| PrefacePancreatic cancer is a clinically common malignant tumor which is difficult to be detected at early stage and often encroach upon neighbour cells, blood vessels and nerves, even metastasize to lymphatics at advanced stage. During the proliferation and metastasis of malignant cells, VEGF is regarded as one of the most important factors. Not only does it directly contribute to the increase of va-sopermeability and the proliferation of vascular endothelial cells, it is also an indispensable pro - vasculogenesis factor. Considerable amount of investigations showed that VEGF is expressed by almost all malignant tumours and is rarely seen in surrounding normal tissues. High VEGF expression is significant correlated to the infiltrative growth of tumors, plasma membrane involvement, lymphatic and liver metastasis, high recurrence rate and bad prognosis.Recent studies, however, suggested that macrophages infiltrated into the tumors ( also known as tumor — associated macrophages, TAMs) are also closely related to lymphatic metastasis. In many tumors, especially solid tumors of epithelial tissue, i e. lung, mammary and stomach, there exist a great number of TAMs which substantially come from peripheral blood mononuclear cells and partly formed by fixed tissue macrophage and which mainly infiltrate the ground substance of tumors and endothelium of vessels. Macrophages have antitumors fuction. Even given the fact that tumors are invaded by a great number of macrophages, they continue to growth. Therefore, TAMs may help to promote the development and advancement of tumors.The exact effects of TAMs on tumors are not very clear. Immunohistochemi-cal staining and microvessel counts show that TAMs may be associated with the formation of tumor blood vessels. Other investigations show that the number of TAMs in oral squamous carcinoma is correlated to VEGF expression and TAMs themselves express VEGF. Schoppman et al. also gave evidence to the expression of VEGF in TAMs in carcinoma of cervix and the relation between TAMs and the density of lymphoducts around tumor and lymphatic metastasis. Thus VEGF produced by TAMs may play an important role in the generation of lymphoducts surrounding tumor. However, to the best of our knowledge, theres no report on the relationship between VEGF and TAMs and lymphatic metastasis in pancreatic cancer. Therefore, in this report, we investigate it with immunohisto-logic methods.Methods and materialsA total of 35 patients who underwent surgical resection for pancreatic cancer in the First Affiliated Hospital of China Medical University and Fengtian Hospital of Shenyang Medical College between 1999 and 2004 were examined. A-mong them (age from 34 to 76, average 54. 2) , 24 were male and 11 were female. None of them underwent any therapy before operation. All surgical specimens were confirmed by pathology review with 19 cases of high - differentiated adenocarcinoma, 9 cases of middle - differentiated adenocarcinoma and 7 cases of low — differentiated adenocarcinoma. According to the clinical TNM staging, 8 were of I stage, 12 of E stage, 10 of H stage and 5 of W stage. 15 cases had lymphatic metastasis and 20 had no lymphatic metastasis. Six 39 to 75 year - old (average age 58. 4) cases of normal pancreatic tissue were collected as negative control with 4 male and 2 female.All specimens went through anhydration, formalin fixation, paraffin imbedding, serial cross -sectioning of 4 |xm. After antigen retrieval, sections of normal pancreatic tissue and pancreatic cancer were stained with regular SP immu-nohistochemical methods. Macrophages were labeled by mouse anti - human CD68 monoclone antibody while VEGF by mouse anti - human VEGF monoclonal antibody. Positive and negative controls and HE staining were included ineach experiment. The TAMs value of a case was defined as the average number of TAMs of 3 random high power field ( x 400) in the same carcinoma tissue under light microscope. VEGF scoring was used for the valuation of VEGF expression as counted by the sum of staining intensity score, with 0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining in the relevant cell types and positive cell score with 0 for positive staining cells < 5% of all cells, 1 for 5 ~ 10% , 2 for 10 ~ 20% , 3 for 20 ~ 50% ,and 4 for > 50% . The cases with VEGF score^2 was defined as VEGF positive cases, those with VEGF score ^2 as VEGF negative cases.Statistical analyses were carried out using SPSS 12. 0 statistical packages. Counts data are shown as means ± SD. Differences between 2 groups were determined by t (t") - test, those between 3 or more groups by one - way ANOVA (Newman - keuls, q - test). Correlation between VEGF score and TAMs counts was also analyzed by analysis of covariance (Pearson correlation). Statistical significance was accepted at the P < 0. 05 level.Results1. Results of immunohistochemical staining;Macrophages are scarcely seen in control group, while cells staining for CD68 show yellow or brown granules located on cell membrane or in cytoplasm extensively and unevenly distributed in carcinoma and sunounding tissues. Cells staining for VEGF also show yellow or brown granules in cytoplasm which mainly distribute in carcinoma cells and less present in normal tissues and tissues surrounding carcinoma.2. Relationship between VEGF expression and clinical data;VEGF positive rate (51% , 18/35) and VEGF score (2.71 ±1.41) of pancreatic cancer group are obviously higher than control group ( P <0. 01) . But there are no significant differences between patients with high, middle and low - differentiated carcinoma (P >0.05) and among patients with different clinical stages (P >0. 05). VEGF expression in the group with lymphatic metastasis is significantly higher than that without lymphatic metastasis (P <0. 05).3. Relationship between TAMs counts and clinical data;Mean counts ofTAMs in pancreatic cancer is 110. 66 ±23. 64. TAMs counts of low - differentiated group is higher than middle - differentiated group, and the latter is higher than high - differentiated group (P < 0. 05 ). TAMs counts of lymphatic metastasis group is also significantly higher than that without metastasis (P <0. 05).4. Correlation between VEGF expression and TAMs counts in pancreatic cancer;In pancreatic cancer, VEGF expression showed a straight - line correlation with TAMs counts (7=0. 663 ,P<0.01),ie, with the increase of TAMs, VEGF expression increases in carcinoma tissue.DiscussionPancreatic cancer is a common malignant tumor and vasculogenesis are prerequisite for the growth and metastasis of it. VEGF is one of the strongest and most specific pro - vasculogenesis factors. Our results show that VEGF is mainly expressed in pancreatic cancer and less in surrounding and normal tissues which may suggest carcinoma cells contain a great amount of VEGF which can promote the tumor to generate new vessels. Theres no significant difference in VEGF expression among different clinical stages and pathological degrees, but it is obviously higher in metastasis group than in non - metastasis group which suggest the association between VEGF expression and lymphatic metastasis in pancreatic cancer and that VEGF may be able to promote the growth of lymph vessel which in turn result in metastasis.Modern researches found that there are a great many macrophages in many kinds of tumors which is also the case in pancreatic cancer in our research. We found that the main part of macrophages infiltration in pancreatic cancer is within the stroma. The mean values of TAMs counts in pancreatic cancer are significantly different among the three different differentiated groups, and is the highest in low - differentiated group and the lowest in high - differentiated group. TAMs counts of metastasis group is also obviously higher than non - metastasis group. Thus TAMs may be closely related to the differentiation and metastasis of pancreatic cancer by secreting some factors or other mechanisms. The higher TAMs counts is, the poorer is pancreatic tumor differentiation, the easier is me-tastasis, and the worse is the prognosis.Our study also show that there is a straight line relationship between VEGF expression and TAMs counts in pancreatic cancer, which suggest the close relationship between VEGF secretion and TAMs infiltration and the association between the former two and the metastasis of pancreatic tumor. Some research show that VEGF is the chemotatic factor to TAMs infiltration. Therefore, cancer cells may produce VEGF and induce vasculogenesis and TAMs infiltration, and TAMs may also produce VEGF to promote tumor growth, which become a infernal circle. But if TAMs in pancreatic cancer could produce VEGF remains to be elucidated. Our study also shows that TAMs counts is related to the stages and grades of pancreatic cancer, but VEGF expression is not. So, VEGF expression is not the same as TAMs expression, and they may separately related to other different factors. TAMs may be more important in the differentiation and metastasis of pancreatic cancer. It may be a promising way to prevent TAMs infiltration in the treatment of pancreatic cancer.ConclusionNot only expression of VEGF but also TAMs counts may relate to the lymphatic metastasis of pancreatic cancer. Moreover, the positive relationship exist between scores of VEGF expression and TAMs counts. TAMs counts are related to the clinical stages and pathological degrees of pancreatic cancer, but VEGF expression is not, which show that TAMs may be more important in the differentiation and metastasis of pancreatic cancer. |
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