Tetraspanin co029 expression as a tumor biomarker for monoclonal antibodies preparation: antigenic assessment in colorectal cancer cells

The coding sequence of human TSP co029 gene (NCBI Reference Sequence, NM_001369760.1, NP_001356689) for a protein of 237 amino acids was obtained from HT29 human colorectal cancer cells (BCRJ).

Primers were designed with attB sites for cloning on Gateway^® system for a N-terminal histidine Tag, according to the manufacturer instructions (Figure 1).

The entry clone was obtained by inserting the gene of interest in the pDNOR plasmid by a BP recombination reaction at 25°C for 16h. Escherichia coli TOP10 (Novagen) bacteria was transformed by thermal shock. Positive colonies selected with specific primers for the gene of interest by PCR were cultured in Luria-Bertani (LB) medium supplemented with 50μg/mL of kanamycin, and the plasmid was purified with plasmid DNA Miniprep Kits (invitrogen), following the instructions. Subsequently, the entry clone containing the gene of interest flanked by the attL sites was combined with pEXP1-DEST vector by LR recombination reaction at 25°C for 16h, generating the final expression clone. Competent E. coli BL21AI (Novagen) bacteria was transformed by thermal shock and cultured in LB medium supplemented with 100μg/ml ampicillin at 37°C for 12h. Gene sequencing was performed after each stage using Sanger sequencing technique in order to confirm the correct insertion of the gene.

One liter of bacteria was monitored by a 600nm spectrophotometer for 0.4-0.6 optimum optical density (OD) and the protein synthesis was induced by the addition of 1mM of isopropyl β-D-thiogalactoside (IPTG) at 37°C for 4h under orbital rotation (225rpm). The bacterial pellet was centrifuged at 12,000g for 45min at 4°C when pellet resuspended in 40mL of lysis buffer [50mM Tris, 0.5M NaCl, 0.2mM EDTA, 3% sucrose, Triton-X 1% and 10mM imidazole] plus protease inhibitors [200ug/ml lysozyme, 1mM PMSF, 20ug/ml DNAse]. Cells were sonicated on ice (3 cycles of 30s, 1min interval), and then centrifuged at 12,000g for 45min at 4°C. The supernatant and pellet were filtered through a 0.22μm filter. Recombinant protein was purified on a HisPur Ni-NTA chromatography cartridge nickel column, concentrated on a 10kDa tube (Vivaspin Millipore) and quantified by a BCA kit (ThermoFisher) according to the manufacturer's instructions.

Recombinant TSP co029 protein production and purification were confirmed by SDS PAGE and western blotting methods. Five ug of the purified TSP co029 protein were add to 12% polyacrylamide gels for Coomassie R-250 (Biorad) staining and PVDF membrane transfer. PVDF membrane was then incubated with a his-tag primary monoclonal antibody (Invitrogen) (1:2,000) and consequently with anti-IgG HRP antibody (Santa Cruz) (1:5,000). Immunoreactive bands were visualized using western blotting ECL kit (GE Healthcare Life Science) according to the manufacturer's instructions and image was captured by Digital Image System LAS 4000 (GE Healthcare Life Science).

Based on Kohler and Milstein (1975) methodology,18 two nine-week-old female BALB/c mice were subcutaneously immunized with 20ug of recombinant TSP co029 protein diluted on 200ul of VacSIM® as adjuvant (EnduraVax).21 A protocol of six inoculations two weeks apart was concluded. Sera were collected immediately after each inoculation for the determination of specific antibodies titers by ELISA. A final boost was intraperitoneally performed and, three days later, the spleen cells were fused with Sp2/0 IL-6 myeloma cells (ATCC) using 37°C PEG/DMSO solution with a 1min incubation. The fused cells were cultured and selected with hypoxanthine-aminopterin-thymidine (HAT) medium and hypoxanthine-thymidine (HT) medium (Sigma) for 7 days on a 37°C 5% CO2 incubator. The initial screen of positive growth wells was done by ELISA. TSP co029 recombinant protein was diluted 10μg/ml in coating buffer [0.05M carbonate-bicarbonate, pH 9.6] and microtiter plates MaxiSorp Surface (NUNC Thermo Scientific) were coated at 4°C for 16h. After blocking, 100μl of culture supernatants of the HAT/ HT-selected hybridomas were added and incubated for 1h. The presence of specific antibodies was detected with peroxidase-conjugated anti-mouse IgG (1:5,000) (Santa Cruz) and TMB (Sigma) under a 450nm spectrophotometer (ThermoScientific). ELISA positive hybridomas were selected for expansion. The selected clones (3.E211B) for mAbs and (3.E2) for pAbs were grown in DMEM (Invitrogen) supplemented with 10% FBS, penicillin (100U/ml) and streptomycin (100mg/ml) (Invitrogen) and insulin-transferrine-sodium selenite (ITS). Culture supernatants were harvested and used for ammonium sulfate precipitation).22 Precipitated proteins were dialyzed against PBS at 4°C. mAbs were purified by two extra protocols including a G protein purification column (Sigma) and an anionic exchange chromatography column HiTrap Q HP (GE Healthcare). At the end, mAbs were concentrated by a 50kDa tube (Vivaspin Millipore), quantified by a BCA kit (ThermoFhisher) and stored at 4°C until use.

Aliquots of the mAbs were conjugated to Alexa Fluor 647 with Fluorochrome Protein Labeling Kit (Invitrogen), according to the manufacture instructions. As described before, pAbs and mAbs production and purification was confirmed by SDS PAGE. Antibodies were added on a 10% polyacrylamide gel stained by coomassie blue.

For the evaluation of the effects of mAbs, human colorectal cancer HT29 cells were plated at a density of 2x10⁵ cells/well in 96-well flat-bottom plates together with 0.10 and 0.20mg/ml of 3.E211B anti-TSP co029-Alexa Fluor 647 conjugated mAb. Incubation was performed for 6h and 16h at 37°C.

Cells were examined on fluorescent microscope (Karl Zeiss AxioStar Plus) and on laser confocal microscope (Nikon C2) at 640-642nm, emission filter LP590. Photographic records were taken with a digital camera and analyzed by Software NIS-Elements (Nikon). Z- Stack Capture mode was used for immunolocalization of staining. Control cultures without mAbs were observed in parallel to exclude possible effects of the antibodies on the cells.

Crude extract of human colon adenocarcinoma HT29 cells was initially prepared. Cells were cultivated in supplemented DMEM medium (20% FCS and 1% penicillin) and after reaching confluence; they were released by trypsin, centrifuged at 200xg for 4min, and resuspended in non-supplemented medium. The cell suspension was sonicated at 40% amplitude, in 5 cycles of 15sec of sonication and 30sec interval, on ice. Subsequently, the cell lysate was centrifuged for 10min to cell debris removal and the cells extract were resuspended in PBS or DMEM.

Basically, 96-well microtiter plates MaxiSorpTM Surface (NUNC) were sensitized with 10ug of recombinant TSP co029, HT cells extract in PBS or HT29 extract in DMEM diluted in buffer 0.05M carbonate-bicarbonate pH 9.6 in triplicate for 16 hours at 4°C. The plates were washed three times with 0.15M phosphate buffer saline pH 7.2 with 0.05% of Tween 20 (LGC Biotechnbology) (washing buffer) and, the non-specific sites were blocked with 2.5% casein protein in washing buffer at 37°C for an hour. After new washing steps, 100μl of mAbs anti-TSP co029 (containing 10ug of antibodies) were added into each well and the plates were incubated at room temperature for an hour. Following, the plates were submitted to washing steps and incubated at room temperature for an hour with conjugated anti-IgG murine antibody (Santa Cruz Biotechnology) diluted 1:10,000 in washing buffer. The plates were washed again and 100ul of substrate TMB (Thermo Fisher Scientific) were added to each well. The reaction was stopped after 20 minutes of incubation in the dark by addition of 50ul/ well of 2N sulfuric acid. The results were obtained as absorbance values at 450nm in microplate reader (BioRad Laboratories 3550). Negative controls were done by using recombinant proteins from Schistosoma mansoni parasite with no similarity to TSP co029: Circulating Cathodic Antigen (CCA, PubMed accession number O02197.1) and Major Egg Antigen (MEA, PubMed accession number AAA29903.1).

Four μm thick histological sections were prepared for immunohistochemical reactions. Novolink Polymer Detection System (Leica Biosystems) anti-mouse/anti-rabbit detection kit was used according to the manufacturer's instructions. For the recovery of TSP co029 receptor antigens, steam heat (Pascal^®) with Dako Cytomation Target Retrieval Solution (Dako) pH 6.0 citrate was used.

Briefly, the slides with histological sections were incubated with the appropriate primary antibody (1:100) for 16h in a humid chamber at 4°C. The immunoreactivity was visualized with the chromogen 3'-diaminobenzidine (DAB) substrate system (Dako) and contrasted with the Mayer's hematoxylin. Colorectal cancer positive tissue fragment samples were used as positive controls for the reactions. For negative controls, the primary antibody was replaced with phosphate-buffered saline (PBS). The slides were comparatively analyzed by AxioVision and ImageJ software, for morphometric characterization and classification of protein expression.

The absorbance values were analyzed with Minitab software by the Kolmogorov-Smirnov normality test. Normally distributed data were analyzed by Student's t-test using p<0.05 as the significance level.

The TSP Co029 gene was expressed in E. coli BL21AI, with 6x his tag at 5′ ends. The optimum condition for expression was achieved after 4 h induction by IPTG (1mM), at 37°C and OD 600 of 0.6. Purification of the recombinant protein was carried out, and SDS PAGE analysis revealed as a major band (~31kDa) in the induced fractions (Figure 2). The approximate yield of purification was approximately 1.63mg/ml culture.

Western blotting analyzes using anti-His-Tag monoclonal antibody confirmed the recombinant protein production (Figure 3).

The titers of antibodies against TSP co029 recombinant protein in the sera of immunized mice showed that all mice were immunized against the antigen, but in one mouse (number 1) increase of antibody titer was more than other and this mouse had higher anti-TSP co029 antibody (OD>2.0) (Figure 4) and was selected for the fusion.

The fusion of immunized mice splenocytes and myelomas yielded 105 HAT-resistant hybridoma pre clones producing specific pAbs. Ten pre clones with high TSP co029 binding, determined by ELISA, were stored in liquid nitrogen and 1 (3.E2) was submitted to cloning step. A new ELISA determined 40 positive clones. Seven clones secreting specific mAbs were also expanded and stored in liquid nitrogen. mAbs were then isotyped and the characterization of the selected pAbs and mAbs specifically reacting with TSP co029 recombinant protein is summarized in Table 1 (supplemental file). Final yield of 4.6mg/ml was obtained for pAbs and 1.1mg/ml was obtained for mAbs.

Human colorectal cancer HT29 cells incubated with different concentrations of 3.E211B anti-TSP co029-Alexa Fluor 647 conjugated mAb showed membrane staining of all viable cells after 6h (Figure 5) and 16h on fluorescent microscopy. Results from both incubation times were the same. Furthermore, cellular localization of TSP co029 was visualized by confocal fluorescence microscopy (Figure 6). The HT29 cells treated with 3.E211B anti-TSP co029-Alexa Fluor 647 conjugated mAb demonstrated cell membrane labeling.

Z-Stack Series mode analysis confirmed the exclusive membrane staining with no fluorescent moieties in the cytoplasm of the cells, as can be confirmed in supplementary material section.

Figures 7 and 8 present the specificity of produced mAbs anti-TSP co029 respectively by ELISA and immunohistochemistry assays. mAbs were able to detect the recombinant TSP co029 and TSP co029 presented in HT29 cells extracts by ELISA (p<0.05) when comparing to the blank and negative controls.

Immunohistochemistry analyses using fragments of tissue extracted from a colorectal cancer patient showed the stained tumor cells when mAbs anti-TSP co029 were used.

Research has endeavored to identify innovative biomarkers to initiate their use on cancer diagnosis and clinical management, in order to help physicians and the health team in choosing the best individual option of chemotherapy treatment. In this work, we identified a transmembrane protein called tetraspanin co029 by producing specific antibodies in the laboratory. Using high technology microscopies, we were able to bind our specific antibodies to this protein on the surface of colorectal cancer cells extracted from adult patients.