TA autoantibody, cell lines, tissues and human serum samples
The mouse B cell clones secreting a monoclonal autoantibody reactive to human hepatoma cells were selected from a B cell hybridoma pool constructed using hepatitis B virus X (HBx)-transgenic (tg) mice, as described previously [28]. The cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines originated from humans, except for HT22, which is a mouse hippocampal neuronal cell line. The cells were cultured in Dulbecco’s modified Eagle medium or RPMI-1640 (Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA). Mouse liver or HCC tissues used for western blotting or immunohistochemistry were obtained from Hras12V- or HBx-tg mice provided by Disease Model Research Laboratory of KRIBB [15, 29]. Exosome-containing conditioned media were prepared from 70 to 80% confluent cancer cells, which were cultured for 48 h with exosome-free fetal bovine serum. Human HCC serum samples for this study were provided by the Ajou Human Bio-Resource Bank (AHBB; Suwon, Korea), a member of the National Biobank of Korea (Additional file 1: Table S1). Normal human serum samples (Non-HCC) were provided by the Korean Red Cross (Seoul, Korea). Serum samples were kept at − 70 °C until use.
Identification of target antigen against XC24 TA autoantibody
XC24, an autoantibody secreted from one of our tumor model mouse B-cell clones, was analyzed in this study. The isotype of XC24 autoantibody was determined using a Pierce Rapid ELISA Mouse mAb Isotyping Kit (Thermo) as immunoglobulin (Ig) M. Identified XC24 autoantibody was purified from ascites fluid or hybridoma cell culture media using protein L-agarose (Thermo).
To examine the reactivity of XC24 autoantibody to human cancer cells, flow cytometric analysis and immunofluorescence microscopy were performed. For flow cytometric analysis, the suspended cells were fixed and permeabilized with BD cytoperm/cytofix solution (BD Bioscience, Franklin Lakes, NJ, USA), followed by an incubation with primary antibody solution and with goat anti-mouse IgG F(ab′)2-FITC. The stained cells were analyzed by FACScalibur (BD) and obtained data were analyzed using CellQuest software (BD). When determining whether the autoantibody-mimotope phage can compete with target cellular antigen for antibody binding, primary antibodies were pre-incubated with each phage at room temperature for 60 min and then used for staining.
To detect the XC24 antibody-specific antigen, western blot analysis of tumor cell lysates was performed. Cell lysates were prepared with NP40 lysis buffer [PBS (10 mM sodium phosphate and 150 mM NaCl, pH 7.4) containing 1% NP-40 and protease inhibitor cocktail (Sigma-Aldrich)]. For the proteomic analysis of the XC24 antigen, HepG2 cell lysates prepared with NP40 lysis buffer were immunoprecipitated with XC24 antibody-conjugated beads. Antibody-conjugated beads were prepared using immunoprecipitation kit (Thermo). The precipitated beads were resolved by 10% SDS-PAGE and analyzed by western blotting or Coomassie Blue staining. XC24 reactive antigen bands were then excised from SDS-PAGE gel and in gel-digested with trypsin (Trypsin Gold, Mass Spectrometry Grade; Promega, Fitchburg, Wisconsin, USA). The peptide extracts of in-gel digestion were analyzed by nano-liquid chromatography electrospray ionization–tandem mass spectrometry (nano-LC–ESI–MS/MS), as previously described [28].
To confirm the result of mass spectrometric analysis, HepG2 cells were transfected with AccuTarget™ siRNA against human SF3B1 or HYOU1 (Bioneer Corporation, Daejeon, Korea; si-SF3B1: sense 5′-CGA AGA UCG CCA AGA CUC A(dTdT)-3′, antisense 5′-UGA GUC UUG GCG AUC UUC G(dTdT)-3′; si-HYOU1: sense 5′-CAG AGA UGG ACC AGA UCU U(dTdT)-3′, antisense 5′- AAG AUC UGG UCC AUC UCU G(dTdT)-3′) using Lipofectamine RNAiMAX reagent (Thermo), and then reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were performed 72 h after transfection. AccuTarget™ Negative Contol siRNA (Bioneer) was used as negative control. Total RNA was extracted from cells using Qiagen RNA extraction kit (Qiagen, Hilden, Germany) and the first-strand cDNA was synthesized using Superscript III (Thermo). RT-PCR was performed using the following primer pairs (Bioneer; SF3B1: forward 5′-CTC GAG ATG GCT GCA TTG CGT CAG ATT AC-3′, reverse 5′-CTC GAG CAT TAC TGA GTC CTC TGT AGT-3′; GAPDH forward 5′-AGA GAC TGG AGC CAT TAC TTC-3′, reverse 5′-CAA CCT CAG CAG ACT GTG TG-3′). To further confirm whether XC24 antigen is SF3B1, the immunoprecipitated complex with anti-SF3B1 antibody (Bethyl Laboratories, Montgomery, TX, USA) was probed with XC24 antibody or vice versa.
For the immunofluorescence microscopy analysis, cells plated on 18 × 18-mm glass coverslips in 6-well plates were treated with BD cytofix/cytoperm solution to fix and permeabilize the cells. Then, cells were incubated with antibodies (XC24 or anti-SF3B1 antibody) diluted in BD cytoperm/wash solution (5 μg/mL) and with FITC-conjugated secondary reagents. The immuno-stained coverslips were mounted with Vectashield medium containing DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed using a Zeiss LSM510 Meta microscope (Carl Zeiss MicroImaging, Jena, Germany).
Subcellular localization of SF3B1 or XC24 antigen was determined by western blotting on subcellular fractionated cell lysates. Cells were washed twice with PBS and lysed using NE-PER nuclear and cytoplasmic extraction reagents (Thermo). Membranes were probed with one of the following antibodies: anti-Lamin B1 (nuclear marker; Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA), anti-GAPDH (cytoplasm marker, Santa Cruz), and anti-SF3B1 antibody or XC24 monoclonal autoantibody.
To evaluate the expression of SF3B1 in HCC tissues, cell lysates prepared using RIPA buffer as previously described [28] or tissue lysates prepared by homogenization and sonication were analyzed by western blot analysis. Exosomes collected from the cell culture media by ultracentrifugation [30] was resolved in RIPA buffer and analyzed by western blotting. The protein concentration was determined by the Bradford method (Bio-Rad, Hercules, CA, USA), and equal amounts of protein (50 μg) were resolved by SDS-PAGE and transferred onto a PVDF membrane (Millipore, MA, USA). The membranes were probed with XC24 monoclonal antibody or anti-SF3B1 antibody; β-actin was probed as a loading control (Abcam) and an exosome marker ALIX was detected as with anti-ALIX antibody (Millipore). Calexin, an endoplasmic reticulum marker, was probed (Abcam) as cell contamination marker. Positive bands were detected by horseradish peroxidase (HRP)-linked anti-mouse IgG/M/A antibody (Abcam) or other corresponding secondary reagents, followed by enhanced chemiluminescence reagents (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Band intensity was quantified using Image J (NIH, USA) and the relative intensity compared to β-actin was calculated.
For the staining of mouse tissue, 4-μm-thick sections were cut from the formaldehyde-fixed and paraffin-embedded tissue specimens and mounted on charged glass slides as described previously [15]. Tissue section samples were incubated overnight at 4 °C with anti-SF3B1 antibody. The sections were then incubated with HRP-conjugated secondary antibody for 30 min at room temperature, and 3,3′-diaminobenzidine (DAB) substrate chromogen solution was applied. Finally, the sections were counterstained with hematoxylin, dehydrated using graded alcohol and xylene series, and mounted with Permount (VWR International, Strasbourg, France). The photomicrographs were acquired at 200× or 400× magnification. DAB intensity was quantified using Image J (NIH) and plotted.
Evaluation of anti-SF3B1 autoantibody in human patients with hepatocellular carcinoma
We screened the conformational epitopes against XC24 antibody with the cyclic peptide library Ph.D.™-C7C [New England Biolabs (NEB), Ipswich, MA, USA] to use them as detection antigens of SF3B1 autoantibody instead of using a recombinant SF3B1 protein. Screening of conformational epitopes was already proven to be successful in the study of anti-FASN and anti-CK8/18 autoantibody biomarkers [28, 31]; Panning was repeated four times, and sequencing of selected mimotope phages was performed following the manufacturer’s instructions. To confirm the specificity of selected epitopes displayed on M13 phages, phage ELISA was performed [28]. Briefly, the ELISA plate (MaxiSorp™; Thermo) was coated with a selected phage (1010 pfu/well) in 0.1 M sodium carbonate buffer (pH 8.6). After the wells were blocked with protein-free blocking buffer (Thermo), primary antibody solution (100 ng purified XC24 or XC20 monoclonal antibody in 100 μl blocking buffer) was added and incubated at room temperature for 2 h. HRP-linked anti-mouse IgG/M/A antibody (Sigma-Adrich; 1:2000 diluted in blocking buffer) was used as a secondary reagent. Tetramethylbenzidine (TMB)-based substrate solution (Thermo) was used for color development. When evaluating the effect of cyclic conformation on peptide antigenicity, cyclic phages were reduced and alkylated and then coated onto the ELISA plates described previously [28]. For the competitive FACS analysis to confirm the specificity of selected epitopes, the primary antibody XC24 or XC20 was pre-incubated with the indicated epitope-displaying phages (indicated pfu/100 μl reaction) and used for cell staining. Competitive western blotting was also performed. The HepG2 or other cell lysates were resolved by SDS-PAGE and western blotted with primary antibody XC24 or XC20, which was pre-incubated with epitope-displaying phages (1011 pfu of indicated phages/100 μl reaction).
For the preparation of epitope-display coating antigens for human serum ELISA, we constructed XC24p11 epitope-fused streptavidin expression vector. The streptavidin-coding region was PCR-amplified from Streptomyces avidinii (ATCC 27419) and cloned into a NotI/XhoI-digested pET28a (+) vector. The DNA coding the cyclic peptide XC24p11 epitope sequence (-CDATPPRLC-) with restriction enzyme sites NdeI and NotI was synthetized (Bioneer; foward 5′-tatg ggt ggt gcg TGC GAC GCG ACC CCG CCG CGT CTG TGC ggt gga ggt tcg gcc-3′, reverse 5′-ggc cgc cga acc tcc acc GCA CAG ACG CGG CGG GGT CGC GTC GCA cgc acc acc ca-3′; underlined sequences correspond to XC24p11 epitope sequence) and cloned into the streptavidin-cloned pET28a (+) vector. The cyclic epitope gene was separated from the N-terminal His-tag and streptavidin gene by a linker encoding the amino acids GSGSA. DsbA, a bacterial thiol disulfide oxidoreductase (TDOR), was also cloned into the cyclic epitope-streptavidin cloned-pET28a (+) vector to catalyze the intrachain disulfide formation of the cyclic epitope. In addition, for the effective disulfide bond formation, SHuffle® T7 (NEB) was used as a host cell, which is an E. coli K12 strain suitable for T7 protein expression with enhanced capacity to correctly fold proteins with multiple disulfide bonds in the cytoplasm.
Transformants of E. coli strain SHuffle® T7 with XC24p11 epitope-fused streptavidin expression vector were grown overnight at 30 °C in 2× YT broth containing kanamycin (50 μg/ml). The culture was diluted 100-fold into fresh medium and grown in a shaking incubator at 30 °C. When the culture attained an absorbance at 600 nm of 4, isopropyl-β-d-thio-galactopyranoside (Sigma-Aldrich) was added to a final concentration of 1 mM, and incubation was continued overnight at 25 °C. Cells were harvested at 24–26 h post-inoculation by centrifugation, washed with TBS [10 mM Tris–HCl and 150 mM NaCl (pH 7.4)], and pelleted by centrifugation. The cells were resuspended in an ice-cold solution of 5% glycerol, 50 mM Tris–HCl (pH 7.4), and 2.0 mg/ml lysozyme, placed on ice for 30 min, and the cell suspension was sonicated to obtain the cell lysate. The lysate solution was centrifuged (10,000×g for 90 min, 4 °C) to pellet the cellular debris. The supernatant was decanted and filtered through a 0.2-μm filter. The lysate solution, to which NaCl and imidazole were added to a final concentration of 500 and 10 mM, respectively, was then applied over a Talon affinity column (Takara Bio, Mountain View, CA, USA) equilibrated in 50 mM Tris–HCl (pH 7.4) containing 0.25 M NaCl, 10 mM imidazole, and 5% glycerol. The column was washed with 10 column volumes of equilibration buffer and then eluted with an imidazole gradient up to 0.5 M in equilibrium buffer. The eluate fractions containing streptavidin antigen were pooled and concentrated using Amicon® Ultra Centrifugal Filters (Merck Millipore, Darmstadt, Germany) down to 1 ml. The concentrate was applied to a HiLoad 16/600 Superdex 200 prep grade column (GE) equilibrated with PBS containing 1 mM β-mercaptoethanol. Each fraction obtained by size-exclusion chromatography was analyzed by SDS-PAGE and western blotting, and fractions containing tetrameric epitope-fused streptavidin were pooled and used for further analysis and human serum ELISA.
The ELISA plate, MaxiSorp-, or biotin-coated plates (Thermo) were coated with the indicated amounts of epitope-fused streptavidin in 0.1 M sodium carbonate buffer (pH 8.6) overnight at 4 °C. After the wells were blocked with protein-free blocking buffer, XC24 primary antibody solution (containing the indicated amount of purified monoclonal antibody in 100 μl blocking buffer) was added and incubated at room temperature for 2 h. HRP-linked anti-mouse IgG/M/A antibody (Sigma-Aldrich; 1:2000 diluted in blocking buffer) was used as a secondary reagent. TMB solution was used for color development. For the detection of reactivity of patient sera to XC24p11-streptavidin, the MaxiSorp plates were coated with XC24p11 epitope-fused streptavidin at 500 ng/well, and after blocking with protein-free blocking buffer as described above, the plates were treated with albumin-depleted human sera (1:1000 diluted in blocking buffer) and detected by HRP-conjugated anti-human IgG/M/A antibody (1:2000 diluted in blocking buffer). Albumin depletion of the human serum was performed using Affi-Gel® Blue Gel following the manufacturer’s instructions (Bio-Rad). Empty-streptavidin (Eph) without a peptide epitope insert was used as control coating antigen. Alpha-fetoprotein (AFP) levels in human sera were evaluated with Human alpha-Fetoprotein Quantikine ELISA Kit (R&D systems, Minneapolis, MN, USA).
Statistical analysis
Data are presented as the mean ± SD. The two-tailed Student’s t-test was used to evaluate significance; p values < 0.05 were considered statistically significant. The sensitivity and specificity of anti-XC24p11 autoantibody or AFP for the diagnosis of HCC was evaluated using receiver-operating characteristics (ROC), leading the estimates of the area under the curve (AUC), with 95% confidence intervals. Statistical analysis was carried out using Prism 7 software (GraphPad Software, La Jolla, CA, USA). ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.