Esophageal carcinoma is a common kind of malignant tumor and about 90% of which is esophageal squamous cell carcinoma (ESCC), and it has a high incidence in China.1–3 In recent years it has been argued that angiogenesis plays a key role in tumor growth and metastasis and it is a complex process influenced by many factors.4–7 Vascular endothelial growth factor (VEGF) can strongly stimulate the growth of vascular endothelial cells and it is one of the most potent factors with such function.8–11 PC cell-derived growth factor (PCDGF or progranulin) is newly discovered and closely correlated with the growth, development and metastasis of carcinoma.12 It was reported recently that PCDGF may help the vessel formation of tumors by up-regulating the expression of VEGF. We have failed to find any articles about the relationship among PCDGF, VEGF and microvessel density (MVD). Our work on this subject should help to explain ESCC's mechanism of growth and provide clues for preventing and treating this disease.
Tumor samples were collected from 50 patients who had surgical removal of ESCC in the Chest Surgery Department of the First Affiliated Hospital of Zhengzhou University between July 2005 and May 2006. Among them, 27 were male and 23 were female with a median age of 60.50 years old (45–80). No patients received any treatment such as radiation or chemotherapy before operation and all the samples were identified as ESCC by a pathological assessment. Classification tumor, nodes, metastasis (TNM) staging was as follows: 6 in I, 30 in II, 14 in III and IV. Seventeen were well differentiated tumors, 24 moderately differentiated and 9 poorly differentiated. Twenty normal control samples were procured from the same operations with a distance of 5 cm away from the edge of the tumor and were all confirmed as normal by light microcopy and special tissue staining. Briefly, 5 μm thickness sections were cut from each tumor and were transferred onto the glass slides. One was for hematoxylin and eosin (HE) staining and the other 3 for immunohistochemical staining.
Mouse polyclonal antibody of PCDGF was purchased from Wuhan Sanying Biotechnology Company (Wuhan, China). Mouse monoclonal antibody against VEGF (working concentration 1: 80), mouse monoclonal antibody against CD105 (working concentration 1: 100), EDTA repairing solution, SP kit and DAB were purchased from Beijing Zhongshan Golden Bridge Company (Beijing, China).
Each paraffin-embedded tissue section was deparaffinized, hydrated, and incubated in 3% H2O2 for 3 minutes to block endogenous peroxidase activity. The sections were incubated with 10% serum blocking solution to block nonspecific binding, then the sections were incubated with the primary antibodies in ahumid chamber overnight in a 4°C refrigerator. The sections were then incubated with the biotinylated secondary antibody and with the third antibody labeled with peroxidase for 30 minutes at room temperature. After rinsing with phosphate buffered saline Tween-20 (PBST) for 5 minutes × 3, the sections were stained with DAB and counterstained with hematoxylin. Then anhydration, transparence, mounting and examination under the microscope were carried out routinely. Confirmed positive sections were used as positive controls. Phosphate buffered solution (PBS) in place of the primary antibody was used as a negative control.
PCDGF: There were deep yellow or brown particles in the nuclei or cytoplasm with positive staining, and we made an overall judgment about staining density and area according to Serrero's standards.13 We counted the positive cells under 400×vision, >5% as positive section and <5% as negative. The staining intensity was classified into 3 grades: faint yellow, yellowish brown and brown. The positive intensity of partly scattered faint yellow was weak, partly or diffusely scattered yellowish brown was media, diffusely scattered brown was strong.
VEGF: tumor cells or vascular endothelial cells with yellowish brown color particles in the cytoplasm were counted as positive. According to Zhong,14 for each section, 5 fields of 400× were randomly selected, counting 200 cells in every field (1000 cells together) and calculated the percentage of positive cell, <10% as negative and >10% as positive. MVD test in the tumor: labeling CD105 showed the newly formed vessels inside the tumor, and calculated the stained microvessels and small vessels. According to Weidner's technique,15 single or clusters of endothelial cells of yellowish brown were thought as a capillary vessel, and we did not count the capillary vessel which is weak in color, thick in muscular layer and the diameter of its lumin greater than 8 erythrocytes.
This was how we counted: first, use a low power lens (10×10) to scan the whole section, to locate 3 fields with dense capillary vessels in the area of tumor infiltration then counted the stained capillary vessel with high power len within the field. The number of microvessel on the slide was the average number of the counting result of the 3 fields.
Data analysis was made with SPSS13.0. χ2 test was used to analyze numeration data, t test and SNK (student-Newman-Keuls) were used to judge the difference of average between groups of measurement data and mean comparison of groups was analyzed with ANOVA (analysis of variance). P values <0.05 were considered statistically significant.
Expression of PCDGF, VEGF and MVD in tissue
PCDGF showed strong positive staining in ESCC. Deep yellow or brown particles could be found in both plasma and nuclei (Figures 1–3). Sporadic interstitial cells, some esophageal glands and vascular endothelial cells were also positive (Figure 4). The positive and strongly positive rates were 94.0% (47/50) and 48.0% (24/50), respectively. In the well differentiated carcinoma with PCDGF positive, the central horny pearl showed obvious light staining compared with the tumor nest, and in the poorly differentiated ones the nest was stained relatively uniformly. In normal esophageal endothelium, PCDGF was mostly negative. Some had light positive stain between the epithelium basal layer and prickle cell layer, and the positive and strongly positive rates were 40.0% (8/20) and 0% (0/20), respectively.
VEGF positive mainly showed brown or deep yellow particles in the cytoplasm in ESCC. Besides, endothelium of blood vessels could also be positive with the positive rate of 58.0% (29/50). In VEGF positive well differentiated ESCC, the cells around the nest had a heavy staining, while the cells around the horny pearl showed light staining. In the poorly differentiated ESCC, diffused positive cells could be seen. In the normal endothelium, VEGF was mainly negative. Some showed a weak positive at the basement of the endothelium with the positive rate of 25.0% (5/20).
The positive expression of CD105 was mainly located in the cytoplasm and membrane of the vascular endothelial cells and the nest could also be positive. The CD105 labeled vessels were stained with buffy or brown color, the MVD was 35.72±10.10. In normal mucosa, the MVD was 16.31±6.74, there was significant difference between the two groups (t=3.56, P=0.00087). The vessels had a small diameter, thin membranes and little smooth muscle. Sometimes clear lumin can be seen as various shapes or concreted dots, bud shapes or cords. The vessels were mainly distributed randomly at the edge of the cancer nests with different sizes. The vessels in the normal esophageal endothelium were lightly stained a pale yellow or buffy color. The positive vessels had bigger diameters.
Relationship between PCDGF, VEGF expression and clinicopathologic factors
From Table 1, we can infer that the positive rate of PCDGF and VEGF was correlated with the infiltration depth, lymph node metastasis and TNM staging of ESCC; PCDGF group: χ2=5.059, P=0.025; χ2=5.055, P=0.025; χ2=4.365, P=0.031; respectively. VEGF group: χ2=4.428, P=0.035; χ2=4.918, P=0.027; χ2=8.890, P=0.012; respectively. The expression of PCDGF was positive and significantly correlated with the differentiation of ESCC (χ2=12.489, P=0.002), and the expression of VEGF was correlated with the long distance metastasis of ESCC (χ2=4.937, P=0. 021).
Relationship between the expression of PCDGF, VEGF and MVD in ESCC
Table 2 shows the MVD in the PCDGF positive group and in the VEGF positive group were 40.58±11.07 and 39.34±10.70, respectively, which were significantly higher than in the control groups’ 31.23±6.59 and 30.71±6.65 (PCDGF positive group: t=3.662, P=0.006, VEGF positive group: t=3.263, P=0.002). The expression of PCDGF and VEGF were both significantly correlated with MVD, respectively (q=4.934, PAB=0.0085, q=9.717, PAC=0.00094, q=7.907, PBC=0.0045).
Relationship between the expression of PCDGF and VEGF
Both PCDGF and VEGF were expressed in 31 of 50 samples and they tested as significantly correlated (χ2=4.42, P=0.031).
Many studies have confirmed that the formation of new blood vessels is a condition for tumor growth and metastasis.16–19 It is a complex course regulated by many angiogenesis factors and anti-angiogenesis factors. The up-regulation of growth factors and down-regulation of inhibiting factor can both trigger angiogenesis and activate the sleeping vascular endothelial cells to help in the formation of tumor blood vessels.
VEGF and its receptor are one of the most important angiogenetic factors of the many factors described. It was proven to be a highly specific mitogen for vascular endothelial cells and VEGF can link the endothelial cell receptor and promote angiogenesis directly and enhance the permeability of the vascular membrane under physical or pathological conditions. Therefore, it is closely correlated with tumor growth, infiltration and metastasis.20 In this research, our results support the points above: VEGF is closely correlated with ESCC infiltration depth, lymph node metastasis and tumor stage (P <0.05). MVD in the VEGF positive group is obviously higher than that in the negative group, which can be used as another proof of VEGF's importance in tumor growth, infiltration and metastasis.
PCDGF is a 88 kD glycoprotein coming from the mouse PC cell line and it promotes cell growth by autocrine function.21 Because its protein hydrolysate belongs to the granulin-epithelin family, rich in a specific and highly conventional 12 aminothiopropionic acid motif, PCDGF can also be called a granulin-epithelin precursor (GEP or progranulin).22 PCDGF has been proven to be the only purified growth factor that can promote the mouse fibroblast to grow when they lack IGF-IR.23 In recent years, this growth regulating polypeptide was found correlated with the existence and development of many epithelium and lobus intermedius derived tumors, such as breast cancer,13 ovarian cancer and prostatic adenocarcinoma24,25 These reports were usually within adenocarcinomas but seldom in squamous cell carcinomas.
In Kong's study26 on throat squamous cell carcinoma, PCDGF had a higher level of expression in cancer of the larynx and its precancerous lesions than in normal tissue. In this research, the positive and strongly positive rates of PCDGF in ESCC is 94.0% (47/50) and 48.0% (24/50), both significantly higher than in normal esophageal mucous membranes, 40.0% (8/20) and 0 (0/20). And the level of expression is significantly correlated with ESCC infiltration depth, lymph node metastasis and tumor TNM stage (P <0.05), consistent with Kong’ conclusion.
A lot of in vivo or in vitro study has proven that over expression of PCDGF can promote tumor invasion and metastasis and thus plays a key role in the tumor development and progression.27–29 So we can infer that PCDGF can do the same in ESCC. It should be noted that tumor tissues are positive for PCDGF labeled immunohistochemistry staining. In our experiment, we traced the line-shaped tumor infiltration and diffused tumor cells in interstitium by PCDGF expression. The positive tumor nest is clearly different from the peripheral negative interstitium. This makes PCDGF a possible marker for ESCC.
Researches in recent years showed that PCDGF may also play an important role in angiogenesis. Tangkeangsirisin and Huang30,31 both found in human breast cancer cell lines that the expression of PCDGF could affect the expression of VEGF and their expression changed in the same way. Our results are similar to their findings. The PCDGF or VEGF positive group had much higher MVD than in the negative group (P <0.01), suggesting that PCDGF and VEGF both regulate angiogenesis. MVD of the PCDGF (+++)/VEGF (+) group (group A) was significantly higher than the control group, which indicated that they may work together when promoting angiogenesis.
The expression of PCDGF has a significant positive correlation with that of VEGF (P <0.05). With the fact that VEGF is one of the most important angiogenic factors known, we think PCDGF can act as an upstream protein that can regulate angiogenesis by altering the expression of VEGF. From past research, we see the three kinase pathways activated by PCDGF in regulating tumor growth are also involved in VEGF promotion of vessel growth.
PCDGF can bind with IGF-IR to regulate cell growth. But it still promotes cell proliferation very much without IGF-IR. We can presume that there must be other pathways, for which two questions must be answered: whether PCDGF must bind along with IGF-IR to affect the expression of VEGF or whether PCDGF still affect cell growth, apoptosis and angiogenesis without the change of VEGF expression?
Until now, the mechanism of how PCDGF can affect VEGF is not clear yet. In our experiment, some vascular epithelial cells inside the ESCC tissue were clearly stained positive, so whether vascular epithelial cells have PCDGF ligand associated and their binding can also activate VEGF signal transducting pathway and stimulate cell growth and angiogenesis is not known. Whether this ligand is specific to PCDGF or not is a question worthy of consideration.
By now, no ideal prognosis and long term survival can be obtained for patients with progressing ESCC and no breakthroughs have occurred in treatment. PCDGF is closely correlated with vessel formation in the tumor and in some epithelium tumor cells have a malignant phenotype; such as uncontrollable proliferation, escape from apoptosis and tissue invasion, and they play an important role in tumor growth and progression. For clinical consideration, PCDGF may be a potent biomedical marker for ESCC.
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