Study Of Homing Peptide Tmtp2 Binding To Vegfr-3 And Its Tumor Targeting Therapy

Background and Objective In recent years, VEGF-C/VEGFR-3 signaling pathway have been confirmed play an important role in mediated solid tumors and intratumoral lymphangiogenesis process in many studies. The signaling pathway is pay a very important role in lymphangiogenesis and tumor cell mobile,invasion and metastasis.The clinical and pathological studies indicate that the expression of VEGF-C or VEGF-D in tumor cells is directly related to a variety of tumor metastasis. Recent studies have shown that the transfer of soluble VEGFR-3 can be blocked metastasis to the lymph nodes by using recombinant adeno-associated virus gene therapy in mice model of melanoma. These findings suggest that blocking VEGFR-3 signaling pathway can inhibit lymph node metastasis effectively, and may reduce the incidence of distant metastasis. Therefore, based on the importance of this pathway in process of tumor metastasis and lymphangiogenesis. These will be expected to become an effective means of anti-tumor lymph node metastasis by blocking the signaling pathways in cancer therapy. We obtained a period of five peptide sequences TMTP2 via using the extracellular domain of recombinant active human protein VEGFR-3 by display random peptide library screening in the previous study.The lymphatic vessel and lymphangiogenesis was selected as targets in this study. Establishment of the model of lymphangiogenesis and tumor-related model to explore the the specific binding capacities of peptide TMTP2 and lymphatic endothelial cells (LECs. The inhibit effect of peptide TMTP2-DKK on cancer metastases were estimated in breast cancer metastasis model in vivo. The aim was to bring the new theoretical basis and practical experience in the application of the peptide carrier. The peptide carrier may carry the chemotherapy drug in targeting tumor lymphangiogenesis and metastasis therapy and expected to provide an early diagnosis and control in tumor metastasis. And bring new opportunities and hope for the molecular therapy of tumors.Methods We establish the tube formation lymphatic vessels by the method of three-dimensional culture in vitro, and analysis those tube-like structure by related software. We also transplant TC-1 cells in C57BL/6 mouse cornea micropocket by the correspondding tumor, tumors and tumor lymphatic were detected through the HE and immunofluorescence staining. The establishment of the transfer model of spontaneous lung and other organs through injected the 4T1 cells in BALB/c mouse mammary subcutaneous fat pad. After transfer 2-3 weeks, the lung tissue observed under the stereo microscope, and further validation by HE staining respectively. The breast cancer xenograft model in BALB/c-nu mice are inject human breast cancer cell line MDA-MB-231in situ mammary fat pad subcutaneous, the inside slice of tumor lymphangiogenesis and metastasis by further staining. The fluorescently labeled peptide injected into mice or zebrafish via the tail vein injection or microinjection in vivo. After inject the organization of mouse were removed at different time and the effect of target was tested by using immunofluorescence assay. The effect of target of peptides in tumor and lymphatic were dectect in further validation. tumors and tumor lymphatic were detected through the HE and immunofluorescence staining. The targeting effect of peptide was validated in the process of lymphangiogenesis by use breast cancer orthotopic transplantation model, corneal micropocket tumor model and the zebrafish model in vivo. We injected peptide with fluorescent labeled through the tail vein or microinjection into mice or zebrafish in vivo. And further to test the expression of peptide at different time points by immunofluorescence. we used those model to validate the inhibit effect of peptide TMTP2-DKK in vivo and in vitro. The 4T1 cells treated with different concentrations of TMTP2-DKK in vitro, and the biological behavior changes of cells after TMTP2-DKK treatment were assessed by migration and invasion through transwell assay. The inhibit effect of peptide TMTP2-DKK on cancer metastases were estimated in breast cancer metastasis model in vivo. The mouse metastatic tumor of lungs were calculated using microscopy, and the structure of tissue is further validation by HE staining.Results The human lymphatic endothelial cells culture in three-dimensional after 6h, compare with blank control group, the group of VEGF-C protein have more tube-like structure formation. The complex three-dimensional tube final form closed into lymphatic network with increasing incubation time. The corneal micropocket mice transplant with a small piece of tumor tissue appear visible changes of angiogenesis from five days, and more from 7 days until 14 days growth of vessels. After 4 weeks transplanted with 4T1 tumor cells, all mice grown and occurrence of large primary tumors and visible multiple metastatic nodules in the lungs of BALB/C mouse in 4T1 breast cancer metastasis model. The tumor growth in different degrees after transplant in mouse breast cancer orthotopic xenograft models.The synthesized TMTP2 could specific binding to the high expression of VEGFR-3 LECs by immunofluorescence assay.It were seen a strong green fluorescence In the cytoplasm of the experimental group, also part of the nucleus. whereas it could not recognize fluorescence in the cytoplasm except visible blue nuclear in control peptide group. The tissue of slices in mouse breast cancer and whole corneal showed strong fluorescence, yet there is not expression of fluorescent in the group control peptide. After 8 hours injected TMTP2 in the yolk sac of zebrafish, the distribution of fluorescence was observed under the microscope in the thoracic duct.Compare with untreated group and VEGFC group, tubular structure formation reduce in groups of TMTP2-DKK and TMTP2-DKK+VEGFC in vitro. The differences between those groups with specific statistical significance(P<0.05), but the control and TMTP2 peptide did not inhibit LEC tubular structure formation.Compared with the control group, tumor volume and weight were significantly smaller in the group of TMTP2-DKK treated in breast cancer tumor-bearing mice and in corneal tumor formation, and the number of internal lymphatic vessels were less than the control group(p<0.05). Moreover, compared with the control group, the pericardial edema and thoracic duct separation after treated with TMTP2-DKK in zebrafish experiment. The zebrafish lack of thoracic duct was observed under high power field in the treat group(P<0.05).In transwell assays, compared with the control group, the number of cells accross into the bottom clozet was decreased in TMTP2-DKK treated cells (P<0.05). The volume and weight of tumor in TMTP2-DKK treated mice is smaller than the control group, and the incidence of lung metastasis also lower than in the control group(P<0.05). There are a large number of visible metastases in lung by HE staining.Conclusion This study established the three-dimensional lymphangiogenesis model in vitro, and the corneal micro-pocket tumor model, breast cancer metastasis model and orthotopic xenograft tumor model in vivo. This study will provide the ideal animal model for lymphangiogenesis and lymphatic pathway and for studying this complex biological process. The peptide TMTP2 was acquired by a random 12-mer cyclic peptide display library could specific binding to the high expression of VEGFR-3 LECs. The peptide TMTP2 with DKK can inhibit lymphangiogenesis in vitro and tumor growth and tumor intratumoral lymphatic vessels in vivo. The peptide TMTP2 will greatly improve the targeting and efficiency in early tumor treatment if conjugated with anticancer drugs such as doxorubicin. It also may become a significant tool for the diagnostic and therapeutic in targeting repressor lymphatic formation and inhibition of tumor metastasis in clinical.