| Background and Objective:Esophageal cancer is one of the most common malignant tumors in the world. Especially the north of Henan province, such as Linzhou, Anyang, Huixian and so on, where is the highest morbidity and mortality region in the world, new incidence up to 250 thousand every year. More and more researchers have paid attention to the research and prevention of esophageal cancerLymphatic metastasis plays an important role in esophageal cancer. On the one hand, lymph node metastasis may be caused by the invasion of tumor cells in pre-existing lymphatic vessels in the tumor periphery; On the one hand, through the induction of lymphangiogenesis via growth factor production, promoting the generation of lymphatic endothelial cells and the lymphatic invation and metastasis of tumor cells. Therefor, it will be significant scientific value and clinical prospects to identify the specific biomarkers of the lymphatic endothelial cells, and being a target for inhibiting the production of lymphatic vessel endothelial cells and lymphatic metastasis.Vascular endothelial growth factor-C(VEGF-C) is the most important lymphangiogenesis inducers involved in tumor metastasis, which plays an important role of angiogenesis and lymphangiogenesis.VEGF?C is predominantly combined with VEGF receptor-3(VEGFR-3), which is mainly expressed in lymphatic capillaries. Several studies have reported that not only tumor cells can secrete VEGF-C, TAMs can also involvement of lymphangiogenesis and regional lymph node metastasis via VEGF-C/VEGFR-3 axis, which affects the occurrence, progression and metastasis process of tumor.The emerging evidence suggests that using small interfering RNA(si RNA) can against tumor cells development in vitro. Nonetheless, lymphangiogenesis is not significantly influcenced in transplanted tumor nude mice model. So it is conferred that inhibition the expression of VEGF-C to suppress lymphangiogenesis is impracticable. In order to research that is there any synergistic effects of esophageal carcinoma cells and TAMs on lymphatic endothelial cells proliferation and ring formation, then explore its possible mechanism. In this study we inhibit VEGF-C expression in esophageal cancer cells along with TAMs. Then, LEC were cultured in conditioned medium before and after transfection. We measured VEGF-C/VEGFR-3 level using ELISA,Western blot,q(RT)PCR methods. Meanwhile, comparied LEC proliferation, migration and tube formation by CCK-8, Transwell chamber cell migration assay, cells Matrigel three-dimensional culture technologies. To explore whether it is possible to inhibit LEC proliferation and lymphangiogenesis synergisticly in order to suppression lymphatic metastasis of tumor. In search of new methods for esophageal cancer diagnosis and gene therapy targeting VEGF-C/ VEGFR-3.Materials and methods1. Isolation and induction of M2 macrophages in vitro Heparinized blood samples were obtained from healthy adult individuals, the separation of human peripheral blood mononuclear cells(PBMCs) from 50 ml fresh blood. Monocytes were isolated by anti-CD14 microbeads. M2 macrophages were obtained after culturing in medium and supplemented with macrophage colony stimulating factor(M-CSF) and interleukin 4(IL-4), respectively.2. The synergistic effects of esophageal squamous carcinoma cells KYSE150 and M2 macrophages on esophageal squamous carcinoma associated LEC proliferation, migration and tube formation.2.1 Conditioned medium and cell culturesEsophageal carcinoma KYSE150 and M2 macrophages are cultured in RPMI-1640 medium containing 10 % fetal bovine serum(FBS)and replacing serum free medium overnight when cells fusion reached 70%~80%. Collected the supernatant after 24~48h and Stored in the-80℃ refrigerator.Esophageal cancer related lymphatic endothelial cells were divided into four groups on the basis of two kinds of conditioned medium, meanwhile established control groups. Collected cells after 24 h. Cells are divided as follows:1) A: KYSE150 and M2 macrophages medium and intermingle with each other. As conditioned medium develop esophageal cancer related LEC;2) B: M2 macrophages medium only and culture esophageal cancer related LEC;3) C: KYSE150 medium and culture esophageal cancer related LEC;4) D: Primary medium to culture esophageal cancer related(LEC)(RPMI-1640 medium containing 10 % FBS).2.2 Esophageal cancer related LEC proliferation, migration and tube formation The esophageal cancer related LEC proliferation were detected by CCK-8 assay; Esophageal cancer related LEC migration was meared by Transwell chamber; Lymphangiogenesis was tested by network formation of endothelial cells on matrigel.2.3 Determination of VEGF-C concentration and the expression of VEGFR-3 The culture supernatant fluid was collected, the ELISA was employed to study the VEGF-C content secreted by each group of cells; The expression of VEGFR-3 proteins were verified by Western Blot analysis;The q(RT)-PCR was used to analyze the m RNA level of VEGFR-3.observing the effects on esophageal squamous cancer related LEC after transfection 3. VEGF-C si RNA transfection of KYSE150 cells and M2 macrophages,3.1 Construction and transfection of plasmids Three sequences of si RNA oligonucleotides targeting VEGF-C were synthesized using the plasmids : S1:VEGF-C si RNA-1;S2:VEGF-C si RNA-2;S3: VEGF-C si RNA-3.Transfection si RNA into KYSE150 and M2 macrophages. In order to inhibit the expression of VEGF–C. The optimal concentration of si RNA was observed by fluorescence microscope and flow cytometry.3.2 Cells grouping after transfection Divided esophageal squamous cancer related LEC into 4 groups after transfection of VEGF-C si RNAs, meanwhile established control groups. The culture supernatant and cells were collected after 24 h. Cells are divided as follows:1) Z1: Mixing KYSE150 and M2 macrophages medium after transfected by VEGF-C si RNAs. As conditioned medium develop esophageal cancer related LEC;2) Z2: Esophageal cancer related LEC grow in M2 macrophages medium after transfected by VEGF-C si RNAs;3) Z3: Esophageal cancer related LEC are cultured in KYSE150 medium after transfected by VEGF-C si RNAs;4) Z4: Primary medium to culture esophageal cancer related LEC(RPMI-1640 medium containing 10 % FBS).3.3 Esophageal cancer related LEC proliferation, migration and tube formation after transfection Detection esophageal squamous cancer related LEC development, proliferation, migration, tube formation and according to the method 2.2.3.4 Determination of VEGF-C and expression of VEGFR-3 after transfection The expression of VEGF-C and VEGFR-3 are measured according to the method 2.3.4. Statistical analysis The SPSS 17.0 statistical software package was used for data analysis. Each experiment is presented as mean ± standard error(at least n = 3) and multiple sets of mean comparison analyzed by ANVOA. With P<0.05 considered to represent a statistically significant difference.Results1. Isolation and induction of M2 macrophages in vitro Peripheral blood monouclear cells ratio was 94.63 + 3.46% after sorted by immunomagnetic beads. The proportion of CD163+ macrophages(M2 macrophages) was 81.3 + 2.66%, which could be used for the next experiment.2. The synergistic effects of KYSE150 and M2 macrophages on esophageal squamous carcinoma associated LEC proliferation, migration and tube formation.2.1 Proliferation Each group cells were seeded onto 96-well tissue-culture microplates for 6, 12, 24, 36, 48 hours, detection of absorbance at 450 nm. Campared with control group(D), the proliferation ability of group A, B and C was significantly increased, the difference is statistically significant(P<0.05); the proliferation ability of the cells in group A was the strongest, which was significantly stronger than group B and group C(P<0.05); but the proliferation ability of the cells in group B was a little weaker than group C, there was no significant difference(P>0.05).2.2 Cellular migration Direct microscopic counting at ×200 magnification of cells that have migrated to the lower side of the membrane was performed and a mean value for each view was calculated. The number of migrating cells in group A was 628±19.03, group B was 506±18.68, group C was 474±8.73, group D was 358±29.51. Campared with control group(D), the number of migrating cells in group A, B, C was significantly increased, the difference is statistically significant(P<0.05); the number of migrating cells in group A was the most, which was significantly more than group B and C(P<0.05); but the number of migrating cells in group B was a little less than group C, there was no significant difference(P>0.05).2.3 Tube formation Tube formation was examined on an inverted microscope. A mean value for each view was calculated. The number of tubular structures in group A was 20.00±1.53, group B was 16.00±2.31, group C was 16.00±0.58, group D was 12.00±2.08. Campared with control group(D), the number of tubular structures in group A, B, C was significantly increased, the difference is statistically significant(P<0.05); the number of tubular structures in group A was the most,which was significantly more than group B and C(P<0.05); but the number of tubular structures in group B was a little less than group C, there was no significant difference(P>0.05).2.4 VEGF-C concentration in conditioned media The supernatants were collected and subjected to a VEGF-C ELISA. Campared with control group(D), the VEGF-C concentration in group A, B, C was significantly increased, the difference was statistically significant(P<0.05); VEGF-C concentration in group A was the highest, which was significantly higher than group B and C(P<0.05); whereas no significant difference was observed between group B and C(P>0.05).2.5 The expression of VEGFR-3 m RNA Campared with control group(D), the level of VEGFR-3 m RNA in group A, B, C was significantly increased. The expression of VEGFR-3 m RNA in group A was about four-fold higher than control group(P<0.01); meanwhile that was about two-fold higher in group B and group C(P<0.05); the level of VEGFR-3 m RNA in group A was significantly increased compared with the level in group B and group C(P>0.05).2.6 VEGFR-3 protein levels in each group Campared with control group(D), the expression of VEGFR-3 protein in group A, B, C was increased, the difference is statistically significant(P<0.05). the expression of VEGFR-3 protein in group A was the strongest, which was significantly higher than group B and C(P<0.05); whereas no significant difference was observed between group B and C(P>0.05).3. Effects on esophageal squamous cancer related LEC after transfection3.1 Detection the transfection efficiency by flow cytometry and Fluorescence microscope The transfection efficiency was 62.5 + 3.03% at 8nmol/L; The transfection efficiency was 88.97±3.62% at 12nmol/L; The transfection efficiency was 60.00±7.44% at 20nmol/L.So select 12nmol/L.3.2 Gene silencing efficacy Campared with control group, the level of VEGFR-3 m RNA in S3 was decreased more than 50%(P<0.05). So S3 silencing efficacy is better than other groups.3.3 Proliferation Campared with control group(Z4), the proliferation ability of group Z1, Z2 and Z3 was significantly decreased, the difference is statistically significant(P<0.05); the proliferation ability of the cells in group Z1 was the least, which was significantly weaker than group B and C(P<0.05); but the proliferation ability of the cells in group Z2 was a little stronger than group Z3, there was no significant difference(P>0.05).3.4 Cellular migration A mean value for each view was calculated. The number of migrating cells in group Z1 was 122±20.00, group Z2 was 228±22.60, group Z3 was 216±16.65, group Z4 was 358±29.51. Campared with control group(Z4), the number of migrating cells in group Z1, Z2, Z3 was significantly decreased, the difference is statistically significant(P<0.05); the number of migrating cells in group Z1 was the least, which was significantly less than group B and C(P<0.05); but the number of migrating cells in group Z2 was a little more than group Z3, there was no significant difference(P>0.05).3.5 Tube formation The number of tubular structures in group Z1 was 5±2.03, group Z2 was 9±1.52, group Z3 was 10±1.53, group Z4 was 14±1.53. Campared with control group(Z4), the number of tubular structures in group Z1, Z2, Z3 was significantly decreased, the difference is statistically significant(P<0.05); the number of tubular structures in group Z1 was the least,which was significantly less than group Z2 and Z3(P<0.05); the number of tubular structures in group Z2 was a little more than group Z3, there was no significant difference(P>0.05).3.6 VEGF-C concentration in conditioned media The supernatants were collected and subjected to a VEGF-C ELISA. Campared with control group(Z4), the VEGF-C concentration in group Z1, Z2, Z3 was significantly decreased, the difference was statistically significant(P<0.05); VEGF-C concentration in group Z1 was the lowest, which was significantly lower than group Z2 and Z3(P<0.05); whereas no significant difference was observed between group Z2 and Z3(P>0.05).3.7 The expression of VEGFR-3 m RNA Campared with control group(Z4), levels of VEGFR-3 m RNA in group Z1, Z2 and Z3 was significantly decreased. The expression of VEGFR-3 m RNA in group Z1 was about reduced by 77% than control group(P<0.01); meanwhile that was about reduced by 45% in group Z2 and group Z3(P<0.05); the level of VEGFR-3 m RNA in group Z1 was significantly increased compared with the level in group Z2 and group Z3(P>0.05).3.8 VEGFR-3 protein levels in each group Campared with control group(Z4), the expression of VEGFR-3 protein in group Z1, Z2 and Z3 was decreased, the difference is statistically significant(P<0.05). the expression of VEGFR-3 protein in group Z1 was the weakest, which was significantly lower than group Z2 and Z3(P<0.05); whereas no significant difference was observed between group Z2 and Z3(P>0.05).Conclusions1. M2 macrophages could promote the proliferation, invasive and tube formition ability of tumor associated lymphatic endothelial cells in vitro.2. Esophageal squamous carcinoma cells and M2 macrophages can accelerate the proliferation and tube formition of LEC.3. VEGF-C si RNA can inhibit the expression of VEGF-C and VEGFR-3 on esophageal squamous carcinoma cells and M2 macrophages, thereby influence the the inhibition effects of tumor associated lymphatic endothelial cells proliferation, invasive and tube formation. |
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