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Effects of the fibroblast activation protein on the invasion and migration of gastric cancer
Experimental and Molecular Pathology 95 (2013) 350–356
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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Effects of the fibroblast activation protein on the invasion and migration of gastric cancer Rui-Fen Wang a, Li-Hong Zhang b, Li-Hui Shan c, Wen-Guang Sun d, Cui-Cui Chai c, Hong-Mei Wu c, Juan C. Ibla e, Li-Feng Wang a,⁎, Jia-Ren Liu e a
Department of Pathology, Xin Hua Hospital affiliated to Shanghai JiaoTong University School of Medicine, Shanghai 200092, People's Republic of China Department of Radiology, The First Clinical College of Harbin Medical University, 23 YouZheng Street, NanGang District, Harbin 150001, People's Republic of China Department of Pathology, The First Clinical College of Harbin Medical University, 23 YouZheng Street, NanGang District, Harbin 150001, People's Republic of China d Department of Clinic Nutrition, Affiliated Sixth People's Hospital of Shanghai JiaoTong University, 600 Yi-shan Road, Shanghai 200233, People's Republic of China e Boston Children's Hospital and Harvard Medical School, 300 Longwood Ave, Boston, MA 02115, United States b c
a r t i c l e
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Article history: Received 25 September 2013 and in revised form 17 October 2013 Available online 25 October 2013 Keywords: Gastric cancer Fibroblast activation protein (FAP) Cancer-associated fibroblasts (CAFs) MGC-803 cells
a b s t r a c t Objective: Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor microenvironment. CAFs are believed to play an important role in tumor invasion and metastasis. Recently, fibroblast activation protein (FAP), a type II integral membrane glycoprotein belonging to the serine protease family, has emerged as a specific marker of CAFs. FAP was overexpressed in stromal fibroblasts of solid malignancies, however, the role of FAP on the process of invasion and metastasis of gastric carcinomas is still unknown. Methods: Expression of FAP level was detected by immunohistochemistry in 60 gastric cancer surgical specimens (28 with omentum metastasis and 32 without), 20 normal human gastric tissues and omentum of 10 nonneoplastic gastric diseases. Fibroblasts were isolated from patient's tissues in the distal normal zones and tumor zones respectively, which were correspondingly designated as normal zone fibroblasts (NFs) and cancer-associated fibroblasts (CAFs). To explore the effects of FAP on NFs or CAFs, fibroblasts were co-cultured with human gastric cancer cell line MGC-803 cells. The ability of invasion and migration of MGC-803 cells was evaluated after transfecting FAP siRNA into CAFs of gastric carcinomas. Results: We investigated the level of expression of FAP in surgical specimens, and found overexpressed in CAFs and non-expressed in NFs. Expression of FAP level in CAFs is significantly associated with Lauren classification, the degree of differentiation, depth of tumor invasion and TNM stage, but it is not correlated to age and gender in gastric carcinoma patients. There was positive correlation between the FAP level with metastasis to the omentum (p b 0.05, R2 = 0.2736, p b 0.05, R2 = 0.1479). In addition, the invasion and migration abilities of MGC-803 cells were significantly increased when cells were co-cultured with CAFs. On the other hand, invasion and migration abilities were significantly decreased by 46.9 and 50.3%, respectively, after knocking down FAP in CAFs. Further, NFs did not have appreciable effect on the invasion and migration of MGC-803 cells. Conclusions: Our findings showed that FAP was overexpressed in CAFs of gastric carcinomas, and siRNA-mediated knock down of FAP significantly suppressed invasion and migration of MGC-803 cells. FAP may be an important regulator in the invasion and migration of gastric cancer and may provide a novel therapeutic target in gastric carcinomas. © 2013 Elsevier Inc. All rights reserved.
Introduction Gastric cancer is the second leading cause of death due to cancer worldwide. The 5-year survival rate is 30–40%, with a poor prognosis for advanced tumor (Cappellani et al., 2010; Kim et al., 2009). The high mortality is closely related to tumor invasion and metastasis. Generally, the complex process of metastasis formation can be divided into several stages: a) migration from the primary tumor, invasion of the ⁎ Corresponding author. E-mail addresses: [email protected] (L.-F. Wang), [email protected] (J.-R. Liu). 0014-4800/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexmp.2013.10.008
surrounding tissue and its extracellular matrix (ECM); b) intravasation into the circulation or the lymphatic system via transmigration through the endothelial lining and the basement membrane; and c) extravasation and metastasis formation at target sites (Hugo et al., 2007). Previous studies have indicated that tumor progression is not controlled by tumor cells independently, and it is also closely related to the tumor stroma. As the most abundant cells in tumor stroma, cancer-associated fibroblasts (CAFs) have distinctly different morphological and biological characteristics compared with normal fibroblasts (NFs). CAFs may promote tumorigenesis and progression through multiple mechanisms, including increased angiogenesis, proliferation, invasion, and inhibition of tumor cell death. These effects are mediated
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through the expression and secretion of numerous growth factors, cytokines, proteases, and extracellular matrix proteins (Brennen et al., 2012; Ishii et al., 2010; Orimo et al., 2005). A key characteristic of CAFs is the expression of fibroblast activation protein (FAP). FAP is a type II integral membrane serine protease of the prolyl oligopeptidase family and classified into the dipeptidyl peptidase (DPP) subfamily. The crystal structure analysis of FAP has documented that the enzyme exists as a homodimer and that dimerization is necessary for enzymatic function (Aertgeerts et al., 2005). It has been reported that FAP is overexpressed in cancer-associated fibroblasts in many types of carcinomas, including colorectal, ovarian, breast, bladder, and lung cancer (Cheng et al., 2002; Tahtis et al., 2003). FAP overexpression has been associated with an overall poorer prognosis in many cancers, including pancreatic (Cohen et al., 2008), hepatocellular (Ju et al., 2009), colon (Saigusa et al., 2011), ovarian (Zhang et al., 2007), and gastrointestinal carcinomas (Saadi et al., 2010). However, the effect of FAP on gastric cancer progression still remains unclear. Our previous study reported a high level of FAP in the invasion front of gastric cancer (Shan et al., 2012). Thus, the objective of this study was to further explore the effects of FAP on the invasion and migration of gastric cancer by the genetic knock-down FAP in CAFs. Materials and methods Tissue specimens All samples used in this study were surgical specimens from patients treated at the First Clinical College of Harbin Medical University (Harbin, China) from 2008 to 2010 after informed consent. Patients who had received preoperative radiotherapy or chemotherapy were excluded from this study. Sixty cases of gastric cancer were selected as the experimental group (28 with omentum metastasis and 32 without omentum metastasis), 20 normal gastric tissues and 10 non-neoplastic gastric diseases were selected as control groups. The patients' mean age was 51.5 (range, 21–72) years. Of the 60 gastric cancers, the histologic classification was characterized according to Lauren classification, and the TNM stage was done according to AJCC/UICC (American Joint Cancer Committee/Union for International Cancer Control, 2009 edition). Immunohistochemistry (Liu et al., 2008) Tissue sections (4 μm) were prepared from formalin-fixed paraffinembedded blocks. Fibroblast activation protein (FAP) was determined by immunostaining. Briefly, the deparaffin sections were quenched with 3% H2O2 for 10 min. After washing with phosphate-buffered saline (PBS), the sections were incubated in 5% bovine serum albumin for 20 min, followed by incubation with FAP primary monoclonal antibody (2 μg/mL) (R&D Systems, Minneapolis, MN) overnight at 4 °C and secondary antibodies for 40 min at room temperature. The location of FAP was visualized by 3,3-diaminobenzidine (Slack et al., 2007) solution for 3–5 min. Finally, the slides were washed with water before being counterstained with hematoxylin. PBS was substituted for primary antibody as a negative control. FAP expression was quantified as the relative percentage of the stroma area with positive staining area in slides. The degree of FAP staining in gastric cancer stroma was classified into three groups (Sugiura et al., 2009): +++, strong staining in N 50% of stroma fibroblasts; ++, moderate staining in N50% of stroma fibroblasts; and +, faint or weak staining in N50% of stroma fibroblasts. Isolation of primary CAFs and NFs Tissue samples corresponding to the tumor zone (tissue within the tumor boundary) and the normal zone (more than 5 cm far from the tumor zone) were minced into 1–2 mm3 fragments, washed twice in antibiotic-containing phosphate-buffered saline (PBS) and disaggregated overnight in serum-free DMEM with 0.1% collagenase at 37 °C
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on a rotator. After 24h, the epithelial cells were separated from the stromal cells by differential centrifugation, as described by Speirs et al. (1996). Stromal cells were washed twice in antibiotic-containing PBS and plated in 35 mm dishes at 37 °C in an incubator containing 5% CO2. The CAFs were isolated from tumor zone and NFs from normal zones. All experiments were performed using fibroblast cultured for 3–10 passages. Characterization of primary fibroblasts (Zhang et al., 2012) Cultured cells were plated in 24-well glass chambers, rinsed twice with cold PBS, fixed in 4% paraformaldehyde for 20min at room temperature and permeabilized with 0.1% Triton X-100 for 30 min on ice. Primary FAP antibody solutions (R&D Systems, Inc.) were added to each chamber and the slides were incubated overnight at 4 °C. After washing with PBS, the cells were incubated with a FITC-conjugated second antibody (1:100) and a TRITC-conjugated second antibody (1:100) for 1h at room temperature. The cells were then covered with a coverslip. The slides were observed under an immunofluorescent microscope. Small interfering RNA transfection CAFs and NFs were recovered and cultured in DMEM containing of 20% FBS without penicillin–streptomycin in 24-well plates (0.5 × 105 cells/well). The cells were incubated overnight. The following day, 10 nmol/L siRNA of FAP-2252 was transiently transfected into cells using siLentFect™ Lipid Reagent (BIO-RAD) in Opti-MEM. Nontargeting siRNA labeled by FAM was regarded as the negative control. No siRNA was transfected into cells in blank control group. The efficiency of transfection was evaluated by counting the number of cells labeled by FAM in a fluorescence microscope after 24 h. Migration and invasion assays (Li et al., 2011) The gastric cancer cell line MGC-803 was purchased from the China Center for Type Culture Collection, Harbin Medical University (Harbin, China). For cell migration assay, MGC-803 cells (2 × l04 cells/well) were seeded on transwell inserts with 8-μm pores (Corning Incorporated, Corning, NewYork, USA) and cultured in serum-free DMEM. NFs or CAFs (5 × l04 cells/well) were transiently transfected with siRNA of FAP-2252 or without siRNA for 24 h and were placed in the lower chamber as chemoattractant. After 24 h of incubation, the cells which did not migrate through the pores were removed by scraping the membrane with a cotton swab. Cells which migrated through the membranes were fixed in 95% ethanol and stained with crystal violet. For cell invasion assay, cells must migrate through an extracellular matrix (ECM) barrier by enzymatic degradation. Thus, the transwell inserts were coated with a uniform layer of 50μL of a 1:3 dilution of Matrigel basement membrane matrix (BD Biosciences, Bedford, Massachusetts, USA) per well. MGC-803 cells were seeded on transwell inserts (2 × l04 cells/well) and cultured for 24 h. Invasive cells that penetrated through the pores and migrated to the underside of the membrane were stained with crystal violet. The number of cells was counted under a microscope (×200) in five random fields by two independent observers with double-blinded samples. Western blotting (Dong et al., 2013) NFs or CAFs were cultured to 100% confluence, transfected, washed, and then serum-starved in RPMI with 0.1% bovine serum albumin for 24 h. The cells were lysed in ice-cold radioimmunoprecipitation assay buffer. Cell lysates were clarified by centrifugation at 12,000 rpm for 10 min and protein concentrations were measured using the BCA™ Protein Assay Kit. A total of 30 μg of protein was separated by an 8% SDS-Page gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 90 min with 5% milk in
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tris-buffered saline containing 0.05% Tween-20 (TBS-T). The primary antibody against FAP (1:50) was incubated overnight at 4 °C, and the secondary antibody was applied (1:2000) and incubated for 60 min at room temperature. Then proteins were visualized by chemiluminescence detection reagent.
Table 1 Association between FAP expression in CAFs and clinicopathological variables of gastric cancer. Variable
n
FAP staining area (%)
p value
Mean ± S.D.
Statistical analysis All data are expressed as the mean ± standard deviation (S.D.). The differences were performed by using the two-tailed Student's t test and one-way analysis of variance (ANOVA). The intensity of FAP staining in relation to various clinicopathological factors was assessed with the χ2 test. The Spearman rank correlation test was conducted to investigate the relationships between FAP value in each primary tumor and a corresponding omentum metastatic lesion. Data analyses were generated using SPSS for Windows version 13.0 (SPSS Inc., Chicago, IL). Statistical significance was set at p b 0.05 and all p values were unadjusted for multiple comparisons. Results FAP expression in gastric tissues In order to understand the overall FAP expression in gastric cancer, 60 gastric cancer and 20 normal gastric tissues were analyzed by immunohistochemistry. The results demonstrated that no FAP expression was detected in both stroma fibroblasts and epithelial cells of normal gastric tissue samples (Fig. 1A). However, FAP overexpression was found in fibroblasts of gastric cancer stroma and no expression of FAP in cancer cells of gastric cancer tissue samples (Fig. 1B, C, E and F). The negative control was shown in Fig. 1D.
FAP staining intensity
p value
+
++
0.228
13 11
7 13
8 8
0.426
22.08 ± 7.78 24.00 ± 6.86
0.499
10 8
12 5
14 11
0.574
32 28
18.12 ± 6.44 27.82 ± 5.01
0.000
13 9
15 5
4 14
0.004
Differentiation degree High 29 Low 31
18.38 ± 6.37 27.21 ± 5.93
0.000
9 9
16 8
4 14
0.017
TNM stage I–II III–IV
24 36
17.25 ± 6.54 26.25 ± 5.90
0.000
14 6
6 16
4 14
0.003
Depth of tumor invasion Serosal invasion 37 No serosal invasion 23
24.80 ± 7.30 17.46 ± 5.54
0.000
6 13
17 5
14 5
0.008
Lymph node metastasis Present 33 Absent 27
24.69 ± 5.71 19.77 ± 8.46
0.010
5 12
15 5
13 10
0.014
Omentum metastases Present 28 Absent 32
27.80 ± 5.78 18.97 ± 6.48
0.000
4 7
7 17
17 8
0.020
Age b55 ≥55
28 32
21.46 ± 7.07 23.83 ± 7.95
Gender Male Female
36 24
Lauren classification Intestinal Diffuse
+++
FAP: fibroblast activation protein. TNM: tumor lymph node metastasis. TNM stage: TNM Classification of Malignant Tumors (TNM).
Association of FAP and clinicopathologic variables The relationship between the quantitative levels of FAP in gastric cancer stroma and clinic-pathologic characteristics was assessed in this study. As shown in Table 1, the staining area of FAP in gastric cancer CAFs correlates with Lauren classification, the degree of differentiation, depth of tumor invasion and TNM stage. The FAP staining area was
highly expressed in advanced-stage disease with 26.3 ± 5.9% and 17.3 ± 6.5% for III–IV stage and I–II stage, respectively (p = 0.000). The FAP expression area was also higher in patients with lymph node and metastases to omentum than that in those without metastases. In addition, analysis of the relationship between FAP staining intensity and
Fig. 1. Expression of fibroblast activation protein (FAP) in human gastric tissues. Panels A and D show no FAP expression in stroma fibroblasts of normal gastric tissues (×200). Panels B and C show FAP overexpression in almost all fibroblasts embedded in stroma of intestinal type gastric cancer tissues (×200 and × 400). Panels E and F show FAP overexpression in almost all fibroblasts embedded in stroma of diffuse type gastric cancer tissues (×200 and × 400). Scale bar = 100 μm.
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clinic-pathological factors also showed a significant association with Lauren classification, degree of differentiation, depth of tumor invasion, and TNM stage. No correlation was found between the staining intensity of FAP and age or gender (Table 1).
Identification of primary NFs and CAFs The fibroblasts from the normal and tumor zone tissues were designated to NFs and CAFs. In our study, NFs and CAF populations were successfully isolated from gastric tissues of the same patient. Fibroblasts had the typical characteristics: a long spindle-like morphology, strong expression of the fibroblastic marker (vimentin) but negative expression for epithelial marker (cytokeratin) (Fig. 2A). To identify the NFs and CAFs, FAP expression was determined by immunofluorescence. The results showed that FAP protein was overexpressed in CAFs when
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compared to NFs (Fig. 2B). Additional results from flow cytometry also showed an overexpression of FAP in CAFs (Fig. 2C). FAP siRNA inhibited the invasion and migration of gastric cancer cells in vitro MGC-803 cells co-cultured with CAFs and NFs showed increased invasion and migration in comparison with MGC-803 cells alone. CAFs significantly increased the invasion and migration of MGC-803 cells when compared to NFs (Fig. 3A–D) (p b 0.05). In addition, when siRNA against FAP was transfected into CAFs, MGC-803 cells were decreased by 46.9% when co-cultured with the CAFs in comparison with the control group. NFs transfected with FAP siRNA did not increase MGC-803 cell invasion when compared to the control group (Fig. 3A and B). The same pattern was also observed in the migration of MGC-803 cells. Migration rate of MGC-803 cells co-cultured with CAFs was decreased by 50.3% when compared to the control group (Fig. 3C and D) (p b 0.05). Western blot analysis using mAb anti-FAP was performed on samples that were suspended in SDS sample buffer but not boiled to preserve the 170 kDa FAP dimer. Dimer (170 kDa) of FAP is essential to its enzymatic activity. Our result showed the high expression of FAP in CAFs. The expression of FAP (170 kDa and 97 KDa dimer) was not detected in CAFs or NFs when transfected with FAP-2252 siRNA (Fig. 3E and F) (p b 0.05). FAP expression in omentum To explore the relationship between FAP overexpression and metastases to omentum, FAP expression intensity in omentum was measured in 50 patients, including 10 non-neoplastic gastric diseases, 20 gastric cancers with omentum metastasis, and 20 gastric cancers without omentum metastasis. No FAP expression was detected in stromal fibroblasts of omentum tissue samples in the non-neoplastic gastric diseases (Fig. 4A). In omentum tissue samples with gastric cancer cell metastasis, the tumor cells formed solid invading nests, surrounded by omentum adipose and CAFs which was expressed as FAP protein. With the omentum metastasis enlarged, the stroma cells were found to have with high expression of FAP protein (Fig. 4B). One striking finding was that some omentum tissue samples free of gastric metastasis also demonstrated the overexpression of FAP protein (Fig. 4C). The correlation of FAP overexpression in primary tumor CAFs and corresponding omentum metastasis lesions was also analyzed in this study. Our results showed significant positive correlation between FAP expression in primary tumor CAFs and corresponding omentum with/without gastric cancer metastasis (Fig. 4D–E). Discussion
Fig. 2. Characteristics of primary fibroblasts from the different zones of human gastric tumor tissue. (A) NFs and CAFs were examined expression of vimentin and cytokeratin by immunocytochemistry. The fibroblasts from NFs and CAFs had a long, spindle-like morphology and the immunostaining of vimentin was positive. These cells were negative cytokeratin expression. (B) NFs and CAFs were universally expressed FAP by immunofluorescence. FAP was significantly overexpressed in CAFs compared with the levels in NFs. (C) Flow cytometry results showed that FAP was variably expressed in the isolated NFs and CAFs, with the higher expression in CAFs. Bar = 100 μm.
CAFs play important roles in tumor invasion and metastasis (Cirri and Chiarugi, 2011; Kunz-Schughart and Knuechel, 2002a; KunzSchughart and Knuechel, 2002b). A key characteristic of CAFs is the expression of FAP (O'Brien and O'Connor, 2008). Although FAP was found to be overexpressed in stroma of multiple cancers, there is little information about FAP expression in gastric cancer. In the present study, FAP expression was detected in gastric cancer stroma and its correlation explored with clinic-pathological characteristics. Our results showed no FAP expression in stroma of normal gastric tissue samples, however there was overexpression in CAFs of gastric cancer tissue samples. FAP immunostaining intensity was associated with Lauren classification, degree of differentiation, depth of tumor invasion and TNM stage. Our previous findings showed that stroma fibroblasts from gastric cancer invasion front (the interface zone fibroblasts, INFs) had strong positive FAP expression. The degree of FAP expression was higher in INFs than that in NFs and CAFs. In addition, the degree of FAP expression in INFs was significantly related to Lauren classification, degree of differentiation, depth of tumor invasion and TNM stage (Shan et al., 2012). It
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Fig. 3. Effects of FAP siRNA in CAFs or NFs on invasion and migration of MGC-803 cells. Invading and migrating cells on the reverse side of the membranes were stained by crystal violet. CAFs transfected with FAP-2252 siRNA showed a suppressed invasion and migration of MGC-803 cell when compared to the control group (p b 0.05). However, NFs transfected with FAP2252 siRNA did not increase invasion and migration of MGC-803 cell (Fig. 3A and C, ×200). These results showed that the FAP siRNA in CAFs remarkably suppressed invasion and migration of MGC-803 cells compared with control (Fig. 3B and D). Both CAFs and NFs increased invasion and migration of MGC-803 cells; the CAFs had a far greater potential to increase invasion and migration of MGC-803 cells, compared with NFs (Fig. 3A–D) (p b 0.05). The results also revealed that expression of FAP level in CAFs is stronger than those in NFs. FAP (170 kDa and 97 KDa dimer) were not detected in CAFs or NFs transfected with FAP-2252 siRNA when compared to the control group (Fig. 3E, 3F) (p b 0.05).
suggested that CAFs from the gastric cancer invasion front may supply their own optimal microenvironment for tumor cells invasion and migration.
CAFs and NFs were isolated from the tumor zone and normal zone respectively of the same patients with gastric cancer. FAP was found to be overexpressed in CAFs in comparison with NFs. CAFs significantly
Fig. 4. FAP expression in omentum tissues of surgical specimens by immunostaining. (A) No FAP expression in the fibroblasts of normal omentum tissues. (B) Omentum tissues implanted with gastric cancer cells showed a large number of CAFs which was overexpressed as FAP (magnification × 200, inset: magnification ×400); (C) Omentum tissue free detectable cancer cells in patients with gastric carcinoma also have CAFs with FAP overexpression (×200 and × 400); scale bar = 100 μm. (D–E) There was a significant positive correlation between FAP expression in primary tumor and corresponding omentum with/without metastasis (p b 0.05).
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promoted the invasion and migration of MGC-803 cells when compared to NFs. Our previous study also found that INFs isolated from the gastric cancer invasion front had a high expression of FAP protein when compared to CAFs and NFs. This indicated that INFs have a promoted effect on invasion and migration of MGC-803 cells. To explore whether FAP was a regulator of gastric cancer cell invasion and migration, FAP siRNA was transfected into CAFs by genetic knock-down FAP. It was found that the invasion and migration of MGC-803 cells were significantly suppressed. We demonstrated that CAFs could promote the invasion and migration of gastric cancer cells by up-regulating FAP which may be a crucial factor in tumor invasion and migration of gastric cancer. Other studies also have shown that FAP increased the invasion, proliferation and migration of HO-8910PM ovarian cancer cells (Chen et al., 2009). FAP-expressing cells were an immune-suppressive component of the tumor microenvironment (Kraman et al., 2010). In another study, knock-down of FAP expression by siRNA lentiviral vector transfection resulted in a low or no expression of FAP in CAFs to inhibit cell growth. These results indicate that FAP was an important regulator of the microenvironment in tumor formation (Lai et al., 2012). In the present study, a seemly paradox phenomenon regarding FAP overexpression appeared in the gastric cancer tissue of surgical specimens and NF from primary cell culture in vitro. We found that FAP expression was negative in fibroblasts of normal gastric tissues of surgical specimens, weakly positive expression in NFs from primary cell culture, and high expression of FAP in CAF. The ability of invasion and migration did not change when MGC-803 cells were co-cultured with NF knock-down FAP expression. In the present study, we describe an interesting phenomenon in which CAFs with FAP overexpression not only existed in omentum with gastric cancer cells metastasis, but also existed in omentum of some patients without metastasis. This suggested that CAFs arrive at target organs before the implantation of cancer cells. The CAFs in omentum may promote cancer cell adherence, allowing for further growth, invasion, and metastases. We also found a significant positive correlation between FAP expression in primary tumor CAFs and corresponding omentum lesions with/without gastric cancer metastasis. A high concordance of FAP expression in pairs of primary tumor and their corresponding omentum metastasis may suggest that individual cancer in metastases sites will produce their own optimal microenvironment to support migration. It has also been demonstrated that CAFs existed not only in omentum with epithelial ovarian cancer (EOC) metastasis but also in omentum of patients without metastasis (Zhang et al., 2011). Another study showed that TGF-β1 (transforming growth factor-β1), HGF (hepatocyte growth factor) and MMP-2 (matrix metalloproteinase-2) may be involved in the adhesion and invasion of EOC cells, indicating that activated fibroblasts in omentum form an optimal environment to promote EOC cells implantation (Cai et al., 2012). In the present study, FAP expression in stroma of gastric cancers with omentum metastases and lymph node metastases was much higher than that of without metastases. This may be related to the collagenolytic activity of FAP resulting in increased invasion and metastases by regional blood vessels and lymph nodes (Forssell et al., 2007; Malik et al., 2011). In summary, CAFs can promote gastric cancer cell invasion and migration by up-regulating FAP. Therefore, inhibition of FAP expression could be used as a new potential therapeutic strategy against gastric cancer. The FAP inhibitor PT630 has been found to have potent anticancer effects in several mouse models (Santos et al., 2009). Another anti-FAP antibody FAP5-DM1 can induce long-lasting inhibition of tumor growth (Ostermann et al., 2008). FAP-based pre-clinic drug strategy has been shown as promising in achieving targeted delivery of anticancer agents (Liu et al., 2012). However, the effects of FAP on tumor growth and invasion, and the exact molecular mechanisms remain to need further study.
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Conflict of interest statement They declared no conflicts of interests. Acknowledgments This work was supported by Natural Science Foundation of HeiLongJiang Province (D201103) and National Natural Science Foundation of China (No. 81072296). Li-Feng Wang and Jia-Ren Liu are cocorresponding authors. Rui-Fen Wang and Li-Hong Zhang are co-first authors. References Aertgeerts, K., et al., 2005. Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein alpha. J. Biol. Chem. 280, 19441–19444. Brennen, W.N., et al., 2012. Rationale behind targeting fibroblast activation proteinexpressing carcinoma-associated fibroblasts as a novel chemotherapeutic strategy. Mol. Cancer Ther. 11, 257–266. Cai, J., et al., 2012. Fibroblasts in omentum activated by tumor cells promote ovarian cancer growth, adhesion and invasiveness. Carcinogenesis 33, 20–29. Cappellani, A., et al., 2010. Clinical and biological markers in gastric cancer: update and perspectives. Front. 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Effects of the fibroblast activation protein on the invasion and migration of gastric cancer Rui-Fen Wang a, Li-Hong Zhang b, Li-Hui Shan c, Wen-Guang Sun d, Cui-Cui Chai c, Hong-Mei Wu c, Juan C. Ibla e, Li-Feng Wang a,⁎, Jia-Ren Liu e a
Department of Pathology, Xin Hua Hospital affiliated to Shanghai JiaoTong University School of Medicine, Shanghai 200092, People's Republic of China Department of Radiology, The First Clinical College of Harbin Medical University, 23 YouZheng Street, NanGang District, Harbin 150001, People's Republic of China Department of Pathology, The First Clinical College of Harbin Medical University, 23 YouZheng Street, NanGang District, Harbin 150001, People's Republic of China d Department of Clinic Nutrition, Affiliated Sixth People's Hospital of Shanghai JiaoTong University, 600 Yi-shan Road, Shanghai 200233, People's Republic of China e Boston Children's Hospital and Harvard Medical School, 300 Longwood Ave, Boston, MA 02115, United States b c
a r t i c l e
i n f o
Article history: Received 25 September 2013 and in revised form 17 October 2013 Available online 25 October 2013 Keywords: Gastric cancer Fibroblast activation protein (FAP) Cancer-associated fibroblasts (CAFs) MGC-803 cells
a b s t r a c t Objective: Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor microenvironment. CAFs are believed to play an important role in tumor invasion and metastasis. Recently, fibroblast activation protein (FAP), a type II integral membrane glycoprotein belonging to the serine protease family, has emerged as a specific marker of CAFs. FAP was overexpressed in stromal fibroblasts of solid malignancies, however, the role of FAP on the process of invasion and metastasis of gastric carcinomas is still unknown. Methods: Expression of FAP level was detected by immunohistochemistry in 60 gastric cancer surgical specimens (28 with omentum metastasis and 32 without), 20 normal human gastric tissues and omentum of 10 nonneoplastic gastric diseases. Fibroblasts were isolated from patient's tissues in the distal normal zones and tumor zones respectively, which were correspondingly designated as normal zone fibroblasts (NFs) and cancer-associated fibroblasts (CAFs). To explore the effects of FAP on NFs or CAFs, fibroblasts were co-cultured with human gastric cancer cell line MGC-803 cells. The ability of invasion and migration of MGC-803 cells was evaluated after transfecting FAP siRNA into CAFs of gastric carcinomas. Results: We investigated the level of expression of FAP in surgical specimens, and found overexpressed in CAFs and non-expressed in NFs. Expression of FAP level in CAFs is significantly associated with Lauren classification, the degree of differentiation, depth of tumor invasion and TNM stage, but it is not correlated to age and gender in gastric carcinoma patients. There was positive correlation between the FAP level with metastasis to the omentum (p b 0.05, R2 = 0.2736, p b 0.05, R2 = 0.1479). In addition, the invasion and migration abilities of MGC-803 cells were significantly increased when cells were co-cultured with CAFs. On the other hand, invasion and migration abilities were significantly decreased by 46.9 and 50.3%, respectively, after knocking down FAP in CAFs. Further, NFs did not have appreciable effect on the invasion and migration of MGC-803 cells. Conclusions: Our findings showed that FAP was overexpressed in CAFs of gastric carcinomas, and siRNA-mediated knock down of FAP significantly suppressed invasion and migration of MGC-803 cells. FAP may be an important regulator in the invasion and migration of gastric cancer and may provide a novel therapeutic target in gastric carcinomas. © 2013 Elsevier Inc. All rights reserved.
Introduction Gastric cancer is the second leading cause of death due to cancer worldwide. The 5-year survival rate is 30–40%, with a poor prognosis for advanced tumor (Cappellani et al., 2010; Kim et al., 2009). The high mortality is closely related to tumor invasion and metastasis. Generally, the complex process of metastasis formation can be divided into several stages: a) migration from the primary tumor, invasion of the ⁎ Corresponding author. E-mail addresses: [email protected] (L.-F. Wang), [email protected] (J.-R. Liu). 0014-4800/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexmp.2013.10.008
surrounding tissue and its extracellular matrix (ECM); b) intravasation into the circulation or the lymphatic system via transmigration through the endothelial lining and the basement membrane; and c) extravasation and metastasis formation at target sites (Hugo et al., 2007). Previous studies have indicated that tumor progression is not controlled by tumor cells independently, and it is also closely related to the tumor stroma. As the most abundant cells in tumor stroma, cancer-associated fibroblasts (CAFs) have distinctly different morphological and biological characteristics compared with normal fibroblasts (NFs). CAFs may promote tumorigenesis and progression through multiple mechanisms, including increased angiogenesis, proliferation, invasion, and inhibition of tumor cell death. These effects are mediated
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through the expression and secretion of numerous growth factors, cytokines, proteases, and extracellular matrix proteins (Brennen et al., 2012; Ishii et al., 2010; Orimo et al., 2005). A key characteristic of CAFs is the expression of fibroblast activation protein (FAP). FAP is a type II integral membrane serine protease of the prolyl oligopeptidase family and classified into the dipeptidyl peptidase (DPP) subfamily. The crystal structure analysis of FAP has documented that the enzyme exists as a homodimer and that dimerization is necessary for enzymatic function (Aertgeerts et al., 2005). It has been reported that FAP is overexpressed in cancer-associated fibroblasts in many types of carcinomas, including colorectal, ovarian, breast, bladder, and lung cancer (Cheng et al., 2002; Tahtis et al., 2003). FAP overexpression has been associated with an overall poorer prognosis in many cancers, including pancreatic (Cohen et al., 2008), hepatocellular (Ju et al., 2009), colon (Saigusa et al., 2011), ovarian (Zhang et al., 2007), and gastrointestinal carcinomas (Saadi et al., 2010). However, the effect of FAP on gastric cancer progression still remains unclear. Our previous study reported a high level of FAP in the invasion front of gastric cancer (Shan et al., 2012). Thus, the objective of this study was to further explore the effects of FAP on the invasion and migration of gastric cancer by the genetic knock-down FAP in CAFs. Materials and methods Tissue specimens All samples used in this study were surgical specimens from patients treated at the First Clinical College of Harbin Medical University (Harbin, China) from 2008 to 2010 after informed consent. Patients who had received preoperative radiotherapy or chemotherapy were excluded from this study. Sixty cases of gastric cancer were selected as the experimental group (28 with omentum metastasis and 32 without omentum metastasis), 20 normal gastric tissues and 10 non-neoplastic gastric diseases were selected as control groups. The patients' mean age was 51.5 (range, 21–72) years. Of the 60 gastric cancers, the histologic classification was characterized according to Lauren classification, and the TNM stage was done according to AJCC/UICC (American Joint Cancer Committee/Union for International Cancer Control, 2009 edition). Immunohistochemistry (Liu et al., 2008) Tissue sections (4 μm) were prepared from formalin-fixed paraffinembedded blocks. Fibroblast activation protein (FAP) was determined by immunostaining. Briefly, the deparaffin sections were quenched with 3% H2O2 for 10 min. After washing with phosphate-buffered saline (PBS), the sections were incubated in 5% bovine serum albumin for 20 min, followed by incubation with FAP primary monoclonal antibody (2 μg/mL) (R&D Systems, Minneapolis, MN) overnight at 4 °C and secondary antibodies for 40 min at room temperature. The location of FAP was visualized by 3,3-diaminobenzidine (Slack et al., 2007) solution for 3–5 min. Finally, the slides were washed with water before being counterstained with hematoxylin. PBS was substituted for primary antibody as a negative control. FAP expression was quantified as the relative percentage of the stroma area with positive staining area in slides. The degree of FAP staining in gastric cancer stroma was classified into three groups (Sugiura et al., 2009): +++, strong staining in N 50% of stroma fibroblasts; ++, moderate staining in N50% of stroma fibroblasts; and +, faint or weak staining in N50% of stroma fibroblasts. Isolation of primary CAFs and NFs Tissue samples corresponding to the tumor zone (tissue within the tumor boundary) and the normal zone (more than 5 cm far from the tumor zone) were minced into 1–2 mm3 fragments, washed twice in antibiotic-containing phosphate-buffered saline (PBS) and disaggregated overnight in serum-free DMEM with 0.1% collagenase at 37 °C
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on a rotator. After 24h, the epithelial cells were separated from the stromal cells by differential centrifugation, as described by Speirs et al. (1996). Stromal cells were washed twice in antibiotic-containing PBS and plated in 35 mm dishes at 37 °C in an incubator containing 5% CO2. The CAFs were isolated from tumor zone and NFs from normal zones. All experiments were performed using fibroblast cultured for 3–10 passages. Characterization of primary fibroblasts (Zhang et al., 2012) Cultured cells were plated in 24-well glass chambers, rinsed twice with cold PBS, fixed in 4% paraformaldehyde for 20min at room temperature and permeabilized with 0.1% Triton X-100 for 30 min on ice. Primary FAP antibody solutions (R&D Systems, Inc.) were added to each chamber and the slides were incubated overnight at 4 °C. After washing with PBS, the cells were incubated with a FITC-conjugated second antibody (1:100) and a TRITC-conjugated second antibody (1:100) for 1h at room temperature. The cells were then covered with a coverslip. The slides were observed under an immunofluorescent microscope. Small interfering RNA transfection CAFs and NFs were recovered and cultured in DMEM containing of 20% FBS without penicillin–streptomycin in 24-well plates (0.5 × 105 cells/well). The cells were incubated overnight. The following day, 10 nmol/L siRNA of FAP-2252 was transiently transfected into cells using siLentFect™ Lipid Reagent (BIO-RAD) in Opti-MEM. Nontargeting siRNA labeled by FAM was regarded as the negative control. No siRNA was transfected into cells in blank control group. The efficiency of transfection was evaluated by counting the number of cells labeled by FAM in a fluorescence microscope after 24 h. Migration and invasion assays (Li et al., 2011) The gastric cancer cell line MGC-803 was purchased from the China Center for Type Culture Collection, Harbin Medical University (Harbin, China). For cell migration assay, MGC-803 cells (2 × l04 cells/well) were seeded on transwell inserts with 8-μm pores (Corning Incorporated, Corning, NewYork, USA) and cultured in serum-free DMEM. NFs or CAFs (5 × l04 cells/well) were transiently transfected with siRNA of FAP-2252 or without siRNA for 24 h and were placed in the lower chamber as chemoattractant. After 24 h of incubation, the cells which did not migrate through the pores were removed by scraping the membrane with a cotton swab. Cells which migrated through the membranes were fixed in 95% ethanol and stained with crystal violet. For cell invasion assay, cells must migrate through an extracellular matrix (ECM) barrier by enzymatic degradation. Thus, the transwell inserts were coated with a uniform layer of 50μL of a 1:3 dilution of Matrigel basement membrane matrix (BD Biosciences, Bedford, Massachusetts, USA) per well. MGC-803 cells were seeded on transwell inserts (2 × l04 cells/well) and cultured for 24 h. Invasive cells that penetrated through the pores and migrated to the underside of the membrane were stained with crystal violet. The number of cells was counted under a microscope (×200) in five random fields by two independent observers with double-blinded samples. Western blotting (Dong et al., 2013) NFs or CAFs were cultured to 100% confluence, transfected, washed, and then serum-starved in RPMI with 0.1% bovine serum albumin for 24 h. The cells were lysed in ice-cold radioimmunoprecipitation assay buffer. Cell lysates were clarified by centrifugation at 12,000 rpm for 10 min and protein concentrations were measured using the BCA™ Protein Assay Kit. A total of 30 μg of protein was separated by an 8% SDS-Page gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 90 min with 5% milk in
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tris-buffered saline containing 0.05% Tween-20 (TBS-T). The primary antibody against FAP (1:50) was incubated overnight at 4 °C, and the secondary antibody was applied (1:2000) and incubated for 60 min at room temperature. Then proteins were visualized by chemiluminescence detection reagent.
Table 1 Association between FAP expression in CAFs and clinicopathological variables of gastric cancer. Variable
n
FAP staining area (%)
p value
Mean ± S.D.
Statistical analysis All data are expressed as the mean ± standard deviation (S.D.). The differences were performed by using the two-tailed Student's t test and one-way analysis of variance (ANOVA). The intensity of FAP staining in relation to various clinicopathological factors was assessed with the χ2 test. The Spearman rank correlation test was conducted to investigate the relationships between FAP value in each primary tumor and a corresponding omentum metastatic lesion. Data analyses were generated using SPSS for Windows version 13.0 (SPSS Inc., Chicago, IL). Statistical significance was set at p b 0.05 and all p values were unadjusted for multiple comparisons. Results FAP expression in gastric tissues In order to understand the overall FAP expression in gastric cancer, 60 gastric cancer and 20 normal gastric tissues were analyzed by immunohistochemistry. The results demonstrated that no FAP expression was detected in both stroma fibroblasts and epithelial cells of normal gastric tissue samples (Fig. 1A). However, FAP overexpression was found in fibroblasts of gastric cancer stroma and no expression of FAP in cancer cells of gastric cancer tissue samples (Fig. 1B, C, E and F). The negative control was shown in Fig. 1D.
FAP staining intensity
p value
+
++
0.228
13 11
7 13
8 8
0.426
22.08 ± 7.78 24.00 ± 6.86
0.499
10 8
12 5
14 11
0.574
32 28
18.12 ± 6.44 27.82 ± 5.01
0.000
13 9
15 5
4 14
0.004
Differentiation degree High 29 Low 31
18.38 ± 6.37 27.21 ± 5.93
0.000
9 9
16 8
4 14
0.017
TNM stage I–II III–IV
24 36
17.25 ± 6.54 26.25 ± 5.90
0.000
14 6
6 16
4 14
0.003
Depth of tumor invasion Serosal invasion 37 No serosal invasion 23
24.80 ± 7.30 17.46 ± 5.54
0.000
6 13
17 5
14 5
0.008
Lymph node metastasis Present 33 Absent 27
24.69 ± 5.71 19.77 ± 8.46
0.010
5 12
15 5
13 10
0.014
Omentum metastases Present 28 Absent 32
27.80 ± 5.78 18.97 ± 6.48
0.000
4 7
7 17
17 8
0.020
Age b55 ≥55
28 32
21.46 ± 7.07 23.83 ± 7.95
Gender Male Female
36 24
Lauren classification Intestinal Diffuse
+++
FAP: fibroblast activation protein. TNM: tumor lymph node metastasis. TNM stage: TNM Classification of Malignant Tumors (TNM).
Association of FAP and clinicopathologic variables The relationship between the quantitative levels of FAP in gastric cancer stroma and clinic-pathologic characteristics was assessed in this study. As shown in Table 1, the staining area of FAP in gastric cancer CAFs correlates with Lauren classification, the degree of differentiation, depth of tumor invasion and TNM stage. The FAP staining area was
highly expressed in advanced-stage disease with 26.3 ± 5.9% and 17.3 ± 6.5% for III–IV stage and I–II stage, respectively (p = 0.000). The FAP expression area was also higher in patients with lymph node and metastases to omentum than that in those without metastases. In addition, analysis of the relationship between FAP staining intensity and
Fig. 1. Expression of fibroblast activation protein (FAP) in human gastric tissues. Panels A and D show no FAP expression in stroma fibroblasts of normal gastric tissues (×200). Panels B and C show FAP overexpression in almost all fibroblasts embedded in stroma of intestinal type gastric cancer tissues (×200 and × 400). Panels E and F show FAP overexpression in almost all fibroblasts embedded in stroma of diffuse type gastric cancer tissues (×200 and × 400). Scale bar = 100 μm.
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clinic-pathological factors also showed a significant association with Lauren classification, degree of differentiation, depth of tumor invasion, and TNM stage. No correlation was found between the staining intensity of FAP and age or gender (Table 1).
Identification of primary NFs and CAFs The fibroblasts from the normal and tumor zone tissues were designated to NFs and CAFs. In our study, NFs and CAF populations were successfully isolated from gastric tissues of the same patient. Fibroblasts had the typical characteristics: a long spindle-like morphology, strong expression of the fibroblastic marker (vimentin) but negative expression for epithelial marker (cytokeratin) (Fig. 2A). To identify the NFs and CAFs, FAP expression was determined by immunofluorescence. The results showed that FAP protein was overexpressed in CAFs when
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compared to NFs (Fig. 2B). Additional results from flow cytometry also showed an overexpression of FAP in CAFs (Fig. 2C). FAP siRNA inhibited the invasion and migration of gastric cancer cells in vitro MGC-803 cells co-cultured with CAFs and NFs showed increased invasion and migration in comparison with MGC-803 cells alone. CAFs significantly increased the invasion and migration of MGC-803 cells when compared to NFs (Fig. 3A–D) (p b 0.05). In addition, when siRNA against FAP was transfected into CAFs, MGC-803 cells were decreased by 46.9% when co-cultured with the CAFs in comparison with the control group. NFs transfected with FAP siRNA did not increase MGC-803 cell invasion when compared to the control group (Fig. 3A and B). The same pattern was also observed in the migration of MGC-803 cells. Migration rate of MGC-803 cells co-cultured with CAFs was decreased by 50.3% when compared to the control group (Fig. 3C and D) (p b 0.05). Western blot analysis using mAb anti-FAP was performed on samples that were suspended in SDS sample buffer but not boiled to preserve the 170 kDa FAP dimer. Dimer (170 kDa) of FAP is essential to its enzymatic activity. Our result showed the high expression of FAP in CAFs. The expression of FAP (170 kDa and 97 KDa dimer) was not detected in CAFs or NFs when transfected with FAP-2252 siRNA (Fig. 3E and F) (p b 0.05). FAP expression in omentum To explore the relationship between FAP overexpression and metastases to omentum, FAP expression intensity in omentum was measured in 50 patients, including 10 non-neoplastic gastric diseases, 20 gastric cancers with omentum metastasis, and 20 gastric cancers without omentum metastasis. No FAP expression was detected in stromal fibroblasts of omentum tissue samples in the non-neoplastic gastric diseases (Fig. 4A). In omentum tissue samples with gastric cancer cell metastasis, the tumor cells formed solid invading nests, surrounded by omentum adipose and CAFs which was expressed as FAP protein. With the omentum metastasis enlarged, the stroma cells were found to have with high expression of FAP protein (Fig. 4B). One striking finding was that some omentum tissue samples free of gastric metastasis also demonstrated the overexpression of FAP protein (Fig. 4C). The correlation of FAP overexpression in primary tumor CAFs and corresponding omentum metastasis lesions was also analyzed in this study. Our results showed significant positive correlation between FAP expression in primary tumor CAFs and corresponding omentum with/without gastric cancer metastasis (Fig. 4D–E). Discussion
Fig. 2. Characteristics of primary fibroblasts from the different zones of human gastric tumor tissue. (A) NFs and CAFs were examined expression of vimentin and cytokeratin by immunocytochemistry. The fibroblasts from NFs and CAFs had a long, spindle-like morphology and the immunostaining of vimentin was positive. These cells were negative cytokeratin expression. (B) NFs and CAFs were universally expressed FAP by immunofluorescence. FAP was significantly overexpressed in CAFs compared with the levels in NFs. (C) Flow cytometry results showed that FAP was variably expressed in the isolated NFs and CAFs, with the higher expression in CAFs. Bar = 100 μm.
CAFs play important roles in tumor invasion and metastasis (Cirri and Chiarugi, 2011; Kunz-Schughart and Knuechel, 2002a; KunzSchughart and Knuechel, 2002b). A key characteristic of CAFs is the expression of FAP (O'Brien and O'Connor, 2008). Although FAP was found to be overexpressed in stroma of multiple cancers, there is little information about FAP expression in gastric cancer. In the present study, FAP expression was detected in gastric cancer stroma and its correlation explored with clinic-pathological characteristics. Our results showed no FAP expression in stroma of normal gastric tissue samples, however there was overexpression in CAFs of gastric cancer tissue samples. FAP immunostaining intensity was associated with Lauren classification, degree of differentiation, depth of tumor invasion and TNM stage. Our previous findings showed that stroma fibroblasts from gastric cancer invasion front (the interface zone fibroblasts, INFs) had strong positive FAP expression. The degree of FAP expression was higher in INFs than that in NFs and CAFs. In addition, the degree of FAP expression in INFs was significantly related to Lauren classification, degree of differentiation, depth of tumor invasion and TNM stage (Shan et al., 2012). It
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Fig. 3. Effects of FAP siRNA in CAFs or NFs on invasion and migration of MGC-803 cells. Invading and migrating cells on the reverse side of the membranes were stained by crystal violet. CAFs transfected with FAP-2252 siRNA showed a suppressed invasion and migration of MGC-803 cell when compared to the control group (p b 0.05). However, NFs transfected with FAP2252 siRNA did not increase invasion and migration of MGC-803 cell (Fig. 3A and C, ×200). These results showed that the FAP siRNA in CAFs remarkably suppressed invasion and migration of MGC-803 cells compared with control (Fig. 3B and D). Both CAFs and NFs increased invasion and migration of MGC-803 cells; the CAFs had a far greater potential to increase invasion and migration of MGC-803 cells, compared with NFs (Fig. 3A–D) (p b 0.05). The results also revealed that expression of FAP level in CAFs is stronger than those in NFs. FAP (170 kDa and 97 KDa dimer) were not detected in CAFs or NFs transfected with FAP-2252 siRNA when compared to the control group (Fig. 3E, 3F) (p b 0.05).
suggested that CAFs from the gastric cancer invasion front may supply their own optimal microenvironment for tumor cells invasion and migration.
CAFs and NFs were isolated from the tumor zone and normal zone respectively of the same patients with gastric cancer. FAP was found to be overexpressed in CAFs in comparison with NFs. CAFs significantly
Fig. 4. FAP expression in omentum tissues of surgical specimens by immunostaining. (A) No FAP expression in the fibroblasts of normal omentum tissues. (B) Omentum tissues implanted with gastric cancer cells showed a large number of CAFs which was overexpressed as FAP (magnification × 200, inset: magnification ×400); (C) Omentum tissue free detectable cancer cells in patients with gastric carcinoma also have CAFs with FAP overexpression (×200 and × 400); scale bar = 100 μm. (D–E) There was a significant positive correlation between FAP expression in primary tumor and corresponding omentum with/without metastasis (p b 0.05).
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promoted the invasion and migration of MGC-803 cells when compared to NFs. Our previous study also found that INFs isolated from the gastric cancer invasion front had a high expression of FAP protein when compared to CAFs and NFs. This indicated that INFs have a promoted effect on invasion and migration of MGC-803 cells. To explore whether FAP was a regulator of gastric cancer cell invasion and migration, FAP siRNA was transfected into CAFs by genetic knock-down FAP. It was found that the invasion and migration of MGC-803 cells were significantly suppressed. We demonstrated that CAFs could promote the invasion and migration of gastric cancer cells by up-regulating FAP which may be a crucial factor in tumor invasion and migration of gastric cancer. Other studies also have shown that FAP increased the invasion, proliferation and migration of HO-8910PM ovarian cancer cells (Chen et al., 2009). FAP-expressing cells were an immune-suppressive component of the tumor microenvironment (Kraman et al., 2010). In another study, knock-down of FAP expression by siRNA lentiviral vector transfection resulted in a low or no expression of FAP in CAFs to inhibit cell growth. These results indicate that FAP was an important regulator of the microenvironment in tumor formation (Lai et al., 2012). In the present study, a seemly paradox phenomenon regarding FAP overexpression appeared in the gastric cancer tissue of surgical specimens and NF from primary cell culture in vitro. We found that FAP expression was negative in fibroblasts of normal gastric tissues of surgical specimens, weakly positive expression in NFs from primary cell culture, and high expression of FAP in CAF. The ability of invasion and migration did not change when MGC-803 cells were co-cultured with NF knock-down FAP expression. In the present study, we describe an interesting phenomenon in which CAFs with FAP overexpression not only existed in omentum with gastric cancer cells metastasis, but also existed in omentum of some patients without metastasis. This suggested that CAFs arrive at target organs before the implantation of cancer cells. The CAFs in omentum may promote cancer cell adherence, allowing for further growth, invasion, and metastases. We also found a significant positive correlation between FAP expression in primary tumor CAFs and corresponding omentum lesions with/without gastric cancer metastasis. A high concordance of FAP expression in pairs of primary tumor and their corresponding omentum metastasis may suggest that individual cancer in metastases sites will produce their own optimal microenvironment to support migration. It has also been demonstrated that CAFs existed not only in omentum with epithelial ovarian cancer (EOC) metastasis but also in omentum of patients without metastasis (Zhang et al., 2011). Another study showed that TGF-β1 (transforming growth factor-β1), HGF (hepatocyte growth factor) and MMP-2 (matrix metalloproteinase-2) may be involved in the adhesion and invasion of EOC cells, indicating that activated fibroblasts in omentum form an optimal environment to promote EOC cells implantation (Cai et al., 2012). In the present study, FAP expression in stroma of gastric cancers with omentum metastases and lymph node metastases was much higher than that of without metastases. This may be related to the collagenolytic activity of FAP resulting in increased invasion and metastases by regional blood vessels and lymph nodes (Forssell et al., 2007; Malik et al., 2011). In summary, CAFs can promote gastric cancer cell invasion and migration by up-regulating FAP. Therefore, inhibition of FAP expression could be used as a new potential therapeutic strategy against gastric cancer. The FAP inhibitor PT630 has been found to have potent anticancer effects in several mouse models (Santos et al., 2009). Another anti-FAP antibody FAP5-DM1 can induce long-lasting inhibition of tumor growth (Ostermann et al., 2008). FAP-based pre-clinic drug strategy has been shown as promising in achieving targeted delivery of anticancer agents (Liu et al., 2012). However, the effects of FAP on tumor growth and invasion, and the exact molecular mechanisms remain to need further study.
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Conflict of interest statement They declared no conflicts of interests. Acknowledgments This work was supported by Natural Science Foundation of HeiLongJiang Province (D201103) and National Natural Science Foundation of China (No. 81072296). Li-Feng Wang and Jia-Ren Liu are cocorresponding authors. Rui-Fen Wang and Li-Hong Zhang are co-first authors. References Aertgeerts, K., et al., 2005. Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein alpha. J. Biol. Chem. 280, 19441–19444. Brennen, W.N., et al., 2012. Rationale behind targeting fibroblast activation proteinexpressing carcinoma-associated fibroblasts as a novel chemotherapeutic strategy. Mol. Cancer Ther. 11, 257–266. Cai, J., et al., 2012. Fibroblasts in omentum activated by tumor cells promote ovarian cancer growth, adhesion and invasiveness. Carcinogenesis 33, 20–29. Cappellani, A., et al., 2010. Clinical and biological markers in gastric cancer: update and perspectives. Front. 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