The relationship between tumor-associated macrophages and lymphat ic metastasis in oral squamous cell carcinoma
Angiogenesis is an important component of tumor growth and probably takes place in the cascade of biological events involved in tumor metastasis and less favorable prognosis[1]. Angiogenesis is believed to be induced by increased angiogenic factors/conditions such as vascular endothelial growth factor (VEGF), decreased production of angiogenic inhibitors caused by cancer cells, vascular endothelial cells and other stromal cells. One of the most interesting and functionally diverse stroma cells is reactive macrophages, the so-called tumor associated macrophages (TAMs)[2], that can infiltrate tumors,. The function of TAMs in oral squamous-cell carcinomas (OSCC) is still enigmatic. The aim of this study is to explore a possible relationship among angiogenesis, VEGF expression, and TAMs in oral squamous-cell carcinomas (OSCC), so as to provide more information about TAMs' function for further study of OSCC.
1 MATERIALS AND METHODS
1.1 Clinicopathological parametersA total of 42 tissue specimens were obtained, formalin fixed, paraffin embedded, cut continuously to slices (thickness 4μm), and subjected to pathological diagnosis. Of the specimens, 34 were human oral squamous-cell carcinomas (OSCC), including 27 males and 7 females, with an average age of 52 years (37-69 years), and 23 were highly, 10 miderately and 1 lowly differentiated. The other 8 cases were normal oral mucosa specimens used as control. Among the tested OSCC patients,23 cases had negative and 11 cases had positive lymph nodes metastasis.
1.2 Immunohistological staining Immunohistological analysis was carried out on paraffin-embedded sections of 42 specimens to determine microvessel counts, VEGF expression and macrophages using a standard avidin-biotin (SP)labelling technique. Small blood vessels were visualized by staining endothelial cells for CD31 detection(Dako, Copenhagen, Denmark, clone JC/70A). Three areas of the highest neovascularization (hot spot) were chosen in each tumor by scanning the section with low amplification.Individual microvessels in 400× fields were counted. Any CD31 positive endothelial cell or endothelial cell cluster separated from an adjacent cluster was considered a single countable microvessel[3]. The average microvessel counts were recorded for subsequent statistical analysis.Anti-VEGF polyclonal antibody (BioGenex Laboratories, Inc., San Ramon, Calif.; code PU360-UP) was used to determine VEGF expression. Five cancerous fields of each tumor were detected by automated image analysis quantitatively. After the positive subjects were marked, the positive area ratio and average luminosity were obtained.Positive index (PI) was calculated by multiplying the two numbers together, which reflected VEGF expression in OSCC. To detect macrophages, sections were stained using monoclonal CD68 antibodies (Dako, code KP1). Macrophage numbers in three hot areas of each section were counted in 400×fields of microscope. The average counts were recorded for subsequent statistical analysis [4]. Double immunohistological staining (Zymed, LAB-SA system) method was used to detect VEGF expression in macrophages in OSCC cases. The first antibody was anti-VEGF antibody, which was stained as indigo, and the second was CD68 antibody stained as scarlet.The numbers of total macrophages and VEGF-containing macrophages were counted in three hot areas of 400×field of each section. The ratio was calculated.
1.3 Statistical analysis Statistical analysis was performed with SPSS Package Release 10.0 for PC using t tests and Pearson correlation coefficients tests.
2 RESULTS
2.1 Immunohistochemical analysis The clinicopathological parameters and the immunohistochemical analysis results of the 34 patients are summarized in Table 1.Tab.1 Clinicopathological characteristics,microvessel counts,VEGF expression,
and macrophages counts of specimens studied（略）
2.2 Microvessel and macrophage counts The microvessel hot spots occurred anywhere in the tumor but most frequently at the invasive edges of the tumor (Fig.1). Immunoreactivity of CD68 showed in cytoplasm. All tumors had diffuse infiltration of TAMs in stroma (Fig.2). TAMs were very often on the invasive margins of the tumor or around the foci of necrosis.
3 DISCUSSION
Our previous study revealed a significant positive correlation between the microvessel counts and neck lymphatic metastases in OSCC. Tumor cells can penetrate adjacent lymphatics that form concomitantly with blood capillaries or, malignant cells can pass from the blood stream into the lymphatic via lymphaticovenous junctions[5]. In our sample, microvessel counts correlated with the tumor pathological grades and with a significant increase in the number of microvessels in high differentiated cases. It is obvious that the growth of solid tumors depends on angiogenesis (especially in the early phases of tumor development when stabile vascular network is forming), because newly formed blood vessels supply the tumor with nutrients and oxygen and dispose its metabolic waste products.In this study, we demonstrated a clear positive correlation between VEGF expression and microvessel counts in OSCC, which is consistent with some studies published previously[6]. VEGF is a multifunctional glycoprotein involved in endothelial cell proliferation and migration, vasculogenesis, vascular permeability and stromal degradation by activation of some proteolytic enzymes involved in tumor invasiveness and angiogenesis [7]. The results of this investigation indicated that VEGF is, probably, a product of the most important angiogenic pathway in OSCC. Furthermore, in this investigation, no association between VEGF and histological types was found. However, may be, the evidence is not enough for such a conclusion. It has been reported that VEGF has other pro-tumorous effects, such as induction of plasminogen activator and interstitial collagenase expression, which may alter the tumorous extracellular matrix and facilitate tumor progression and spread to lymph nodes[7].One of these pro-tumorous functions of VEGF is the chemotactic effect of VEGF on monocytes/macrophages[8]. We found a significantly positive relationship between increased VEGF expression and TAMs infiltration. Prior to our report, Leek and his collaborator [9] investigated this. Our finding is in agreement with their results, and in addition, we demonstrated that TAMs secret VEGF with double staining method. Some authors,have discovered a positive relationship between TAMs and tumor grades [10] suggesting a reciprocal interaction between TAMs and tumor cells. The tumor produces VEGF (and other chemokines) that can enhance the attraction to macrophages to the tumor locus, and TAMs in turn may provide optimal conditions for tumor proliferation and growth. Furthermore,results of this investigation showed that the high VEGF expression was significantly associated with the neck lymphatic metastases indicating that VEGF expression is associated with tumor progress, spread, and poor prognosis, probably by stimulating angiogenesis, macrophage infiltration, and remodeling of tumor tissue. These findings are consistent with some studies published previously [6,10] in other tumors. We found that microvessel counts correlated with TAMs counts, being significantly higher in OSCC with diffuse macrophage infiltration than in normal oral mucosa. This indicates an important role of TAMs (and probably other stromal cells) in angiogenesis. After all, VEGF immunoreactivity in OSCC was revealed not only in carcinoma cells but also in tumor-associated macrophages.Double staining confirmed that many of the TAMs secreted VEGF suggesting that TAMs might also be a source of angiogenic impulses. Macrophage is the major terminally differentiated cell type of the mononuclear phagocyte system, and is also one of the key angiogenic effector cells, producing a number of growth stimulators and inhibitors. A number of angiogenic cytokines are known to be produced by macrophages: transforming growth factor-alpha (TGF-a)[11], tumor necrosis factor alpha (TNF-a)[11,12], platelet-derived endothelial cell growth factor/thymidine phosphorylase (PDECGF/TP)[13], interleukin-8 (IL-8)[14], and vascular endothelial growth factor (VEGF)[9]. Macrophages are able to modulate events in the extracellular matrix either via the direct secretion of degrading enzymes or via extracellular matrix-modulating cytokines[15].Hence, macrophages are expected to influence every stage of angiogenesis.In conclusion, the results of this investigation confirm a strong connection among VEGF expression, angiogenesis, and macrophage infiltration in OSCC, which represents an important synergistic inter-relation between tumor cells and stroma in the micro-ecosystem of human OSCC. TAMs, which can comprise up to one-half of the mass of breast carcinoma, are biologically extremely important stromal cells, but the number of infiltrated macrophage in OSCC need further investigations. However, these parameters can provide additional information and guidance to evaluate the risk of the neck lymphatic metastases for OSCC patients. Since a broad panel of angiogenic and anti-angiogenic peptides may be produced by OSCC, further investigations should be carried out in order to define the value of TAMs in anti-angiogenic therapies.
