Experimental Study On The Molecular Imaging And Therapy For VEGF Receptor-3 Binding Peptide Targeting Lymphangiogenesisa In Mouse With Ovarian Cancer

3Background for research:Ovarian cancer is one of common genital malignant tumors for the female. It is severely malignant ,easily infiltrates into the tissue nearby,and metastasizes through the lymph-vessel. Furthermore it has badly responded to the chemotherapy. So it is crucial to search a special target of ovarian carcinoma-associated for diagnosis and therapy. Studies reveal that there are lymphangiogenesis in tumor tissue and around it. .Tumors can activity induce the formation of lymphatic vessel 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-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 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 and therapy study. We can obtain peptide by screening phage display peptide library.We screened and identified a VEGFR-3 binding peptide (SHSWH WLPNL RHYAS) using phage display technology in our early study. Its can competitive inhibition the combination of VEGF-D and VEGFR-3.In our early study indicate that the peptide in the nude mice with ovarian cancer possess selectively accumulative function, has target tropism distribution. And then, in this research we further study the inhibition effect of LECs in vitro and the growth of transplanted tumor in the nude mice in vivo with 131I-peptide. We also observe its ability to provide an accurate image and therapy of tumors lymphatic capillary in nude bearing ovarion tumors. We did so in order to provide a foundation for future studies the radioimmunoimaging and radioimmunotherapy of ovarion tumor.Objective: We sought to study the ability of the peptide labeled with 131I specificity target tropism kill and wound LECs in vitro and performed preliminary studies examining the role of this peptide in the imagine of tumor lymphangiogenesis with single photon emission computed tomography (SPECT) and therapy of tumor in ovarian-tumor-bearing nude mice.Materials and methods:1, Preparation of immune target compound and detection the inhibition effect of LECs with MTT colorimetric method.Peptides and -anti-VEGFR-3 monoclonal antibody was labeled with 131I by the means of Iodogen. 131I-peptide and 131I -anti-VEGFR-3 monoclonal antibody was separated from excess 131I and Herception on a Sephadex G50 minicolumn and the quality control was carried out by paper Chromatography assay. In vitro, 131I-peptide and 131I -anti-VEGFR-3 monoclonal antibody co-culture with LECs respectively. The inhibition effect of LECs was evaluated with MTT colorimetric method.2, SPECT planar imagind and body distribution of the peptide in nude mice bearing ovarian tumors.We subcutaneously injected 200μl of the SKOV3 human ovarian cancer cell suspensions (which contained 1.0～3.0x107/ml cells) into the right fore leg armpit of four- to- six week- old female 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 1.0cm3, the mice were used in our experiments. The peptide and -anti-VEGFR-3 MAb was labeled with 131I by the means of Iodogen. We determined the labeling efficiency by paper chromatography. Then we selected 24 ovarian-tumor-bearing nude mice foe the imaging and bio-distribution experiment. The tumor-bearing nude mice had 3.7MBq of 131I-labeled peptide and 131I-labeled anti-VEGFR-3 monoclonal antibody injected into their caudal vein and was anesthetized using 0.1% pentobarbital. Next, the image of the tumors was acquired using SPECT. At 24h,48h,96h and 168h after injection (six mice at erery time point), the mice were sacrificed and the blood, heart, liver, kidney, intestines, muscles, hind leg bones, brain and tumor tissues were weighted and assessed for radioactivity using a gammar counter. The bio-distribution counts were calculated as percentage of the injection radioactive dose per gram (%ID/g) of wet tissue weight and the T/NT ratio was also calculated.3, Evaluation the targeted therapeutic efficiency of 131I labeled peptide high-binding VEGF receptor 3 in nude mice with ovarian cancer.Ovarian cancer tumor cells were injected by subcutaneous inoculation into 20 nude mice. The peptide and -anti-VEGFR-3 MAb was labeled with 131I by the means of Iodogen. We determined the labeling efficiency by paper chromatography. After tumor formation 2 weeks,the 20 mice were divided into four groups,including peptide(4.4μg),131I -peptide(7.4MBq per mouse),131I -anti-VEGFR-3 MAb(7.4MBq per mouse),and control group. Each drug was injected by the tail vein of the mouse respectively. Observed the toxic response in every day and the size of the tumor was measured weekly and the mice were killed in four weeks after treatment. The inhabited ratios of tumor were calculated and tumors were pathological stained.Results:1, Preparation of immune target compound and detection the inhibition effect of LECs with MTT colorimetric method.The labeling rate of 131I labeled peptide was 82% and the radiochemical purity was 97%, radioactive concentration was 39.96 MBq/ml;The labeling rate of 131I labeled anti-VEGFR-3 monoclonal antibody was 61% and the radiochemical purity was 87%, radioactive concentration was 22.57 MBq/ml. By MTT colorimetric method, the inhibition effect of 131I labeled peptide group on LECs was significantly higher than 131I labeled anti-VEGFR-3 monoclonal antibody group and peptide group(P<0.05)at 48h,72h and 96h.The inhibition ratio of 131I-peptide group on LECs reach top at 72h, and 131I- anti-VEGFR-3 monoclonal antibody group reach top at 96h.2, SPECT planar imagind and body distribution of the peptide in nude mice bearing ovarian tumors.At 24 h post intravenous injection, 131I labeled SHSWH WLPNL RHYAS accumulated at the tumor site in the right foreleg. Significant radioactivity accumulation was also observed in the kidney, liver, and bladder, but the other organ did not exhibit obvious accumulation. As time goes on, transplanted tumor part accumulated radioactivity increase gradually. We observed the most distinct image of the tumor at 96h post injection and after that time point, the amount of 131I labeled SHSWH WLPNL RHYAS accumulated in the tumors gradually decreased but until 168h after injection, we still can get a distinct image; But in the control groups which were injected 131I labeled anti-VEGFR-3 monoclonal antibody, we can also see the imagine at 24h after injection, but the imagine intensity apparently lower than 131I labeled peptide. As time ran off, this imagine became indefinites, we almost can not see the outline of the tumor at 168h after injection. The percentage of the injected radioactive dose per gram (%ID/g) of the kidney is the highest and the brain is lowest at 24 h. in the 131I labeled peptide group, the rate of uptake get its peak at 96h after injection is 39.83%, compared with 23.18% in the 131I labeled anti-VEGFR-3 monoclonal antibody. The result suggests that 131I labeled peptide was mainly distributed to the tumor tissues, liver and kidney 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 low in the liver and kidney, which related with the clearance pathway of the peptide.3, Evaluation the targeted therapeutic efficiency of 131I labeled peptide high-binding VEGF receptor 3 in nude mice with ovarian cancer.The tumor formation rate is 100%. The volme is no significantly different between each group before treatment. After treatment the volume of the tumor in each group was (723±164),(291±68),(457±88),(792±112) mm3 respectively with peptide,131I -peptide,131I -anti-VEGFR-3 MAb and control group. The average volumes of the tumors of the 131I -peptide and 131I -anti VEGFR-3 MAb were significantly less than that of the control group(P<0.05).The growth of implanted tumor was inhibited by 63% and 44% at groups treated with 131I -peptide and 131I -anti VEGFR-3 MAb respectively. Peptide group did not show difference in tumor volume compared to the control group. The transplantation tumor tissue's immunohistochemisty results indicate that tumor tissue has abundant newborn lymphangiogenesis. 131I–peptide has strong apoptosis effect on lymphatic endothelial cells and tumor cells.Conclusion: 1, Peptides and -anti-VEGFR-3 MAb was labeled with 131I by the means of Iodogen. This method is very convenience, can get highly labeling rate and radiochemical purity; 131I labeled peptide has a very apparent inhibition and killer effect to lymphatic endothelial cells.2, 131I labeled peptide can accumulates in tumor tissues in ovarian-tumor-bearing nude mice.A distinct image of the ovarian-tumor-bearing nude mice can get by injecting 131I labeled peptide.3, In vivo, 131I labeled peptide can inhibit mach more the growth of transplantation tumor. Effects surpass than 131I -anti VEGFR-3 MAb and control group. Thus , this peptide could be used in radionuclide imaging and therapy of ovarian cancer and hope to be used as a carrier molecule in targeted diagnosis and therapy of ovarian cancer.