Identification of Soluble NH₂-Terminal Fragment of Glypican-3 as a Serological Marker for Early-Stage Hepatocellular Carcinoma

Serum Samples.

Serum samples were collected at Tokyo University Hospital with informed consent from 69 patients with HCC and 38 patients with LC, defined according to the following criteria: patients with a pathological diagnosis of HCC after surgery or with evidence of tumor stain on computed tomography or angiography were diagnosed with HCC; and patients diagnosed with LC were limited to those who had no history of HCC and no ultrasound evidence of tumor for more than 6 months from the day of serum collection.

Purification of Recombinant GPC3 Proteins.

For protein expression, we used modified pCXN vector that contained dihydrofolate reductase expression unit as a selection marker. Original pCXN vector (20) was generously provided by J. Miyazaki (Osaka University Medical School, Osaka, Japan). An expression vector for GPC3 that lacks the COOH-terminal hydrophobic glycosylated phosphatidylinositol (GPI)-anchoring domain, GPC3ΔGPI, was constructed by introducing cDNA corresponding to amino acid residues 1–563 of GPC3 into modified pCXN with a FLAG tag added at the COOH terminus. An expression vector for GPC3ΔGPI without heparan sulfate, GPC3ΔGPIΔHS, was constructed by changing Ser⁴⁹⁵ and Ser⁵⁰⁹ to Ala to abolish the heparan sulfate attachment site. These constructs were stably transfected into Chinese hamster ovary cells deficient in the dihydrofolate reductase gene. Culture media containing GPC3ΔGPI-FLAG or GPC3ΔGPIΔHS-FLAG recombinant proteins were collected and loaded to DEAE ion-exchange chromatography DEAE Sepharose FF (Amersham Bioscience, Tokyo, Japan). After washing, eluted protein solutions were applied to anti-FLAG M2 antibody beads (Sigma, St. Louis, MO). Proteins eluted with solution containing 200 μg/ml FLAG peptide (Sigma) were subjected to gel filtration chromatography with HiLoad 26/60 Superdex200pg (Amersham Bioscience). Finally, recombinant protein was concentrated using DEAE Sepharose FF.

Generation of Anti-GPC3 mAbs.

We used recombinant GPC3ΔGPI as an immunogen. Spleen cells were isolated and fused with mouse myeloma P3-X63Ag8U1 cells (American Type Culture Collection, Manassas, VA). Hybridomas were selected by ELISA against the purified recombinant GPC3ΔGPIΔHS-FLAG, followed by cloning with limited dilution. Three mouse mAbs (A1836A, M18D04, and M19B11) were used in this study. For epitope mapping of these mAbs, a pGEX-5X (Amersham Biosciences) construct for the NH₂-terminal portion of GPC3 (amino acids 25–358) was expressed in Escherichia coli BL21 Codon Plus (DE3) pLys (Stratagene, La Jolla, CA) as a glutathione S-transferase-fusion protein and subject to immunoblotting analysis.

Immunoblotting.

Total cell lysates were obtained after lysis in 10 mm Tris (pH 7.4), 150 mm NaCl, 5 mm EDTA, 1.0% Triton X-100, 1.0% sodium deoxycholate, and 0.1% SDS with protease inhibitor mixture (Sigma). Culture supernatant was obtained from serum-free medium used for culture of hepatoma cells. Proteins were separated with 12% SDS-PAGE and transferred to polyvinylidene difluoride Hybond P membrane (Amersham Biosciences). The membrane was treated with 2% nonfat milk in TBS containing 0.05% Tween 20 (TBST) followed by incubation with anti-GPC3 mAb in TBST and subsequent incubation with horseradish peroxidase-conjugated secondary antibody (dilution, 1:5000; Amersham Biosciences) in TBST. The protein was visualized using the enhanced chemiluminescence plus detection system (Amersham Biosciences).

Immunoprecipitation.

We first prepared antibody beads by covalently linking 25 μl of protein G-Sepharose (Amersham Biosciences) and 50 μg of anti-GPC3 mAb M18D04 or M19B11 with 20 mm dimethyl pimelimidate (ICN Aurora, Aurora, OH). We then added 50 μl of sera from the patients or culture media of HuH7 cells diluted in 250 μl with PBS to 25 μl of antibody beads and incubated them for 2 h at 4°C. After extensive washing with PBS, antibody beads were boiled for 5 min in 50 μl of SDS-PAGE loading buffer containing 10% 2-mercaptoethanol, and subsequently, immunoblotting was performed.

Sandwich ELISA.

One μg of anti-GPC3 mAb A1836A per well was immobilized to 96-well plate Maxisorp (Nalge Nunc International, Roskilde, Denmark) and stabilized with Immunoassay Stabilizer (Advanced Biotechnologies Inc., Columbia, MD). Twenty-five μl of sera or standard were diluted with 100 μl of buffer containing 20% normal rabbit serum (Pel-Freez Biologicals, Rogers, AR), 1% BSA (Oriental Yeast Co., Ltd., Osaka, Japan), and 2% mouse ascites Hyb-3423 (Institute of Immunology, Tokyo, Japan) in 50 mm Tris-Cl (pH 8.0), 0.15 m NaCl, and 1 mm EDTA and incubated at room temperature for 2 h. After washing, 25 μl of biotinylated antibody solution containing anti-GPC3 mAbs M18D04 (1.88 μg/ml) and M19B11 (3.75 μg/ml) and 100 μl of horseradish peroxidase-labeled streptavidin (Vector Laboratories Inc., Burlingame, CA) were added to the plate and incubated twice at room temperature for 30 min. TMB Soluble Reagent and Stop Buffer (Scy Tek Laboratories, Inc., Logan, UT) were added as substrate, and absorbance at 450 nm was read with EIA Reader (Corona Electric Co., Ltd., Ibaraki, Japan). Recombinant GPC3ΔGPI was used as a standard sample in each assay.

Amino Acid Sequence Analysis.

Recombinant GPC3ΔGPI and GPC3ΔGPIΔHS were purified and separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane ProBlott (Applied Biosystems, Foster City, CA). The membrane was stained with CBB R-250, and sections containing bands of M_r 40,000 and M_r 30,000 were cut out separately. These polyvinylidene difluoride membrane sections were washed with a solution including 50% acetonitrile and 0.1% trifluoroacetic acid and applied to an ABI 492 Protein Sequencer (Applied Biosystems) to sequence the NH₂ terminus of the protein. Because the NH₂ terminus of the M_r 40,000 protein was blocked, the membrane was further incubated in acetate with 0.6 mg/ml 3-bromo-3-methyl-2-nitrophenyl-mecapto-3H-indole (ICN Biomedicals Inc., Irvine, CA) at 80°C for 1 h in the dark to chemically cleave the protein at the COOH terminus of tryptophan residues. After washing twice with 80% acetate and once with 10% methanol, the peptide was analyzed using an ABI 492 Protein Sequencer. The detected sequence was aligned using FASTS software available online, 9 and the protein was identified.

The NH₂-Terminal Portion of GPC3 Is Cleaved between Arg³⁵⁸ and Ser³⁵⁹in Vitro.

We have previously generated mAb K6534 raised against a peptide corresponding to amino acids 355–371 of GPC3 protein, and we demonstrated, for the first time, overexpression of its core protein in HCC with immunoblotting using this antibody (15) . Another antibody is required to construct a sandwich ELISA system for serum examination of GPC3, so we started generating high-affinity mAbs using recombinant GPC3ΔGPI as an immunogen. While purifying the immunogen from the culture supernatant of Chinese hamster ovary cells, we observed a M_r 40,000 band (Fig. 1A) ⇓ in addition to the M_r 66,000 band that corresponds to core protein of GPC3 as observed with K6534 (15) .

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Fig. 1.

Characterization of recombinant glypican-3 (GPC3) proteins. A, CBB R-250-stained SDS-PAGE of purified recombinant GPC3. Brace, glycanated GPC3 (smearing), GPC3ΔGPI; closed arrowhead, core protein of GPC3 (M_r 66,000) that lacks heparan sulfate glycosaminoglycan, GPC3ΔGPIΔHS; open arrowhead, sGPC3 (M_r 40,000); arrow, cGPC3ΔGPIΔHS (M_r 30,000). B, schematic diagram of recombinant proteins. Numbers above the boxes indicate amino acid residue number. Note that NH₂-terminal residue 25 is putative and indicated in parentheses. HS, heparan sulfate glycosaminoglican.

Because the NH₂ terminus of this M_r 40,000 band was modified, as revealed by initial amino acid sequencing, we performed sequencing of internal amino acids of the band after cleavage at the COOH terminus of tryptophan residues to verify its origin. We detected six cycles of three amino acid residues VRY, EPX, YES, ITY, LPX, and QSV, each cycle corresponding to the first to sixth residue following tryptophan (W), respectively. After alignment with FASTF algorithm, these sequences matched with the (W)VPETPV (amino acid 51–57), (W)YCSYCQ (amino acid 261–267), and (W)REYILS (amino acid 296–302) partial sequences of GPC3, respectively, indicating that this band is derived from an NH₂-terminal portion of GPC3. We designated this soluble cleaved fragment of GPC3 as sGPC3.

To further characterize sGPC3, we next tried to precisely identify the undetermined cleavage site by sequencing the residual COOH-terminal portion of GPC3 (designated cGPC3). However, the corresponding band was not visible by SDS-PAGE, presumably due to attachment of heparan sulfate glycosaminoglycan, leading to smearing (Fig. 1A) ⇓ . After substituting the two heparan sulfate attachment sites of the expression construct and purifying the resultant GPC3ΔGPIΔHS, we could observe a band of M_r 30,000, as expected (Fig. 1A) ⇓ . The NH₂-terminal sequence of this band was identified as SAYYPEDLF, identical to amino acids 359–367 of GPC3. Thus, the cleavage site was identified as being between Arg³⁵⁸ and Ser³⁵⁹ (Fig. 1B) ⇓ . We do not have precise information on the NH₂-terminal sequence of sGPC3 due to modification, but considering that amino acid 1–24 is a putative signal sequence, sGPC3 is likely to consist of amino acids 25–358 with an estimated molecular weight of 38,100, consistent with the M_r 40,000 band observed in SDS-PAGE (Fig. 1A) ⇓ .

Soluble GPC3 Is a Major Form of GPC3 Specifically Detected in the Sera of Patients with HCC.

We succeeded in generating a number of high-affinity mAbs specific for GPC3 and classified these antibodies into two groups, N-mAbs and C-mAbs, according to their epitopes within amino acids 25–358 or 359–563, respectively (data not shown). These antibodies could also recognize endogenous GPC3 protein in immunoblotting: core protein (M_r 66,000) and glycanated form (smearing) of GPC3 were detected by both N-mAbs and C-mAbs; whereas sGPC3 (M_r 40,000) was detected only by N-mAbs (Fig. 2A) ⇓ . An additional M_r 50,000 band was detected strongly in the cell lysate of HepG2 with both N-mAbs and C-mAbs (Fig. 2A) ⇓ . This band was only weakly detectable in HuH6 cells and was undetectable in five other hepatoma cell lines (Fig. 2, A and C ⇓ ; data not shown), suggesting cell-specific variations in the processing of the protein. In the culture supernatant, sGPC3, rather than a core protein or a glycanated form of GPC3, was the major form of GPC3 detected (Fig. 2A) ⇓ .

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Fig. 2.

Characterization of endogenous glypican-3 (GPC3) proteins with monoclonal antibodies (mAbs). A, representative immunoblotting of endogenous GPC3 in the cell lysate and culture supernatant with N-mAb and C-mAb. HepG2 and HuH6 were analyzed. Note that soluble GPC3 (sGPC3) alone is detected in the culture supernatant. Brace, glycanated GPC3 (smearing); closed arrowhead, core protein of GPC3 (M_r 66,000); open arrowhead, sGPC3 (M_r 40,000); arrow, uncharacterized processed fragment of GPC3 (M_r 50,000). Lys, lysate; Sup, supernatant of culture media. B, immunoblotting analysis with anti-GPC3 antibodies A1836A, M18D04, and M19B11 recognized glutathione S-transferase-sGPC3 but did not recognize glutathione S-transferase. C, detection of sGPC3 alone in the sera of the patients with hepatocellular carcinoma. Sera from two patients with hepatocellular carcinoma and three healthy adults (NL) were analyzed by immunoprecipitation with M18D04 followed by immunoblotting with A1836A. HuH7 cells were analyzed as a reference. Closed arrowhead, core protein of GPC3 (M_r 66,000); open arrowhead, sGPC3 (M_r 40,000).

Based on the above in vitro finding, we speculated that sGPC3, instead of core protein of GPC3, might be the major form of GPC3 in the sera of HCC patients. To avoid possible interference on immunoblotting by significant migration of albumin or immunoglobulin in the serum, we performed immunoprecipitation before immunoblottng using three N-mAbs (Fig. 2B) ⇓ . sGPC3 alone was successfully detected by immunoprecipitation with M18D04 (Fig. 2C) ⇓ or M19B11 (data not shown) followed by immunoblotting with A1836A in the sera of patients with HCC, but not in sera from normal liver (NL). These results clearly demonstrate that sGPC3 is the major diagnostic target specifically detectable in the sera of HCC patients.

Soluble GPC3 Is Useful as a Serological Marker of WD HCC and MD HCC.

We next constructed a sandwich ELISA system with these three antibodies to measure the serum level of sGPC3 (Fig. 3A) ⇓ . To verify the specificity of the assay, we performed immunoblotting of 10 sera samples from HCC with sGPC3 levels ranging from 4.0 to 55.0 ng/ml and 3 samples from NL with sGPC3 levels of <0.1 mg/ml. We detected only sGPC3 in all 10 HCC samples, whereas no band was detected in 3 samples from NL, indicating high sensitivity and specificity of the assay (Fig. 3B) ⇓ . When we examined sera from 69 cases with HCC, 38 cases with LC, and 96 cases with NL, the level of sGPC3 (mean ± SD) was 4.84 ± 8.91 ng/ml for HCC, 1.09 ± 0.74 ng/ml for LC, and 0.65 ± 0.32 ng/ml for NL and was significantly higher in HCC than in NL (P < 0.001, Student’s t test) or in LC (P < 0.01; Fig. 3C ⇓ ).

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Fig. 3.

Evaluation of soluble glypican-3 (sGPC3) as a serological marker of hepatocellular carcinoma (HCC). A, standard curve of sandwich ELISA. B, high specificity of sandwich ELISA. Specific detection of sGPC3 alone solely in the sera with elevated sGPC3 level measured with sandwich ELISA. Sera from 10 patients with HCC and 3 healthy adults (NL) were analyzed by immunoprecipitation with M18D04 followed by immunoblotting with A1836A. Serum sGPC3 level is indicated for each sample. Open arrowhead, sGPC3 (M_r 40,000); closed arrowhead, IgG. C, distribution of sGPC3 in the sera of patients with normal liver, liver cirrhosis (LC), and HCC (surgery and transcatheter arterial chemoembolization subgroup). Mean ± SD (ng/ml) of serum sGPC3 is indicated. Number of samples is indicated as n. D, receiver-operating characteristic curve analysis of sGPC3 (thick line) and α-fetoprotein (thin line). Top panel, all of the 69 HCCs and 38 cases of LC were included in the analysis. Bottom panel, 32 HCCs (including 7 well-differentiated and 25 moderately differentiated HCCs) and 38 cases of LC were analyzed. Area under the receiver-operating characteristic curve is indicated.

We then evaluated sGPC3 as a general marker for HCC in comparison with AFP. Initial analysis of the receiver-operating characteristic curve using the data from 69 cases with HCC and 38 cases with LC suggested that, used in isolation, sGPC3 is not as good as AFP: the calculated area under the receiver-operating characteristic curve was 0.729 for sGPC3 and 0.799 for AFP (Fig. 3D) ⇓ . The sensitivity and specificity of sGPC3 for the diagnosis of HCC (cutoff value, 2.0 ng/ml) were 51% and 90%, respectively, whereas those of AFP measured in parallel (cutoff value, 20 ng/ml) were 55% and 90%, respectively. AFP and sGPC3 were not correlated (r = 0.13), and combination measurement of both markers markedly improved sensitivity to 72%.

HCC may be divided into two subgroups correlating to the extent of disease: (a) one first treated by surgery, mainly with a solitary tumor or few tumors; and (b) the second treated with transcatheter arterial chemoembolization, mostly with multiple and advanced tumors. The serum level of sGPC3 was 2.61 ± 2.69 ng/ml for the former group, significantly higher than that for NL (P < 0.0001, Student’s t test) or LC (P < 0.01), and 8.36 ± 13.3 ng/ml for the latter group (Fig. 3C) ⇓ , suggesting that the serum level of sGPC3 is elevated in an earlier stage and rises as HCC progresses. We then evaluated sGPC3 as a marker for HCC in relatively early-stage disease. When 43 cases treated by surgery were confined to 32 cases with relatively early-stage HCC (7 cases with WD HCC and 25 cases with MD HCC), calculated areas under the receiver-operating characteristic curve for sGPC3 and AFP were 0.726 and 0.710, respectively, indicating that sGPC3 is superior to AFP (Fig. 3D) ⇓ . The sensitivity of sGPC3 and AFP for the diagnosis of WD HCC and MD HCC was 50% and 47%, respectively. Moreover, combination measurement of both markers in WD HCC and MD HCC also markedly improved sensitivity to 72%. These results clearly demonstrate the utility of sGPC3 as a serological marker for HCC, especially for relatively early-stage HCC, and its complementarity to AFP.