Correlations Of Lymph-angiogenesis Related Factors And CD133 Expression With Tumor Invasion And Metastasis In Human Gastric Carcinoma

ObjectiveTissue microarray and Immunohistochemical method were used to investigate the significance and expression of VEGF and VEGFR-1,VEGFR-2,VEGF-C and VEGFR-3,CD133 in gastric carcinoma.Using anti-CD34 and D2-40 antibody marker lymph-angiogenesis to assess the MVD and MLVD.Analysis of its relationship with clinicopathological characteristics of gastric cancer,in order to reveal the role and significance of VEGF,VEGFR-1,VEGFR-2,VEGF-C,VEGFR-3 as well as their relations with invasion and metastasis of gastric carcinoma.Analyse the relationships and meanings between the expression of VEGF,VEGFR-1,VEGFR-2,MVD, VEGF-C,VEGFR-3,and MLVD to explore the lymph-angiogenesis in gastric carcinoma occurrence and development of mutual relations and their significance.Materials and Methods1.Cilnical PathologyThe material studied which from the oncosurgery,No.1 Hospital of China Medical University and Cancer Hospital of Liaoning Province,consisted of 208 biopsy specimens from patients 68 female and 140 male patients.The mean age was 60 years (20-80).The samples contained 139 cases of normal mucosa and 208 cases of primary gastric carcinoma.The carcinoma included 157 cases of tubular adenocarcinoma,26 cases of mucinous carcinoma,19 cases of signet ring-cell carcinoma,3 cases of papillary adenocarcinoma,3 cases of undifferentiated carcinoma;and 96 cases of well-moderately differentiated tubular adenocarcinoma,112 cases of poorly differentiated adenocarcinoma;30 cases were in early stage,178 cases were in advanced stage.With infiltrates serosa was found in 175 cases and 33 cases without infiltrates serosa;And with lymph node metastasis was found in 156 and 52 cases without lymph node metastasis;21 cases with metastasis to organs and 187 cases without metastasis to organs.None of patients received chemiotherapy or radiotherapy before operation.2.Empirical methodTissue microarray and Immunohistochemical method was used to observe the expression with VEGF,VEGFR-1,VEGFR-2,CD34,VEGF-C,VEGFR-3,D2-40, CD133 in gastric carcinoma.The relationships and significance of clinicopathological characteristics of gastric cancer between their expression was analyzed as well.3.Evaluation of stainingVEGF,VEGFR-1,VEGFR-2,VEGF-C,VEGFR-3 are located in cytoplasm; CD133 is located in epicyte and cytoplasm;CD34 is located in vascular endothelial cells;D2-40 is located in lymphatic endothelial cells;and the positive staining was like brown-yelow granules.Semi-quantitative integration was used for the evaluation and two high magnification visual fields were randomly selected by two observer from each section for observation.The number of positive cells 5%scored 0;6%～25%scored 1; 26%～50%scored 2;51%～75%scored 3;and more than＞75%scored 4.Positive intensity was classified as follows:colorless scored 0,yellow scored 1,brownish yellow scored 2,and saddle-brown scored 3.After multiplying two integrals,score 0 was as negative expression(-),1～4 as positive expression(+),and 5～8 as moderate expression(++),9～12 as strong positive expression(+++).Two high magnification visual fields were randomly selected to count the number of CD133 positive cells in 100 similar cells,campared to mean,recorded as the mean±SD.Assessment of MVD was performed as follows.Each slide was first evaluated at low power to capture the areas with the highest vascularization.Vessels with a clearly defined lumen or well-defined linear vessel shape,but not single endothelial cells were considered for microvascular assessment.We quantified positive vessels in three chosen fields(×400) with the highest vascularization,vision for the average,as the organization's microessel-density(MVD).MVD was tatistically recorded as the mean±SD.Lumen diameter of pipe has eight red blood cells or vascular smooth muscle exists not count.Micro-lymphatic density(MLVD) method with microessel-density(MVD) Counting methods.4.Statistical AnalysisStatistical analyses were performed using the SPSS 13.0 software.The x~2 test or Fisher exact test was used to compare the categorical variables.The Student t test or One-Way ANOVA was used to analyze continuous variables.Kendall's tau-b test was used to determine the relationships.Results were considered statistically significant at P＜0.05.Results1.The expression of VEGF,VEGFR-1 and VEGFR-2 were significantly higher in gastric carcinoma(VEGF:73.6%,153/208;VEGFR-1:59.1%,123/208;VEGFR-2: 64.9%,135/208) than in normal mucosa tissue(VEGF:17.3%,24/139;VEGFR-1: 33.1%,46/139;VEGFR-2:23.0%,32/139;P＜0.01).The expression of VEGF, VEGFR-1 and VEGFR-2 in gastric carcinoma with lymph node metastasis(VEGF: 78.8%,123/156;VEGFR-1:64.7%,101/156;VEGFR-2:71.8%,112/156) and infiltrates serosa(VEGF:77.1%,135/175;VEGFR-1:62.9%,110/175;VEGFR-2: 69.7%,122/175) were significantly higher than without lymph node metastasis(VEGF: 57.7%,30/52;VEGFR-1:42.3%,22/52;VEGFR-2:44.2%,23/52;P＜0.01) and no infiltrates serosa(VEGF:54.5%,18/33;VEGFR-1:39.4%,13/33;VEGFR-2:39.4%, 13/33;P＜0.05).There was no significant difference between VEGF,VEGFR-1, VEGFR-2 expression and gender,age,borrmann classification,macroscopical classification,degree of differentiation,histologic types and metastasis to organs(P＞0.05). 2.MVD at intratumoral areas were significantly more prevalent than at normal mucosa gastric tissue(26.19±16.23 vs 16.02±9.88,P＜0.01).MVD with borrmann classificationⅢ～Ⅳwas significant higher thanⅠ～Ⅱ(27.90±15.50 vs 18.31±17.56, P＜0.01);advanced gastric carcinoma higher than early gastric carcinoma(27.75±15.37 vs 16.97±18.52,P＜0.01);poor degree of differentiation higher than well-mod degree of differentiation(28.63±14.51 vs 24.11±17.42,P＜0.05);and lymph node metastasis higher than no lymph node metastasis(27.29±16.33 vs 19.25±14.14,P＜0.01).There was no significant correlated with ender,age,histologic types,infiltrates serosa and metastasis to organs(P＞0.05).3.The expression of VEGF-C and VEGFR-3 were significantly higher in gastric carcinoma(VEGF-C:62.5%,130/208;VEGFR-3:72.6%,151/208) than in normal gastric mucosa tissue(VEGF-C:6.5%,9/139;VEGFR-3:6.5%,9/139;P＜0.01).The expression of VEGF-C in gastric carcinoma with lymph node metastasis(68.6%, 107/156) was significantly higher than that without lymph node metastasis(44.2%, 23/52;P＜0.01);and with infiltrates serosa(67.4%,118/175) was significantly higher than no infiltrates serosa(36.4%,12/33;P＜0.01).There was no significant difference between VEGF-C expression and gender,age,borrmann classification,macroscopical classification,degree of differentiation,histologic types and metastasis to organs(P＞0.05).The expression of VEGFR-3 in gastric carcinoma with well-mod degree of differentiation(78.1%,75/96) was significantly higher than that with poor degree of differentiation(67.9%,76/112;P＜0.01);and with lymph node metastasis(76.9%, 120/156) was significantly higher than with without lymph node metastasis(59.6%, 31/52;P＜0.05).There was no significant difference between VEGFR-3 expression and gender,age,borrmann classification,macroscopical classification,infiltrates serosa, histologic types and metastasis to organs(P＞0.05).4.MLVD at gastric carcinoma were no significantly more prevalent than that at normal gastric mucosa tissue(9.41±9.32 vs 7.70±7.69,P＜0.01).MLVD with lymph node metastasis(9.81±9.97) at gastric carcinoma was higher than that without lymph node metastasis(6.41±7.85,P＜0.01).There was no significant difference between MLVD and gender,age,borrmann classification,macroscopical classification,degree of differentiation,histologic types,infiltrates serosaand metastasis to organs(P＞0.05).5.Tissue microarray and Immunohistochemical method were used to detect stem cell marker CD133 at 4,6-chip tissues of 65 samples.The result showed that CD133 cells in normal gastric mucosa(0.56±1.56) is slightly higher than in gastric carcinoma cells(0.14±0.73,P＜0.05).At the same time,under the microscope also observed that part of a small number of gastric interstitial cells and vascular endothelial cells have the expression of CD133,located in epicyte and cytoplasm.The number of positive interstitial mononuclear cells(normal gastric stromal mononuclear cells:1.28±1.37; gastric stromal mononuclear cells:1.35±1.98) higher than the number of gastric tissue (normal gastric mucosal cells:0.56±1.56;gastric carcinoma cells:0.14±0.73);There was no significant difference between normal gastric stromal and gastric carcinoma in the expression of monocytes(P＞0.05).The relationship between CD133 expression and pathological factors were as follows:in gastric carcinoma cells associated with histologic types(t=3.315,P＜0.05);in gastric stromal mononuclear cells associated with borrmann classification,Ⅲ～Ⅳ(1.57±2.09) higher than theⅠ～Ⅱ(0.27±0.65, t=0.375,P＜0.05).6.In the area with high expression of VEGF,there were significant positive correlation with VEGFR-1(r_k=0.131,P＜0.05) and VEGFR-2(r_k=0.178,P＜0.01). There was significant positive correlation between VEGFR-1 and VEGFR-2(r_k=0.423, P＜0.01).And there was no significant positive correlation between MVD and VEGF, VEGFR-1,VEGFR-2(P＞0.05).Remarkable positive correlation of VEGF-C protein expression with and VEGFR-3(r_k=0.334,P＜0.01) and MLVD could be found in gastric carcinoma(P＜0.01).Conclusions1.VEGF,VGEFR-1 and VEGFR-2 are widely expressed in gastric carcinma tissues,and are positively related to pathological features and MVD of gastric carcinma patients.The expression of VEGF was significantly positively correlated with VEGFR-1 and VEGFR-2 in gastric carcinma tissue,These results raise the possibility that VEGF,VEGFR-1 and VEGFR-2 have an important role in angiogengesis and metastasis of gastric carcinoma,directly or indirectly involved in the formation of gastric carcinoma angiogenesis,for the growth of gastric carcinoma to provide the necessary nutrients and removed the metabolites,and promoted the process of invasion and metastasis of gastric carcinoma occurrence and development.2.The increased expression of VEGF-C and MLVD in gastric carcinoma closely correlates with lymph node metastasis and infiltrates serosa.In gastric carcinoma the expression of VEGFR-3 closely correlates with lymph node metastasis and degree of differentiation.Remarkable positive correlation of MLVD with VEGF-C protein expression and VEGFR-3 could be found in gastric carcinoma.The results suggest VEGF-C,VEGFR-3 may play an important role in tumor tissue lymphatic formation and lymph node metastases.VEGF-C binding with it receptor induced the tumor new micro-lymphatic vessel formation,directly or indirectly promote tumor lymph node metastasis,has accelerated the occurrence and development of tumor invasion and metastasis.3.In this study,Tissue microarray and Immunohistochemical method were used to detect CD133 and found that the expression of CD133 in normal gastric mucosal tissues is sightly higher than in gastric carcinoma;and part of a small number of interstitial mononuclear cells and vascular endothelial cells have the expression of CD133,located in epicyte and cytoplasm.The number of positive cells in interstitial mononuclear cells higher than the normal gastric mucosa and gastric carcinoma cells. CD133 in gastric carcinoma cells expression associated with histologic types;in gastric stromal mononuclear cells associated with borrmann classification,Ⅲ～Ⅳhigher than theⅠ～Ⅱ.These results suggest that gastric carcinoma may originate from bone marrow-derived cells,under the pathological state,bone marrow-derived cells was raised to accelerate the occurrence of gastric carcinoma invasion and metastasis in the evolution of the process of gastric carcinogenesis.Evaluation of CD133 as a marker of stem cells by tissue microarray has some limitations,which may be associated with tissue microarray wearing column of small lesions(diameter about 1 mm),can not fully observe the overall level of lesions.Therefore,although tissue microarray technology have multi-faceted advantages such as,high-throughput,flexibility and economical,but as a technicalness to detect stem cell markers may still have some limitations.