[Application of iPDMS protein microarray in screening of tumor-associated antigen autoantibodies].

The rapid screening of tumor markers is a challenging task for early diagnosis of cancer. This study aims to use highly sensitive chemiluminescent protein microarray technology to efficiently screen a variety of low abundance tumor related markers. A new material, termed integrated polydimethylsiloxane modified silica gel (iPDMS), was obtained by adding a surface polymerization initiator with olefin end to the conventional polydimethylsiloxane, and fixing into the three-dimensional structure of polydimethylsiloxane by thermal crosslinking through silicon hydrogen bonding. In order to make the iPDMS material resistant to non-specific protein adsorption, a poly(OEGMA) polymer brush was synthesized by surface-initiated atom transfer radical polymerization at the active initiation site. Finally, 20 tumor-related antigens were printed into the specific areas of the microarray by high-throughput spray printing technology, and assembled into 48-well detection microtiterplates of the iPDMS microarray. It was found the VEGFR and VEGF121 autoantibodies that obtained from 8 common tumors (breast cancer, lung cancer, colon cancer, gastric cancer, liver cancer, leukemia, lymphoma and ovarian cancer) can be used as potential tumor markers. The chemiluminescence labeled iPDMS protein microarray can be used for the screening of tumor autoantibodies at early stage.

Application of iPDMS protein microarray in screening of tumor-associated antigen autoantibodies / 生物工程学报

The rapid screening of tumor markers is a challenging task for early diagnosis of cancer. This study aims to use highly sensitive chemiluminescent protein microarray technology to efficiently screen a variety of low abundance tumor related markers. A new material, termed integrated polydimethylsiloxane modified silica gel (iPDMS), was obtained by adding a surface polymerization initiator with olefin end to the conventional polydimethylsiloxane, and fixing into the three-dimensional structure of polydimethylsiloxane by thermal crosslinking through silicon hydrogen bonding. In order to make the iPDMS material resistant to non-specific protein adsorption, a poly(OEGMA) polymer brush was synthesized by surface-initiated atom transfer radical polymerization at the active initiation site. Finally, 20 tumor-related antigens were printed into the specific areas of the microarray by high-throughput spray printing technology, and assembled into 48-well detection microtiterplates of the iPDMS microarray. It was found the VEGFR and VEGF121 autoantibodies that obtained from 8 common tumors (breast cancer, lung cancer, colon cancer, gastric cancer, liver cancer, leukemia, lymphoma and ovarian cancer) can be used as potential tumor markers. The chemiluminescence labeled iPDMS protein microarray can be used for the screening of tumor autoantibodies at early stage.

Expression and Characterization of Human Vascular Endothelial Growth Factor Produced in SiHa Cells Transduced with Adenoviral Vector.

The vascular endothelial growth factor (VEGF) is an essential factor to pathologic angiogenesis. Disruption of VEGF/VEGF receptor interaction in cancer patients inhibits the development of new and pre-existing tumor blood vessels. Consequently, VEGF becomes an important therapeutic target for handling solid tumors. In this work, human VEGF was produced in the culture supernatant of SiHa cells transduced with a replication-defective adenoviral vector (pAdhVEGF121) encoding this molecule. The 35 kDa VEGF121 homodimer was obtained from clarified culture media as a glycosylated protein. VEGF121 expression levels were strictly dependent on the adenoviral viral load used. VEGF121 was produced with purity over 98% after a single step chromatography by immobilized metal affinity chromatography. Additionally, VEGF121 binds Bevacizumab antibody with a KD of 7 nM. Biological characterization by mitogenic assay in HUVEC and ECV-304 cells showed that VEGF121 stimulates cell proliferation in a dose-dependent manner in both cells. Finally, the neovascularization activity of VEGF121 was demonstrated by vascular permeability assays in matrigel plug-bearing mice, showing significantly increased vasculature leakage after treatment with VEGF121. Consequently, transduction of SiHa cells with adenovirus is a suitable alternative for manufacture heterologous proteins of therapeutic interest.

Expression and purification of functional human vascular endothelial growth factor-a121; the most important angiogenesis factor.

PURPOSE: Angiogenesis or formation of new blood vessels is an essential process for tumor growth, invasion and metastasis. Vascular Endothelial Growth Factor (VEGF) and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important factor in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, 145, 165, 189 and 206 amino acids (named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206). METHODS: In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl ß-D-1-thiogalactopyranoside) and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells) cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated. RESULTS: SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro. CONCLUSION: Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay.

68Ga-NODAGA-VEGF121 for in vivo imaging of VEGF receptor expression.

PURPOSE: Vascular endothelial growth factor (VEGF) is a crucial regulator of angiogenesis. In this study, we labeled VEGF121 with (68)Ga using a hydrophilic chelating agent, NODAGA and evaluated the resulting (68)Ga-NODAGA-VEGF121 for in vivo imaging of VEGF receptor (VEGFR) expression. METHODS: NODAGA-VEGF121 was prepared and its binding affinity for VEGFR2 was measured using (125)I-VEGF121. (68)Ga-NODAGA-VEGF121 was prepared by labeling NODAGA-VEGF121 with (68)GaCl3 followed by purification using a PD-10 column. Human aortic endothelial cell (HAEC) binding studies of (68)Ga-NODAGA-VEGF121 were performed at 37°C for 4 h. MicroPET imaging followed by biodistribution studies were performed in U87MG tumor-bearing mice injected with (68)Ga-NODAGA-VEGF121. Immunofluorescence staining of the tumor tissues was performed to verify VEGFR2 expression. RESULTS: Binding affinity of NODAGA-VEGF121 for VEGFR2 was found to be comparable to that of VEGF121. (68)Ga-NODAGA-VEGF121 was prepared in 47.8% yield with specific activity of 3.4 GBq/mg. (68)Ga-NODAGA-VEGF121 was avidly taken up by HAECs with a time-dependent increase from 9.88 %ID at 1 h to 20.86 %ID at 4h. MicroPET imaging of mice demonstrated high liver and spleen uptake with clear visualization of tumor at 1h after injection. ROI analysis of tumors revealed 2.53 ± 0.11 %ID/g at 4 h after injection. In the blocking study, tumor uptake was inhibited by 29% at 4 h. Subsequent biodistribution studies demonstrated tumor uptake of 2.38 ± 0.15 %ID/g. Immunofluorescence staining of the tumor tissues displayed high level of VEGFR2 expression. CONCLUSIONS: These results demonstrate that (68)Ga-NODAGA-VEGF121 led to VEGFR-specific distribution in U87MG tumor-bearing mice. This study also suggests that altered physicochemical properties of VEGF121 after radiolabeling may affect biodistribution of the radiolabeled VEGF121.