 2 x 105 to 5 x 105 for six to eight normal liver tissues, respectively. The average cell viability judged by trypan blue exclusion was ~73% to 80% for both TEC and LEC.

Verification of endothelial cells. The cultured endothelial cells from different tumor and liver exhibited a similar morphology when plated on attachment factor-coated plates, displaying a cobblestone appearance after reaching confluence (Fig. 1A). Two endothelium-specific markers, CD31 (Fig. 1B) and VE-cadherin (Fig. 1C), were both expressed on the surface of the isolated endothelial cells. VE-cadherin aggregated at intercellular boundaries and exhibited strong fluorescence at junction sites. Neither NIH3T3 nor MHCC97H cells expressed a fluorescence signal for CD31 and VE-cadherin. More than 95% of the isolated endothelial cells showed uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine acetylated low-density lipoprotein when cultured in wells (Fig. 1D), which is one of the listed specifications of endothelial cells. The control cell line NIH3T3 fibroblast was not labeled, whereas only a few (<1%) within unfractionated cells were positively labeled (see Supplementary Fig. S1). When analyzed by RT-PCR, the endothelium markers CD31, VE-cadherin, and CD146 showed robust amplification only in the endothelial fractions, whereas hepatocellular carcinoma cell-specific transcript {alpha}-fetoprotein was limited to the NC. A high level of pan-leukocyte marker CD45 was found in NC but not in the isolated endothelial cells (Fig. 1E).



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  		Fig. 1. Verification of endothelial cells from different tissues. Representative examples of MHCC97L TECs. A, phase-contrast image of TECs that displayed characteristic cobblestone appearance after reaching confluence. B, CD31 staining was positive on cell membrane (green). C, strong membrane fluorescence at sites of cell-cell contact was observed when stained with anti-VE-cadherin (arrow). D, endothelial cells were seeded in chamber slides and incubated with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine acetylated low-density lipoprotein; positive uptake was observed (red). Bar, 160 µm (A) and 40 µm (B-D). E, RT-PCR analysis used to assess the purity of isolated TEC from MHCC97L tumor. Three endothelial-specific transcripts, CD31, VE-cadherin, and CD146, showed robust amplification only in TEC; in contrast, expression of the hepatocellular carcinoma–specific transcript {alpha}-fetoprotein and pan-leukocyte marker CD45 was limited to CD31-negative cells (NC).

 
Differential gene expression of endothelial cells from hepatocellular carcinoma with different metastatic potential. cDNA array analysis of CD31-positive fractions (TEC and LEC) and CD31-negative fractions (NC) revealed that endothelial markers, including CD105, CD31, and vascular cell adhesion molecule-1, were highly expressed in each endothelial cell group, but expression levels were quite lower in NC. With 2-fold expression as the cutoff point, no significant differences of gene expression level were observed in most of the examined genes. Only seven differentially expressed genes were identified as consistent with hepatocellular carcinoma metastatic potential (Table 1). In particular, we focused on the gene expression of PDGFR{alpha}, which was at a high level in TEC isolated from highly metastatic tumor (MHCC97H), at a much lower level in TEC from low metastatic tumor (MHCC97L) or very low invasive hepatocellular carcinoma (Hep3B), and not detectable in LEC or NC (MHCC97H; see Supplementary Fig. S2).


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  		Table 1. Seven differentially expressed genes consistent with hepatocellular carcinoma metastatic potential

 
Verification of differentially expressed platelet-derived growth factor receptor {alpha} in tumor endothelial cells. To validate the differential gene expression of PDGFR{alpha} in different TEC, RT-PCR and real-time PCR analyses were done. RT-PCR results showed that robust amplification was only found in the TEC isolated from highly metastatic hepatocellular carcinoma (MHCC97H) at 35 cycles. Corresponding to different metastatic potential of primary tumors (MHCC97H > MHCC97L > Hep3B), the expression level of PDGFR{alpha} dropped sharply in TEC from low metastatic hepatocellular carcinoma (Fig. 2A).



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  		Fig. 2. Verification of differentially expressed PDGFR{alpha} in TEC. RT-PCR (A) and real-time PCR (B) analyses confirmed the differential gene expression of PDGFR{alpha} in different TECs, which is consistent with different metastatic potential of primary tumors (MHCC97H > MHCC97L > Hep3B). *, P < 0.05, statistical significance (Student's t test). Double immunofluorescence staining of PDGFR{alpha} and CD31/platelet-endothelial cell adhesion molecule-1 or {alpha}-smooth muscle actin was used to confirm the expression of PDGFR{alpha}. Colocalization of CD31 and PDGFR{alpha} is yellow. C to E, cultured TECs of MHCC97H tumor were positive for anti-PDGFR{alpha} (red) and anti-CD31 (green) staining. F to H, location of PDGFR{alpha} in MHCC97H tumor was consistent with that of CD31. Anti–{alpha}-smooth muscle actin ({alpha}SMA) staining (red) coupled with anti-CD31 (green; I) and anti–{alpha}-smooth muscle actin staining (green) coupled with anti-PDGFR{alpha} (red; J) further confirmed that the location of PDGFR{alpha} was in the tumor endothelium. Bar, 40 µm (C-E), 160 µm (F-H), and 80 µm (I and J).

 
Similar results were obtained in SYBR Green–based real-time PCR analysis. The amplification plot showed that the signal for PDGFR{alpha} arose at least two cycles earlier in TEC of MHCC97H tumor than in others. When normalized to the internal standard control, PDGFR{alpha} mRNA in TEC from MHCC97H tumor was expressed ~16, 45, and 37 times higher than in MHCC97L-TEC, Hep3B-TEC, and MHCC97H-NC, respectively (Fig. 2B).

These findings were verified by analyzing PDGFR{alpha} expression on cultured TEC and frozen tissue sections by immunofluorescence staining. TECs isolated from MHCC97H and MHCC97L tumor were positive for staining with anti-PDGFR{alpha} antibody but negative in TEC of Hep3B tumor or LEC (Fig. 2C-E). Compared with TEC of MHCC97L tumor, a higher intensity of fluorescence signal was observed in TEC of MHCC97H tumor. In frozen tissue sections, positive staining of PDGFR{alpha} was only observed in MHCC97H tumor, located in the endothelium within the tumor nodule, confirmed by costaining with CD31 (Fig. 2F-H). The distribution of PDGFR{alpha} in endothelium but not in pericytes was also observed by costaining with {alpha}-smooth muscle actin (Fig. 2I and J).

STI571 in combination with IFN-{alpha} blocks MHCC97H tumor growth in vivo. Consistent with our previous study (22, 33), single treatment with IFN-{alpha} (1,000 units/mL) alone did not inhibit the proliferation of MHCC97H cells in vitro. The treatment with STI571 (0.1-10 µmol/L) resulted in a mild increase in cytotoxicity compared with control agent (P = 0.062). The combined treatment with STI571 and IFN-{alpha} did not increase the inhibition effect on proliferation compared with STI571 alone (see Supplementary Fig. S3).

Both treatment with IFN-{alpha} alone or STI571 alone significantly inhibited the growth of highly metastatic hepatocellular carcinoma tumor but did not decrease the body weight compared with the control group. STI571 alone inhibited the tumor growth by 27.3%, whereas the combination therapy using STI571 and IFN-{alpha} produced more effective reductions of tumor weight by 81.8% (P < 0.001; Table 2). MVD was statistically different in tumor lesions from control mice and mice treated with STI571, IFN-{alpha}, or STI571 plus IFN-{alpha} (Table 2). All of the treatments produced significant reductions of MVD, with the largest decrease being 70.2% in mice given a combination of STI571 and IFN-{alpha} (P < 0.001). The incidence of lung metastasis was 100%, 66.7%, 50%, or 0% in control, STI571, IFN-{alpha}, or STI571 plus IFN-{alpha} groups, respectively. Statistical differences were found between the STI571 plus IFN-{alpha} group and other groups (Table 2; see Supplementary Fig. S4).


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  		Table 2. Inhibition effect of STI571 and IFN-{alpha} on highly metastatic human hepatocellular carcinoma model

 
Platelet-derived growth factor receptor {alpha} expression is closely associated with recurrence and metastasis in human hepatocellular carcinoma. PDGFR{alpha} expression was detected in the vessel of 17 (22.1%) specimens: 3 of these patients were tumor-free for at least 3 years after curative resection and 14 of them had recurrent tumor within 1 year. The recurrence rate of PDGFR{alpha}-positive cases was significantly higher than that of PDGFR{alpha}-negative cases (82.4% versus 51.7%; P = 0.028). Noncancerous liver tissues with chronic hepatitis or cirrhosis showed no immunostaining (Fig. 3).



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  		Fig. 3. Immunohistochemistry labeling of PDGFR{alpha} and CD34 in human hepatocellular carcinoma specimens. Red, PDGFR{alpha} expression; blue, CD34 expression; purple, coexpression of CD34 and PDGFR{alpha}. Note that CD34 was expressed in the vessel of tumor (A) and noncancerous liver tissue (D), whereas PDGFR{alpha} was positive in some of tumor vasculature (B and C) but not in noncancerous liver tissue (D). Bar, 80 µm.

 
We then analyzed the correlations between PDGFR{alpha} expression and histopathologic variables. PDGFR{alpha} was significantly related to recurrence in the microvessel invasion-absent group. Among 48 cases without microvessel invasion, PDGFR{alpha}-positive cases presented more frequently with recurrence when compared with PDGFR{alpha}-negative cases (80% versus 36.8%; P = 0.029; Table 3).


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  		Table 3. PDGFR{alpha} expression in patients with or without microvessel invasion

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we compared the gene expression profiles of TECs isolated from hepatocellular carcinoma tumors with different metastatic potentials and identified PDGFR{alpha}, one of the differentially expressed genes, related with higher metastatic potential of hepatocellular carcinoma.

It is widely accepted that heterogeneity of tumor vessels does exist. Several specific markers of tumor vascular endothelium have been found in different tumors, a finding that promises a molecular basis for vessel-targeted therapy (12, 34). As a main target of antiangiogenesis therapy, endothelial cells represent only a minor fraction of the total cell population within tumor tissue, and analysis of endothelial cells has long been considered a hard task due to the difficulty of isolation and purification (9, 14). In this study, we isolated endothelial cells from human hepatocellular carcinoma implanted in nude mice by CD31-conjugated magnetic beads followed by a series of verification steps to exclude contamination of tumor cell and leukocytes.

Because microenvironment plays an important role in modifying the endothelial cell phenotypes (35–37), it is not surprising to find dramatic changes in cultured endothelial cells at the transcription and protein levels, although these cells still maintain certain characteristics of endothelial cells (36, 38). A recent study revealed that even very short-term primary culture can lead endothelial cells to rapidly lose their specialized characteristics when they were isolated from their original microenvironment (39). In our study, the gene expression profile did change a lot when TECs were cultured for 5 to 7 days (data not shown). Therefore, we used freshly isolated endothelial cells to perform gene array analysis and subsequent verifications. No significant differences in terms of gene expression level within these groups were observed in most of the examined genes. This finding supports the assumption that endothelial cells, when compared with tumor cells, are genetically stable and less likely to accumulate mutations that allow them to rapidly develop drug resistance (35, 40). However, several differentially expressed genes were observed. The most interesting finding was the gene expression pattern of PDGFR{alpha} in TEC was consistent with metastatic potentials of hepatocellular carcinoma. As a member of the receptor tyrosine kinase family, PDGFR{alpha} is frequently activated in many cancers and has become an important target for their treatment (41). The role of PDGFR{alpha} in tumor angiogenesis is not clear, however, and little is known about the expression of PDGFR{alpha} in hepatocellular carcinoma. The highly positive correlation between its expression level and metastatic potential of hepatocellular carcinoma warrants further investigation.

Recent studies showed that tumor-associated endothelial cells seemed to express phosphorylated PDGFR when they were exposed to tumor cells that express PDGF (32), and PDGFR{alpha} in turn could control the capacity of tumor cells to generate metastases (42). Blocking PDGFR signaling produced substantial therapeutic effects against cancer metastasis (32, 43). In the present study, treatment with STI571, a protein tyrosine kinase inhibitor of PDGFR, was associated with a decrease in tumor size and MVD in mice bearing highly metastatic hepatocellular carcinoma, and a synergistic effect was observed when STI571 and IFN-{alpha} were used in combination. Moreover, this combined treatment was highly effective in preventing lung metastasis. Our previous studies showed that IFN-{alpha} inhibited growth and metastasis in a human hepatocellular carcinoma nude mice model, which may be partly mediated by antiangiogenesis through down-regulation of vascular endothelial growth factor (22, 44). The administration of STI571 alone or STI571 plus IFN-{alpha} did not produce a significant in vitro antiproliferation effect on hepatocellular carcinoma tumor cells. Furthermore, PDGFR{alpha} was only detected in TEC of tumor at the transcript and translation levels. Therefore, STI571 seems to inhibit the hepatocellular carcinoma tumor growth by antiangiogenesis, at least in part, through inactivation of PDGFR{alpha} exclusively expressed in TEC. The significant synergistic effect of STI571 and IFN-{alpha} in vivo indicates that these two drugs may have different targets in the treatment of hepatocellular carcinoma.

Our immunohistochemical analysis on human hepatocellular carcinoma specimens further revealed PDGFR{alpha} expression associated with recurrence of hepatocellular carcinoma. Notably, PDGFR{alpha} was relatively specific for predicting the recurrence of those low-risk patients who did not have microvessel invasion (62.3% of all patients).The recurrence rate in these patients was as high as 45.8%. By analyzing PDGFR{alpha} expression, we identified 36.4% (8 of 22) recurrent cases in this group of patients. Considering the fact that currently there is no such predicative marker for these patients, this result is intriguing and should initiate further investigation.

The results of the current study suggest that differential gene expression does exist in endothelial cells derived from hepatocellular carcinoma with different metastatic potentials. These differentially expressed genes provide a valuable resource for basic and clinical studies of angiogenesis in hepatocellular carcinoma. The relationship between PDGFR{alpha} expression and hepatocellular carcinoma metastatic potential suggests that PDGFR{alpha} may be a predicative biomarker and therapeutic target for highly metastatic hepatocellular carcinoma. The mechanism of PDGFR{alpha} expressed in TEC induced by highly metastatic hepatocellular carcinoma needs to be investigated further.


   Footnotes
 
Grant support: National Natural Science Foundation of China grant 30300400, State Key Basic Research Program of China grant G1998051210, and Foundation of Key Laboratory of Carcinogenesis and Cancer Invasion Grant, Fudan University, Ministry of Education, People's Republic of China.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: T. Zhang and H-C. Sun contributed equally to this work.

Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

Received 5/ 2/05; revised 9/14/05; accepted 9/26/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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P. Stock, D. Monga, X. Tan, A. Micsenyi, N. Loizos, and S. P.S. Monga
Platelet-derived growth factor receptor-{alpha}: a novel therapeutic target in human hepatocellular cancer
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