Isolation And Culture Of Human Lymphatic Endothelial Cells And The Experiment Study On The Molecular Imaging For VEGF Receptor-3 Binding Peptide Targeting Lymphangiogenesisa In Mouse With Ovarian Cancer

Background for research: Ovarian cancer is one of common genital malignant tumors for the female. It is severely malignant, easily infiltrates into the tissues nearby, and metastasizes through the lymph-vessel. Furthermore it has badly respond to the chemotherapy. So it is crucial to search a specific target of ovarian carcinoma-associated for diagnosis and therapy. Studies reveal that there are lymphangiogenesis in tumor tissue and around it. Tumors can actively induce the formation of lymphatic vessels and that tumor lymphangiogenesis is correlated with lymph node metastasis. So lymphangiogenesis plays an important role in the tumor metastasis. Vascular endothelial growth factor C (VEGF-C) and VEGF-D were the main lymphangiogenesis factors. They act predominantly via vascular endothelial growth factor receptor-3(VEGFR-3), which is expressed by lymphatic endothelial cells. VEGFR-3 activation promotes lymphatic endothelial cells (LECs) proliferation, migration, and survival via various molecular pathways. Clinical studies have shown VEGFR-3 can express in LECs of ovarian cancer. So it is possible to study tumor lymphatic capillary molecular imaging if VEGFR-3 act on as a target. Radionuclide trace technique is the most sensitive and specificity in all of tumor molecular imaging techniques. Recently, radionuclide label peptide used to image tumor. Peptide is smaller in molecular weight, shorter half life, stronger in penetrating power, quicker in clearance in vivo. So it is suit to using imagine study. We can obtain peptide by screening phage display peptide library.We screened and identified a VEGFR-3 binding peptide (SHSWHWLPNLRHYAS) using phage display technology in our early study. Now we selected lymphangiogenesis as studying target and studied if the peptide binding with HLECs. We also observed its ability to provide an accurate image of tumors lymphatic capillary in nude mice bearing ovarian tumors. We did so in order to provide a foundation for future studies the lymphangiogenesis and metastasize of ovarian cancers.Objectives: We sought to study the ability of the peptide specific binding with HLECs in vitro and performed preliminary studies examining the role of this peptide in imaging of tumor lymphangiogenesis with single photon emission computed tomography (SPECT) in ovarian-tumor-bearing nude mice.Materials and methods1. Isolation and characterization of LECs from human skin.The foreskins of children were obtained after routine circumcisions and cut into 1mm3 cubes. We digested dermal cubes with IA-S collagenase at 37°C, for 2 h. LECs were isolated and purified using a mouse anti-VEGFR-3 monoclonal antibody and goat anti-mouse IgG microbeads. Thereafter, HLECs were seeded onto 10-cm culture dishes coated with 2% gelatin and were incubated in EGM-2 with 20% fetal bovine serum and 10 ng/ml of recombinant human VEGF-C. Between 24-48 h after plating, HLECs colonies formed and were isolated using cloning cylinders and were then expanded. We used microscopy and transmission electron microscope to observe the cellular morphology and identified the immunophenotype of cells by immunofluorescence.2. Studying the peptide specific binding with HLECs in vitro.The peptide's ability to bind HLECs in vitro was detected using indirect immunofluorescence staining. The peptide was prepared by conventional solid-phase technology using an automatic peptide synthesizer. During synthesis, biotin was linked to the peptide. The process of the indirect immunofluorescence staining was performed in a similar manner as that described in Section 1, but we used 0.1μg/ml purified biotin-labeled peptide as the primary antibody. A monoclonal anti-VEGFR-3 antibody was used as the positive control. PBS was used as the negative control.3. SPECT planar imaging of the peptide in nude mice bearing ovarian tumors.We subcutaneously injected 100μl of the SKOV3 human ovarian cancer cell suspensions (which contained 1.0-3.0×106 cells) into the right fore leg armpit of four- to six- week-old female nude mice in order to establish transplanted tumors. All handling of the mice occurred in a laminar flow hood. The size of the tumor was measured with Vernier calipers. When the tumor reached a size of about 1.0 cm3, the mice were used in our experiments. The peptide was prepared by conventional solid-phase technology using an automatic peptide synthesizer and coupled to NHS-MAG3. Radiolabeling was performed at room temperature and neutral pH by transchelation from 99mTc-tartrate. We determined the labeling efficiency by paper chromatography. Then we selected 15 ovarian-tumor-bearing nude mice for the imaging and bio-distribution experiment. The tumor-bearing nude mice had 100μl of 99mTc-labeled peptide (approximately 100μg peptide and 3700 KBq 99mTc) injected into their caudal veins and were anesthetized using 0.1% pentobarbital. As a control, free 99mTc was also injected intravenously at a dose of 100μCi per mouse. Next, the image of the tumors was acquired using SPECT. At 1, 3 and 6 h after injection (five mice at every time point), the mice were sacrificed and the blood, heart, liver, spleen, lungs, kidney, stomach, intestines, hind leg bones, muscles and tumor tissues were weighed and assessed for radioactivity using a gamma counter. The bio-distribution counts were calculated as percentage of the injected radioactive dose per gram (% ID/g) of wet tissue weight and the T/NT ratio was also calculated.Results1. Isolation and characterization of LECs from human skin.HLECs began to attach to 2% gelatin-coated culture dishes and colonies began to grow after seeding 12 h to 24h. The HLECs attached and grew well in culture dishes, which indicated that collagenase digestion did not impair the viability of endothelial cells. The purified HLECs exhibited the typical cobblestone-like morphology that they typically do in monolayer cultures, which could be propagated for at least seven passages. Using immunofluorescence staining, we found that cultured HLECs expressed podoplanin, D2-40, LYVE-1, and VEGFR-3. A marker specific to blood endothelial cells (BECs), factor VIII, was not expressed by LECs. Of note, the addition of VEGF-C induced a marked increase in cell proliferation.2. Studying the peptide specific binding with HLECs in vitro.Binding of the peptide antibody to VEGFR-3 yielded a green fluorescence pattern that was diffusely distributed in the cytoplasm of HLECs. The monoclonal anti-VEGFR-3 antibody behaved similarly to the peptide. Negative controls that were incubated without the primary antibodies showed no reaction. The staining result indicates that peptide could bind specifically to VEGFR-3 intracellularly in cultured human HLECs in vitro.3. SPECT planar imaging of the peptide in nude mice bearing ovarian tumors.The labeling rate of 99mTc labeled peptide was 95.27% and the radiochemical purity was 96%. At 1 h post intravenous injection, 99mTc labeled SHSWHWLPNLRHYAS accumulated at the tumor site in the right foreleg. Significant radioactivity accumulation was also observed in the kidneys, liver, and bladder, but the other tissues and organs did not exhibit obvious accumulation. The percentage of the injected radioactive dose per gram (% ID/g) of the kidneys is the highest and the lung is lowest. We observed the most distinct image of the tumor at 3 h post injection and after that time point, the amount of 99mTc-SHSWHWLPNLRHYAS accumulated in the tumors gradually decreased and the quality of the tumors images gradually diminished. At 6 h after injection, the image disappeared completely. The highest percentage of the injected radioactive dose per gram (% ID/g) is the kidneys and the blood is lowest. Free 99mTc was used as a control and did not show any specificity for the tumors and did not produce any tumor images. The result suggest that 99mTc-labeled peptide was mainly distributed to the tumor tissues, liver, and kidneys, and the peptide was quickly eliminated from the blood via the liver and kidneys. We also can see that the T/NT ratios were much less than 1 in the liver and kidneys, which related with the clearance pathway of the peptide.Conclusion: The conjugated peptide has high binding affinity for VEGFR-3 in vitro and can accumulates in tumor tissues in ovarian-tumor-bearing nude mice. Thus, this peptide could be used in radionuclide imaging of ovarian cancer and hope to be used as a carrier molecule in targeted diagnosis and therapy of ovarian cancer.