Neovascularization makes a significant contribution to the biological activity and metastatic potential of solid tumors, and in turn is closely interrelated with tumor progression and prognosis. Neovascularization relies on the balance among various molecules that induce or suppress it. Therefore inhibiting neovascularization in tumors will retard tumor growth and metastasis. Our study was intended to (1) analyze the correlation between the sites and intensity of expression of thrombospondin-1 (TSP-1) and microvessel density (MVD) in mucoepidermoid carcinoma using the methods of streptavidin-peroxidase (SP) immunohistochemistry and of MVD counting, and (2) investigate the inhibition of TSP-1 on neovascularization and growth of mucoepidermoid carcinoma by application of various injected doses of recombinant human thrombospondin-1 (rhTSP-1) in the subcutaneous xenotransplanted tumor model of mucoepidermoid carcinoma in nude mice.
The 45 pieces of mucoepidermoid carcinoma tissues in the study were obtained from in-patients in West China Hospital of Stomotology which was affiliated to Sichuan University from 1997 to 2006. Pathological analysis of the imbedded tissues verified that the tissues were from patients with mucoepidermoid carcinoma. Among the 45 patients who had had no anti-cancer therapy, the average age was 45 years (range 27 to 73). Twenty were men and twenty-five were women. According to the TNM staging system recommended by the International Union Against Cancer (UICC) 2002,1 12 were in stage I (T1N0M0), 8 in stage II (T2N0M0), 15 in stage III (10 in T1N1M0 and 5 in T3N1M0), 3 in stage IVA (T1N2M0), and 7 in stage IVC (T2N1M1). Among the group, there were 25 with lymph node metastasis and 7 with distant metastasis and with pathological classification of highly, moderately and poorly differentiated tumors found in 14, 12 and 19 patients respectively.
Forty experimental animals (6-week old nude mice) were provided by the West China Animal Experiment Center. These nude mice served as “tumor models” of subcutaneous xenotransplanted mucoepidermoid carcinoma (the xenografts were tumor tissues from mucoepidermoid carcinoma patients who had been pathologically diagnosed as having poorly differentiated mucoepidermoid carcinoma of the parotid gland). The mouse monoclonal anti-human CD105 antibody, condensed mouse monoclonal anti-human TSP-1 antibody and the immunohistochemical staining SP kit was purchased from ZYMED Company (USA), and the rhTSP-1 was purchased from R&D Systems, Inc. (USA).
This study protocol has been approved by the Ethics Committee of West China College of Stomatology, Sichuan University in Chengdu, China.
CD105 and TSP-1 were detected by the method of SP immunohistochemistry according to the kit manufacturer’s instructions. The working CD105 concentration and working TSP-1 concentration were 1:80 and 1:100, respectively. Triplicate sections of paraffin embedded mucoepidermoid carcinoma tissues, 3 μm thick, were cut; one of which was HE-stained for confirmation of pathological diagnosis. The other two were analyzed for protein expression of TSP-1 and CD105. Consecutive sections of paraffin embedded tumor xenografts from the nude mice were prepared 3 μm thick in duplicate; one of which was HE-stained for verification of mucoepidermoid carcinoma and the other one was analyzed for the protein expression of CD105 by the method of SP immunohistochemistry. The High Pressure Antigen Retrieval Method was applied to the CD105 antigen and the TSP-1 antigen detection. The negative control was PBS replacing the first antibody, and the positive control was the positive sections from the manufacturer.
The necrotic tissue, along with blood and fascia were removed to obtain the parenchymatous tumor tissues. Small pieces of parenchymatous tumor tissues with a diameter of around 3 mm and weight of around 0.06 g were xenotransplanted subcutaneously into forty nude mice (6-week old) in the area of the left ilium. The experiments started 7 days after the transplantation. The 40 nude mice were randomly divided into 3 experimental groups and 1 control group, with 10 mice in each group. The nude mice were injected with 1 ml rhTSP-1 around the tumor in the three experimental groups. The concentrations of 1.25, 0.75, and 0.25 μg/ml were injected respectively in different groups. In the control group, 1 ml PBS replaced the rhTSP-1 and was injected into each mouse. At the 5th week after transplantation, the experiments were stopped to measure the volume and weight of the tumors. The mice were killed by the method of spinal dislocation for harvesting tumor tissues. The volume curves of subcutaneously xenotransplanted tumors with different concentrations of rhTSP-1 were graphically displayed.
Xenotransplanted tumors were ovoid in shape, and the tumor volumes were calculated: volume (mm3) = length (mm) × width (mm)2/2. The length and width of the tumors at one day before beginning treatment were measured. The longest diameter line of the tumor was regarded as the length and the width was measured at the middle point of the long diameter line. In each of the following weeks, the length and width were measured once. The whole measurement procedure was performed as a blind study.
Cells were TSP-1 positive with deposition of buffy-colored granules in the cytoplasm of tumor cells. The average percentage of TSP-1 positive cells was calculated in the microscopic fields. The tumors without TSP-1 positive cells were negative, those with fewer than 10% were weakly positive (+), those with 10%—50% were positive (++), and those with more than 50% were classified as strongly positive (+++). Tumors were reclassified for convenient statistical analysis: TSP-1 negative (-) and weakly positive (+) tumors were included in the TSP-1 negative group, while the TSP-1 positive and strongly positive belonged to TSP-1 positive group. The MVD in mucoepidermoid carcinoma was measured using the method introduced by Takahashi et al.2 The whole sections were observed under 40× light microscope for the areas with the most dense vessels (named hot points). The areas with dense vessels were mostly located at the invasive edge of the tumor and sometimes were found in other areas of the tumors. Under the 100× visual field, the number of microvessels was counted. For each section, three fields were chosen. The average number of microvessels in the three fields was recorded as MVD. The criterion for microvessles was CD105 labeled vascular endothelial cells or vascular endothelial cell clusters stained with a buffy color (Figure 1).
Statistical analysis was accomplished with SPSS 13.0 software. The enumeration data were done by x2 test, Fisher exact test and Yates’s correction. The measurement data were analyzed by t test and analysis of variance (ANOVA). Correlation analysis was done by Spearman rank correlation analysis. There was a significant difference if P <0.05.
The microvessel density in mucoepidermoid carcinoma
Among the 45 cases, vascular endothelial cells stained by CD105 antibody demonstrated heterogeneity of the microvessel distribution. Dense microvessels were found on the tumor invasive edges. The average MVD was 58.17±19.77. In clinical staging of mucoepidermoid carcinoma, the MVDs of four groups were compared, and a statistically significant difference was found (F=20.89, P <0.001). Among them a significant difference was observed between I stage and stages III and IV, and between II stage and stages III and IV. There was no statistically significant difference between stage I and stage II or between stage III and stage IV. Altogether, the 25 cases with lymph node metastasis showed significantly higher values compared with the 20 cases without lymph node metastasis (71.27±13.60 and 41.80±12.81, respectively, t=7.412, P <0.001), 7 cases with distant metastasis had significantly higher values compared with the 38 cases without distant metastasis (87.67±5.64 and 52.74±16.27, respectively, t=5.572, P <0.001). In the pathological grading of mucoepidermoid carcinoma, the MVDs of the three groups were compared, and a statistically significant difference was observed between every pairing of two groups (F=54.915, P <0.001) (Table 1).
The expression of TSP-1 in mucoepidermoid carcinoma and its correlation with clinical and pathological features of mucoepidermoid carcinoma
In 45 cases of mucoepidermoid carcinoma samples, the TSP-1 positive expression rate was 57.78 % (26/45). Most TSP-1 stained granules were found in the cytoplasm, whereas some were found in the extracelluar matrix(ECM) (Figure 2). The study showed that there was no statistically significant differece between the expression of TSP-1 and the clinical staging of mucoepidermoid carcinoma (x2=7.22, P=0.065), while a significant difference was observed between the expression of TSP-1 and the pathological grading (x2=9.628, P=0.008), between the expression of TSP-1 and the lymph node metastasis (x2 =4.377, P=0.036), and between the expression of TSP-1 and the distant metastasis (x2 =4.490, P=0.034). In cases with lymph node metastasis or distant metastasis, the positive expression rate (44.00% and 14.29%, respectively) was significantly low compared with those without lymph node metastasis or distant metastasis (75.00% and 65.79%, respectively). In highly and moderately differentiated mucoepidermoid carcinoma samples, the TSP-1 expression rate (71.43% and 83.33%, respectively) was significant higher than in poorly (31.58%) differentiated carcinomas (Table 1).
Correlation between the expression of TSP-1 and MVD
Among 45 cases of mucoepidermoid carcinoma samples, the expression rate of TSP-1 in the group with a high MVD was 35.29% (6/17), while in the group with a low MVD was 71.42% (20/28). According to the statistical analysis, mucoepidermoid carcinoma tissues with high TSP-1 expression showed low MVD. Thus, the expression of TSP-1 showed negative correlation with the MVD (rs=-0.947, P <0.001) (Tables 1 and 2).
Effect of rhTSP-1 on the growth of subcutaneously xenotransplanted tumors of mucoepidermoid carcinoma in nude mice
The nude mice were divided into 4 groups, including a group treated with 1.25 μg/ml, a group treated with 0.75 μg/ml, a group treated with 0.25 μg/ml, and the control group. One ml was injected each time, twice a week and each week the tumor volume was measured. At the fifth week after the transplantation, the experiments were concluded and the volume and weight of the xenotransplanted tumors were measured. Results showed that the group of nude mice injected with 1.25 μg/ml had the smallest volume and the lightest weight tumors, while the control group of nude mice had the largest volume and the heaviest tumors. The groups that received 1.25 μg/ml, 0.75 μg/ml, and 0.25 μg/ml were, respectively, 36.97%, 53.36% and 73.61% of the control group in volume ((451±92), (651±113), (898±86) and (1220±157) mm3 respectively, F=53.167, P <0.001). And they were, respectively, 35.14%, 51.35%, and 70.27% of the control group in weight ((1.3±0.53), (1.9±0.46), (2.6±0.34) and (3.7±0.71) g respectively, F=62.669, P <0.001). Significant differences were observed among the 3 experiment groups and the control group, implying that TSP-1 could inhibit the growth of mucoepidermoid carcinoma in a concentration dependent manner (Table 3 and Figure 3).
Effect of rhTSP-1 on MVD of subcutaneously xenotransplanted tumors of mucoepidermoid carcinoma in nude mice
The subcutaneously xenotransplanted tumors were probed with immunohistochemical staining of CD105. The microvessels in the tumors were irregular and were composed of buffy vascular endothelial cells or vascular endothelial cell clusters which were annular, slit-shaped or tubular, unrelated to the vessel diameter. The MVDs of the 1.25 μg/ml, the 0.75 μg/ml and the 0.25 μg/ml treatment groups and the control group were, respectively, 15.43±3.45, 28.35±4.24, 44.57±3.35 and 61.73±5.43 per 100 visual fields. The MVDs of the 3 treatment groups were, respectively, 25.00%, 45.93%, and 72.20% of the control group. Significant differences were found among the 4 groups (F=54.582, P <0.001), implying that TSP-1 could inhibit neovascularization in mucoepidermoid carcinoma in a concentration dependent manner (Table 3).
Neovascularization in solid tumors plays an important role in the growth, invasion and metastasis of tumors,3,4 so the detection of MVD in tumors helps the clinician understand the medical history and predict the prognosis. Research has shown that the often used vascular endothelial cell markers, including CD34, can be used to mark all vascular endothelial cells but do not differentiate normal vessels from microvessels in tumors. However, CD105 is specifically expressed on neovascularized vessels and thus can be used as a marker for microvessels in tumors, indicating both clinical treatment and prognosis.5–8 CD105 is expressed in vascular endothelial cells on both small and large vessels. In tissues with neovascularization (such as tissues in the healed wound, psoriasis, embryonic tissues and tumor), CD105 is specifically highly expressed and is an essential part of the neovascularization process. 9,10 Thus, we chose CD105 as a specific marker for vessels in tumors to detect the MVD in mucoepidermoid carcinoma. TSP-1 has not only an important function in regulating neovascularization, but also during the invasion, metastasis, and relapse and for the prognosis of tumors.11 It is still controversial, however, whether it inhibits or accelerates neovascularization. In this research, we (1) investigated the distribution and expression of TSP-1 in mucoepidermoid carcinoma, and (2) analyzed the correlation between the expression of TSP-1 and MVD and the clinical pathological characteristics of mucoepidermoid carcinoma, as well as their effect on neovascularization and growth of mucoepidermoid carcinoma.
TSP-1, also called thrombospondin-1, is one of the strongest negative regulating molecules during neovascularization in tumors.12 TSP-1 mainly exists in platelet alpha-granules and ECM and belongs to the TSP family. It regulates the growth, metastasis, and neovascularization in various tumors and demonstrates significance in predicting tumor malignancy.12 In recent years, Albo and Huang et al13–16 found that TSP exhibited an important role both in inhibiting the adhesion and migration of tumor cells and in inhibiting neovascularization in tumors.
In this study, the positive expression rate of TSP-1 was 57.78% (26/45). Most TSP-1 stained granules were found in the cytoplasm, although some were in the ECM, which is consistent with the literature. Some researchers reported that TSP was distributed in the cytoplasm of tumor cells in thyroid cancer,17 breast cancers,18 and prostatic carcinoma,19 while others showed TSP-1 was mainly expressed in ECM in pancreatic cancer20 and colon carcinoma.21 Thus, the expression mode of TSP-1 in tumors could vary in different tissues or organs. Kasper et al20 observed that the expression of TSP-1 was positively correlated with MVD, while Rice et al,22 Maeda et al,23 Yao et al,24 and Grossfeld et al25 maintained that the expression of TSP-1 was negatively related with MVD in breast cancer, rectal cancer, oral squamous cell carcinomas and bladder cancer. The present results were consistent with these earlier findings. All of the findings above indicate that the function of TSP-1 in neovascularization of tumors possibly relies on the types of tumors and variation in the internal environment. No significant difference was found between the expression of TSP-1 and clinical staging. That finding could result from subjective factors in clinical staging criteria or the limited sample size.
The current study found that in mucoepidermoid carcinoma with lymph node metastasis and distant metastasis, MVD was evidently higher than in tumors without lymph node metastasis or distant metastasis. It is postulated that because neovascularized microvessels in tumors do not grow to maturity, that endothelial cells on microvessels with fractures lack basal membranes. Concurrently, the fibrinolysin excreted by the endothelial cells plays a complementary role. As a result, tumor cells easily enter the microvessels and are transferred to distant organs through the blood. Neovascularized microvessels provide sufficient nutrients to guarantee rapid growth of tumors resulting in malnutrition and thus necrosis in the central area. The necrosis leads to escalated osmotic pressure in the tumor. Consequently, the mobility of the tumor cells increases in turn to advance the tumor cells to peripheral lymphatic capillaries and to regional lymph nodes through lymphatic vessels. Among mucoepidermoid carcinoma with high expression of TSP-1, those without lymph node metastasis and without distant metastasis show higher positive expression rate of TSP-1 than those with either lymph node metastasis or distant metastasis. This indicates that the positive expression rate of TSP-1 is correlated with MVD, which is consistent with the subsequent result that the expression of TSP-1 is negatively correlated with MVD in tumors with high expression of TSP-1, and that MVD is low in contrast. This implies that TSP-1 could inhibit neovascularization in mucoepidermoid carcinoma.
We further found in animal experiments that, after subcutaneous injection of rhTSP-1 at different concentrations at sites around the tumor twice a week for four consecutive weeks, that the nude mice in the group receiving the highest rhTSP-1 concentration, 1.25 μg/ml, demonstrated the smallest volume and lightest weight tumors and also the lowest MVD. These indices were 36.97%, 35.14% and 25.00% of the respective measurements in the control group. Significant differences were found among the four experimental groups and the control group regarding volume, weight and MVD, implying that TSP-1 can significantly inhibit both tumor growth and neovascularization in mucoepidermoid carcinoma. This is consistent with the results of similar studies on breast cancer by Weinstat et al26 and bladder cancer by Campbell et al.27 The mechanism of inhibition of TSP-1 on neovascularization in mucoepidermoid carcinoma possibly involves two aspects. On the one hand, TSP-1 can compete for endothelial cell binding sites with growth factors through competitive inhibition, leading to (1) inhibition of endothelial cell proliferation stimulated by growth factors28–32 and (2) inhibition of endothelial cell mobility and tubular structures formation at some level.33–35 On the other hand, TSP-1 may specifically induce apoptosis of endothelial cells.36–39 When endothelial cells approach the tumor stroma expressing abundant TSP-1, the increased sensitivity to apoptosis may lead to inhibition of neovascularization in tumors.
A variety of protein molecules participate in regulating neovascularization, but in different organs and different types of tumors, various protein molecules function as the major players. Seeking the predominantly expressed genes and relative proteins in different tumors will be of great importance for ultimately understanding the mechanism of specific tumors. Our study showed TSP-1 was an important protein molecule in suppressing neovascularization in mucoepidermoid carcinoma. However, more researches are needed to elucidate the specific mechanism, which remains uncertain, for that function.
We would like to thank Steven Pan and Mary Meyer for the assistance in polishing the paper. Their innovative suggestions and advice contributed significantly to the final version of the paper.
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