VEGFR-3, also known as Flt-4, which acts as a tyrosine kinase receptor for VEGF-C/D, is expressed in liver and spleen blood sinusoids, trauma repair, and in the nascent vascular endothelium of tumor tissues, in addition to being mainly expressed in lymphatic vessel endothelial cells. Xiaoping Ma, Department of Obstetrics and Gynecology, Jiangsu Provincial Hospital of Integrative Medicine Recent findings suggest that VEGFR-3 expression is also present in tumor cells themselves and is involved in the progression of malignancy in several ways. In this study, the expression of VEGF-C, VEGF-D, and VEGFR-3/Flt-4 in early cervical cancer tissues was detected by immunohistochemical SP method, and VEGFR-3/Flt-4-labeled vascular density (MVD) was also detected to analyze its relationship with clinicopathological factors and to explore the role of VEGFR-3/Flt-4 in the progression process in early cervical cancer. The role of VEGFR-3/Flt-4 in the progression of early cervical cancer was investigated. 1. Materials and methods 1.1 Study subjects 41 cases of surgically resected cervical cancer tissues were selected, fixed with 4% paraformaldehyde and embedded in paraffin. No radiotherapy was performed before surgery, and pathology was confirmed after surgery. The clinical stage was according to the standard of the International Federation of Gynecology and Obstetrics (FIGO, 2000), including 7 cases of stage Ia, 14 cases of stage Ib, and 20 cases of stage IIa; histological grading: G17 cases, G2 13 cases, G3 21 cases; histological type of squamous cell carcinoma 37 cases, adenocarcinoma 4 cases; 15 cases with lymph node metastasis, 26 cases without lymph node metastasis; patient age 26-70 years, median age 42 The median age was 42 years, 12 cases were postmenopausal and 29 cases were premenopausal. The cancer cells were found to be lymphatic infiltration in the lumen of the lymphatic vessels under microscopy, 35 cases without lymphatic infiltration and 6 cases with lymphatic infiltration. 1.2 Study methods 1.2.1 Immunohistochemical SP method staining The immunohistochemical SP kit was used and operated strictly according to the instructions. The tissue wax block was 5 μm serially sectioned; xylene was routinely dewaxed and hydrated; microwave antigen thermal repair (95°C, 10 min), and the antigen repair solution was citrate buffer (pH 6.0); 3% hydrogen peroxide-methanol solution was used for 10 min to block endogenous peroxidase; 10% goat serum was used to block non-specific antibodies; primary antibody: rabbit anti-human polyclonal antibody VEGF-C (1:50. ZA-0266); rabbit anti-human polyclonal antibody VEGF-D (1:100, BA1461); rabbit anti-human polyclonal antibody VEGFR-3 (1:200, ab27278), incubate for 60 min; add anti-rabbit biotinylated secondary antibody dropwise, incubate for 30 min; add horseradish peroxide-labeled chain affinity dropwise, incubate for 30 min; DAB microscopic development, hematoxylin PBS buffer was used as negative control instead of primary antibody, and known breast cancer sections were used as positive control. 1.2.2 Determination of results VEGF-C, VEGF-D, VEGFR-3/Flt-4 positive staining was defined as the cytoplasm showing brownish-yellow granules. Referring to the method reported by Jüttner et al, the positive cells were classified according to the percentage of positive cells: – (no positive cells), + (0-5% positive cells), +++ (5%-50% positive cells), and ++++ (>50% positive cells). The MVD was determined by the method reported by Weidner et al. The dense area of positive lumens, i.e. the “hot spot” area, was searched for under low magnification, observed and counted under high magnification, and the MVD was expressed as the average number of positive lumens in five high magnification fields. 1.3 Statistical treatment All data were processed using SPSS13.0 statistical software. The measurement data MVD was expressed as ± s. P<0.05 was considered a statistically significant difference. 2. Results 2.1 Expression of VEGFR-3 in early-stage cervical cancer tissues VEGFR-3/Flt-4 was expressed on lymphatic vessel endothelial cells, in addition to some vascular endothelial cells. Morphologically VEGFR-3/Flt-4 positive vasculature was partly vascular and partly lymphatic. VEGFR-3/Flt-4 protein expression was also seen in some inflammatory cells surrounding the positive vasculature (Figure 1A). Cancer cells were seen in some VEGFR-3/Flt-4 positive vasculature (Figure 1C). VEGF-C, VEGF-D, and VEGFR-3/Flt-4 protein expressions were seen in tumor cells, and their expression positivity rates were 48.7% (20/41), 58.5% (24/41), and 63.4% (26/41), respectively (Figure 1D, E, F). Figure 1 Expression of VEGFR-3/Flt-4 in early-stage cervical cancer tissues 2.2 Relationship between VEGFR-3/Flt-4 protein, MVD and clinicopathological factors and related protein expression in early-stage cervical cancer tissues VEGFR-3/Flt-4 protein expression was not related to menstrual status, histological grade, histological type of cervical cancer patients, and to clinical stage, lymph node metastasis MVD was not related to menstrual status, histological grade, histological type, lymph node metastasis and lymphatic vascular infiltration, but was related to clinical stage and VEGF-C and D proteins. Table 1 Relationship between VEGFR-3/Flt-4 and MVD and clinicopathological factors and VEGF-C and D protein expression in early cervical cancer tissues 3. Discussion Due to the pro-angiogenic or pro-lymphangiogenic properties, the vascular endothelial growth factor family VEGF-A, B, C, D, E and its receptors VEGFR-1, -2, -3 in promoting the growth and metastasis of malignant tumors The mechanism of action of VEGF-A, B, C, D, E and their receptors VEGFR-1, -2, -3 in promoting malignant tumor growth and metastasis and the corresponding gene therapy strategies have been a hot topic of research. Current studies suggest that tumor angiogenesis is mainly regulated by VEGF-A/VEGFR-2, while tumor lymphangiogenesis is mainly regulated by VEGF-C, D/VEGFR-3. The results of this study showed that VEGFR-3/Flt-4 was expressed on some vascular endothelial cells in addition to lymphatic vessel endothelial cells. Morphologically VEGFR-3/Flt-4 positive vasculature was partly vascular and partly lymphatic, and VEGFR-3/Flt-4-labeled positive vascular density MVD correlated with clinical stage and associated proteins VEGF-C and D, but not with other clinicopathological factors such as menstrual status and histological grade. This is different from the results of Yasuoka et al. The mechanism may be that VEGFR-3/Flt-4 plays an important regulatory role in both tumor lymphangiogenesis and angiogenesis. Tumor cells secrete VEGF-C and D, which act as paracrine receptors VEGFR-3/Flt-4 on lymphatic vessel endothelial cells or vascular endothelial cells to mediate the proliferation, differentiation and lumen formation of endothelial cells. On the one hand, the increased blood vessels in tumor tissues provide tumor cells with nutrients necessary for growth, and on the other hand, the increased lymphatic vessels in tumor tissues provide channels for tumor cell infiltration and metastasis, which are conducive to the metastatic spread of tumor cells. However, due to the lack of real endothelial markers of lymphatic vessels, the endothelial cell markers of lymphatic vessels in different types, different sites and even different stages of development in tissues are different, so the exact mechanism of the role of VEGFR-3/Flt-4 in tumor angiogenesis and lymphatic vessel generation needs to be studied in depth. In addition to VEGFR-3/Flt-4 as a receptor for VEGF-C/D, which is mainly expressed in endothelial cells, recent findings suggest that many tumor cells themselves also express Flt-4 and play an important role in the process of tumor progression. Clinical studies of many human malignancies have shown that VEGFR-3/Flt-4 expression in tumor cells correlates with patient clinical stage, cell differentiation, lymph node metastasis, and patient prognosis.Van et al. found that VEGFR-3/Flt-4 is differentially expressed during progression from cervical intraepithelial neoplasia to cervical invasive carcinoma, and therefore hypothesized that it may be involved in pro-lymphangiogenesis during cervical cancer. It is therefore hypothesized that it may be involved in the transformation of the pro-lymphangiogenic phenotype during cervical cancer transformation. The results of in vitro migration and invasion assay studies showed that certain tumor cells with strong invasive ability, such as cervical cancer cell line SiHa, breast cancer cell line MDA-MB-231, and Hs578T, have the expression of Flt-4 in addition to VEGF-C. Human recombinant body VEGF-C protein (Cys156Ser) can promote the migration and invasive ability of tumor cells. In contrast, the application of recombinant Flt-4/Fc to block VEGFR-3/Flt-4, the receptor for VEGF-C, resulted in a significant decrease in cell migration and invasive ability. masood et al. suggested that VEGFR-3/Flt-4 has a role in promoting the growth of malignant mesothelioma. dias et al. showed that VEGFR-3/Flt-4 may also be involved in leukemia cell proliferation, survival, and chemoresistance formation. The results of the present study suggest that in cervical cancer tissues, tumor cell VEGFR-3/Flt-4 expression correlates with lymph node metastasis, lymphovascular infiltration and clinical stage of patients, and with the expression of their ligands VEGF-C and D proteins. The mechanism may be that tumor cells secrete VEGF-C and D, which act on VEGFR-3/Flt-4 on tumor cells themselves in the form of autocrine, promoting tumor cell migration and infiltration, thus leading to tumor lymphovascular infiltration and lymph node metastasis and participating in tumor progression. However, the results of the study are inconsistent because VEGF-C/D receptors are heterogeneous in different tumor cells, and in addition to VEGFR-3/Flt-4, VEGF-C/D receptors include VEGFR-2, NRP-1 and NRP-2, etc. The results of Jüttner et al. concluded that the expression of VEGFR-3/Flt-4 in gastric cancer cells was not associated with lymph node metastasis in patients. In addition, the results of this study also showed that VEGFR-3/Flt-4 protein expression was also seen in some inflammatory cells in the interstitium surrounding the tumor tissue, but its role is not clear. Some studies have shown that inflammatory cells in the interstitium of tumor tissue may also play an important role in the process of tumor lymphangiogenesis. Thus, we can speculate that the role of VEGFR-3/Flt-4 in the progression of cervical malignancies may be multifaceted, and its role may be different for different tumor types, and its exact mechanism still needs further study.