Different Metastatic Potential Of Ovarian Cancer In Nude Mice And Study The Relationship Between Clinical Tissue Lymphangiogenesis And Lymph Node Metastasis Tube Formation

Objective: To establish nude mice footpad xenograft model withovarian cancer cell marked with green fluorescent protein (GFP), dynamicobservation the growth of ovarian cancer and the process of lymphaticmetastasis. Methods: Primary ovarian cancer SKOV3and highly directionallymphatic metastasis ovarian cancer cells SKOV3-PM4were transfected withlentiviral vector contained GFP. Then the ovarian cancer cellsSKOV3-PM4-GFP+and SKOV3-GFP+were respectively injected into thefootpad of nude mice. The tumor metastases location, path and time weredynamically tracked by vivo imaging system. The tumor volume and weight were recorded regularly. The CA125expression in lymph nodes of nude micewere detected by immunohistochemical stanning. Results:After lentiviral vectortransfection and flow cytometry sorting， the SKOV3-GFP+cells andSKOV3-PM4-GFP+cells were cellected. The result showed thatSKOV3-PM4-GFP+group appeared earlier lymphatic metastasis compared withSKOV3-GFP+. SKOV3-PM4-GFP+group showed more lymph nodeenlargement at the same time，and popliteal lymph nodes appear more CA125positive stanning. The lymphatic metastasis rate of SKOV3-PM4-GFP+groupwas higher than SKOV3-GFP+group (P<0.05). However, there is no significantdifference in mice weight and tumor volume between two groups. Conclusion:Successfully established a directional lymphatic metastasis nude mice modelwith Different lymph node metastatic potential ovarian cancer cells containedGFP. Objective: To study the lymphangiogenesis in different lymphaticmetastasis potential ovarian cancer nude mice model and clinical ovarian tumor.Identified the crucial molecules which impacted lymphangiogenesis andlymphatic metastasis. Analysised the role of lymphangiogenesis in directionallymphatic metastasis. Method：1. The expression of VEGF-C and VEGF-D intransplanted tumors were detected by IHC stanning. Lymphatic vessels werelabeled with D2-40IHC positive staining for lymphatic vessels density (LVD)assessment. Microvessel were labeled with CD34Immunohistochemicalstaining for microvessel density density (MVD) assessment.2. Real-timefluorescent quantitative PCR (qRT-PCR) was uesed to detect the mRNA expression of VEGF-C, VEGF-D and VEGFR-3.3. Ultrastructural of thelymphatic endothelial cells were observed by transmission electron microscopy(TEM).4. Respctively analysised the relationship of VEGF-C/VEGF-D andMVD, LVD combined with clinical and pathological data. Identified therelationship of VEGF-C, VEGF-D, MVD, LVD with ovarian cancer lymphaticmetastasis， omental metastasis correlation. Results：1. LVD ofSKOV3-PM4-GFP+group was higher than SKOV3-GFP+group (9.79±1.72vs5.94±0.78, P<0.01). There was no significantly difference in MVD between twogroups.2. The expression of VEGF-C protein and mRNA inSKOV3-PM4-GFP+significantly higher than SKOV3-GFP+,the proteinexpression level of VEGF-C was (253.18±23.6vs104.22±17.4，P<0.01), themRNA expression level of VEGF-C was(2.66±0.30vs1.13±0.33, P<0.01). ThemRNA expression of VEGFR-3in SKOV3-PM4-GFP+significantly higher thanSKOV3-GFP+(2.52±0.18vs0.99±0.53， P<0.01), But the expression ofVEGF-D protein and mRNA expression showed no statisticallysignificant(P>0.05).3. TEM showed SKOV3-PM4-GFP+group forming aclearer lymphatic lumen，the basement membrane dissolved, mitochondriavacuolization and serious chromatin margination, the connections beweenLymphatic endothelial cells were more closely. SKOV3-GFP+lymphatic lumenis not as clear as SKOV3-PM4-GFP+, and partly basement membrane broken,mitochondria cristae disappear, intercellular connections more loosely.4. The positive expression rate of VEGF-C in benign ovarian tumors, borderlineovarian tumors and epithelial ovarian cancer respectively were34.48%,54.50%,72%；The VGF-D positive expression rate respectively were24.13%,36.36%,57.44%. The VEGF-C/VEGF-D positive expression rate in epithelial ovariancancer was higher than benign ovarian tumors(P<0.05). LVD expressed in eachgroup were5.3±2.49,10.2±1.13,14.2±2.26.LVD in epithelial ovarian cancerwas higher than benign ovarian tumors(P<0.05). But MVD showed nodifference among the groups. VEGF-C, LVD were associated with clinical FIGOstage, lymph node metastasis, omental metastases，whereas the expression ofVEGF-D is associated only with clinical FIGO stage, MVD is associated onlywith omental metastasis. VE GF-C in malignant ovarian carcinoma(P<0.01);VEGF-D was also correlated with MVD and LVD(P<0.05). Conclusion：Ovarian cancer lymphangiogenesis were correlated with lymphatic metastasis，It is possible that the high lymphatic metastatic potential of SKOV3-PM4-GFP+was due to its high expression of VEGF-C and the damagement of ultrastructurein Lymphatic endothelial cells.VEGF-C—VEGFR-3signaling axis may be as apotential therapeutic targets in Lymphatic metastasis of ovarian cancer. Lymphatic vessels play a vital role in tumor metastasis. In recent years,a variety of tumor tissue found micro-lymphatic vessels, lymphangiogenesis andlymphatic vessel density significantly affected tumor metastasis. It depends onthe specific tumor lymphatic lymphatic endothelial markers and tumorlymphangiogenesis factor. Antilymphatic treatment forVEGF-C/VEGF-D—VEGFR-3signal axis transduction systems, is expected tobecome an effective way of anti-lymph node metastasis, which could become apart of controlling tumor growth, metastasis, anda tumor therapy potentialtargets.