Brief Article Open Access
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Feb 28, 2012; 18(8): 840-846
Published online Feb 28, 2012. doi: 10.3748/wjg.v18.i8.840
Expression of fibroblast activation protein in human pancreatic adenocarcinoma and its clinicopathological significance
Min Shi, Dang-Hui Yu, Ying Chen, Chen-Yan Zhao, Jing Zhang, Qing-Hua Liu, Can-Rong Ni, Ming-Hua Zhu
Min Shi, Dang-Hui Yu, Ying Chen, Chen-Yan Zhao, Jing Zhang, Qing-Hua Liu, Can-Rong Ni, Ming-Hua Zhu, Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
Author contributions: Shi M, Chen Y, Zhao CY, Zhang J, Liu QH and Ni CR designed, performed the research and analyzed the data; Zhu MH designed the research and analyzed the data; and Yu DH wrote the paper.
Supported by The National Key Project of Scientific and Technical Supporting Programs of China, No. 2006BAI02A14; and National Natural Science Foundation of China, No. 30770996 and No. 81172310
Correspondence to: Ming-Hua Zhu, Professor, Department of Pathology, Changhai Hospital, Second Military Medical University, No. 168 of Changhai Road, Shanghai 200433, China. mhzhu2000@hotmail.com
Telephone: +86-21-81873700 Fax: +86-21-81873700
Received: April 14, 2011
Revised: September 1, 2011
Accepted: October 28, 2011
Published online: February 28, 2012

Abstract

AIM: To examine fibroblast activation protein (FAP) expression in pancreatic ductal adenocarcinoma (PDAC) and to analyze its relationship with the clinicopathology of PDAC.

METHODS: FAP expression was examined in 134 PDAC specimens by immunohistochemistry, and in four pancreatic cancer cell lines (SW1990, Miapaca-2, AsPC-1 and BxPC-3) by Western blotting assay. We also analyzed the association between FAP expression in PDAC cells and the clinicopathology of PDAC patients.

RESULTS: The results showed that the FAP was ex-pressed in both stromal fibroblast cells (98/134, 73.1%) and carcinoma cells (102/134, 76.1%). All 4 pancreatic cancer cell lines expressed FAP protein at different levels. Protein bands corresponding to the proteolytically active 170-kDa seprase dimer and its 88-kDa seprase subunit were identified. Higher FAP expression in carcinoma cells was associated with tumor size (P < 0.001), fibrotic focus (P = 0.003), perineural invasion (P = 0.009) and worse clinical outcome (P = 0.0085).

CONCLUSION: FAP is highly expressed in carcinoma cells and fibroblasts in PDAC tissues, and its expression is associated with desmoplasia and worse prognosis.

Key Words: Pancreatic ductal adenocarcinoma, Cancer-associated fibroblasts, Fibroblast activation protein, Prognosis



INTRODUCTION

Pancreatic carcinoma is one of the most aggressive human malignancies. Currently pancreatic cancer ranks fourth in cancer-related deaths in Western countries and sixth in China[1,2]. As one of the most lethal malignancies, human pancreatic ductal adenocarcinoma (PDAC) results in an overall 5-year survival of the patients of less than 5%, a rate with no substantial improvement over the past 25 years[3,4]. The poor prognosis is mainly associated with early local invasion, a high incidence of recurrence and a poor response to chemotherapy and radiotherapy. Failure of the conventional therapies for PDAC might be due to our limited understanding of the role of tumor-stromal interactions in cancer progression[5].

In many solid tumors, the stroma is increasingly recognized to be important in promoting tumor proliferation, invasion, metastasis, and chemoresistance[6]. PDAC is one of the most highly invasive of the solid cancers and is characterized by an extensive desmoplastic stromal response. Several studies revealed that the elements in desmoplasia played important roles in tumorigenesis of PDAC[7-9] and was associated with a poor prognosis of the patients[10,11]. Mounting evidence suggests that cancer-associated fibroblasts (CAF), the predominant stromal cell type, actively communicate with and stimulate tumor cells, thereby contributing to tumor development and progression.

Fibroblast activation protein (FAP), or seprase, a cell surface glycoprotein belonging to the serine protease family, is a 170 kDa dimer that is catalytically active and has dipeptidase and gelatinase activities. FAP was identified as being expressed in reactive fibroblasts during embryonic development[12], in healing wounds[13], in chronic inflammation, in liver cirrhosis[14], and most importantly, in the CAFs of many human carcinomas[15-19]. As a marker of CAFs, FAP can enhance stromal cell proliferation and invasiveness, affect cell apoptosis, and is closely correlated with poor prognosis of a variety of tumors[20-23]. Recently, Kraman et al[23] reported that stromal cells expressing FAP may be involved in tumor immune system suppression. FAP expression has been described to be present predominantly in the tumor stroma of epithelial malignancies, and its presence has been associated with cancer invasion, tumor angiogenesis, and subsequent growth and metastasis[15-19]. FAP was also reported to be expressed in carcinoma cells of the stomach[20], colorectum[21], breast[22] and uterine cervix[24]. Cohen et al[15] found that FAP was expressed predominantly in the fibroblasts in PDAC, and was shown to be statistically highly correlated with a worse clinical outcome. However, there has been no study of the expression of FAP in pancreatic cancer cells and its role in pancreatic cancer development and progression[20]. In the present study, we aimed to investigate the expression of FAP in PDAC carcinoma cells and its association with desmoplasia and patient survival, which may shed light on the role of FAP in desmoplasia in PDAC, and more importantly, signify a potentially new therapeutic target in PDAC.

MATERIALS AND METHODS
Patients and specimens

The PDAC specimens were collected with informed consent from 134 patients undergoing radical resection of primary PDAC in Changhai Hospital, Second Military Medical University during 2005-2007. Of the 134 specimens, 74 were used for tissue array study, and the remaining 60 were used to prepare standard pathological sections. All the experimental procedures were approved by the Research Ethics Committee of the Second Military University. The medical records of the patients were retrospectively reviewed, and the demographic, clinicopathological and treatment data were also collected. Tumor location and size were derived from the surgical report. The patients receiving preoperative chemotherapy or radiotherapy were excluded from the study. All patients were followed up after the operation, and the follow-up of the survivors at the time of analysis ranged from 1-33 (median 12) mo postoperatively. The patients who were alive at the time of the last follow-up were censored for overall survival analysis.

Cell lines

Four pancreatic cancer cell lines (SW1990, Miapaca-2, AsPC-1 and BxPC-3) maintained in our laboratory were incubated in the presence of 5% CO2 in a humidified atmosphere at 37 °C. AsPC-1, SW1990 and BxPC-3 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories GmbH, Linz, Austria), and Miapaca-2 cells were maintained in DMED supplemented with 10% FBS and 2.5% horse serum (Gibco BRL, Invitrogen Corp, CA, United States).

Assessment of fibrotic focus and perineural invasion

Sixty standard slides were observed under the microscope after hematoxylin and eosin staining. A fibrotic focus was defined as a scar-like fibrosclerotic area located within the tumor containing a number of fibroblasts admixed with collagen fibers. The fibroblasts and collagen fibers were arranged in a storiform-like or irregular pattern[25]. The degree of perineural invasion was determined by two independent observers as described previously[26,27].

Immunohistochemistry

Tissue microarrays were constructed using the 74 paraffin-embedded tumor tissue specimens by a precision arraying instrument (Beecher Instruments, Silver Spring, MD, United States). In each case, three tumor cores and two surrounding non-tumor tissues were selected. The standard slices of the other 60 specimens and the sections from the tissue microarray blocks were deparaffinized, rehydrated and then heated in 0.01 mol/L sodium citrate buffer (pH 6.0) for 8 min at 95 °C. After incubation with 0.3% hydrogen peroxide in methanol for 15 min at room temperature and treatment with normal goat serum (Invitrogen, Carlsbad, CA), the sections were incubated overnight at 4 °C with rabbit anti-human FAP polyclonal antibody (LifeSpan BioSciences Inc, United States) at a dilution of 1:70 in solution (Zymed Labroatories, Invitrogen Corp, CA). Slides were rinsed for 10 min in phosphate buffered saline (PBS) wash solution and incubated for 30 min with the horseradish peroxidase (HRP)-labeled polymer conjugated secondary antibody (EnVision+; DakoCytomation, Carpintera, CA, United States) was added according to the manufacturer’s instructions. A known positive endometrial cancer tissue biopsy sample was used as the positive control. PBS and non-immune serum were used instead of the primary antibody for the blank control and negative control samples, respectively.

Evaluation of FAP staining was carried out by two independent observers, and the stained area and intensity were scored separately. Specifically, a score of 0 was assigned to a stained area with ≤ 10% of the tumor cells, 1 for an area with > 11% to ≤ 25% of tumor cells, 2 for > 26% to ≤ 50% tumor cells, and 3 for > 51% tumor cells. For the staining intensity, a score of 0 was assigned for absent/weak staining (negative control), 1 for a weak staining obviously stronger than the negative control level, 2 for moderately intense staining, and 3 for intense staining. The final grade of the section was derived from the sum of the stained area and intensity scores. A final score ≥ 3 was recognized to indicate positive expression.

Western blotting

Upon reaching 80%-90% confluence, the cultured cells were harvested and lysed in ice-cold lysis buffer (50 mmol/L Tris-HCl (pH8.0), 150 mmol/L NaCl, 0.02% NaN3, 1% NP-40, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 μg/mL aprotinin), and the protein level was quantified according to the manufacturer’s instructions of the BCA protein assay kit (Tiangen Biotech Co, China). Total protein was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk, incubated with 1:1000 rabbit anti-human FAP polyclonal antibody (Abcam, United States) overnight at 4 °C, washed in Tris-buffered saline Tween-20 buffer, and then incubated with HRP-conjugated goat anti-rabbit IgG (MR Biotech, China) for 1 h at room temperature. The protein bands were visualized by chemiluminescence (Tiangen Biotech Co, China).

Statistical analysis

All the analysis were carried out using SPSS 11.5 software. The χ2 test was used to analyze the correlation between FAP expression and clinicopathological features of PDAC. The Spearman correlation was used to evaluate the correlation of fibrotic focus and FAP expression. The Kaplan-Meier method was used for survival analysis and the difference in the survival curves was evaluated using the log-rank test. The Cox proportional hazards regression model in a stepwise manner was used to analyze the independent prognostic factors. All the tests were conducted with a 5% type I error.

RESULTS
Clinical data

We examined a total of 134 specimens. The patients consisted of 92 males and 42 females, age range 29-80 (median, 59) years. The patients’ characteristics are summarized in Table 1.

Table 1 The clinical data of 134 pancreatic ductal adenocarcinoma patients.
ItemsCategoryn (%)
Age (yr) ≤ 6063 (47.0)
> 6071 (53.0)
GenderMale92 (68.7)
Female42 (31.3)
Location of tumorHead97 (72.4)
Body/tail37 (27.6)
Size of tumor (cm)1 ≤ 2.017 (13.2)
> 2.0112 (86.8)
Histological gradeG13 (2.2)
G2116 (86.6)
G315 (11.2)
Perineural invasionAbsent25 (18.7)
Present109 (81.3)
pT1T110 (7.6)
T243 (32.8)
T346 (35.1)
T432 (24.4)
pN1Absent84 (65.1)
Present45 (34.9)
pTNM1I32 (24.8)
II30 (23.3)
III33 (25.6)
IV34 (26.4)
Fibrotic focus in PDAC

A fibrotic focus was detected in 47 (78.3%) of the 60 PDAC specimens; in the fibrotic focus, active fibroblasts and collagen fibers were radially aligned around the tumor cells (Figure 1A), a pattern different from that in the surrounding PDAC stroma, where the fibroblasts and collagen fibers were arranged in an orderly fashion (Figure 1B). There was no significant correlation between the presence of a fibrotic focus and other clinicopathological factors.

Figure 1
Figure 1 Fibrotic focus in pancreatic ductal adenocarcinoma. A: In the fibrotic focus, fibroblasts and collagen fibers are radially aligned around the tumor cells (× 200); B: A pattern different from that in the surrounding pancreatic ductal adenocarcinoma stroma where the fibroblasts and collagen fibers are arranged in an orderly fashion (× 100).
Patterns of FAP expression in PDAC tissues

FAP was strongly expressed not only in activated fibroblasts within the PDAC tissues (98/134, 73.1%), but also in carcinoma cells in 102 of the 134 specimens (76.1%). Three patterns of FAP immunostaining were found in PDAC tissues, which included carcinoma cells (15/134, Figure 2A), stroma cells (11/134, Figure 2B), both carcinoma and stroma cells (87/134, Figure 2C). FAP immunostaining was localized mainly on the cell membrane and in the cytoplasm, and occasionally in the gland lumens. The expression presented with a diffuse or focal distribution in the carcinoma tissues. The FAP-positive fibroblasts were mainly located adjacent to the carcinoma tissue. The adjacent noncancerous pancreatic ducts showed virtually no positive staining for FAP, and the fibroblasts away from the carcinoma tissues rarely expressed FAP (Figure 2D).

Figure 2
Figure 2 Fibroblast activation protein expression in pancreatic ductal adenocarcinoma. A: Fibroblast activation protein (FAP) expression in carcinoma cells is located mainly on the cell membrane and in the cytoplasm (× 200); B: FAP-positive fibroblasts are mainly located adjacent to carcinoma tissue (× 200); C: FAP is expressed in carcinoma cells and in fibroblasts adjacent to carcinoma tissue (× 100); D: The adjacent noncancerous pancreatic ducts show no positive staining for FAP and fibroblasts away from the tumor tissues rarely express FAP (× 200).
FAP expression in pancreatic cancer cell lines

To further investigate the expression of FAP in pancreatic cancer cells, we performed Western blotting assays on cancer cells in vitro. The results showed that all four pancreatic cancer cell lines expressed FAP protein at different levels. Protein bands corresponding to the proteolytically active 170 kDa seprase dimer and its 97 kDa seprase subunit protein were identified (Figure 3). This result suggests that pancreatic cancer cells can secrete FAP by an autocrine mechanism.

Figure 3
Figure 3 Western blotting analysis of fibroblast activation protein in pancreatic cancer cell lines. All four cell lines express fibroblast activation protein (FAP) with protein bands corresponding to the proteolytically active 170-kDa seprase dimer and its 88-kDa seprase subunit protein. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
Correlation of FAP expression with clinicopathological features of PDAC patients

The χ2 test indicated significant correlations of FAP expression in the carcinoma cells from PDAC patients with patient age (P < 0.001), tumor size (P < 0.001), fibrotic focus (P = 0.003) and perineural invasion (P = 0.009) (Table 2). The Spearman correlation revealed that FAP expression was positively correlated with fibrotic focus, with a Pearson correlation coefficient of 0.379 (P = 0.003). We also found that PDAC patients with high expression of FAP in the carcinoma cells had a significantly shorter median overall survival compared to those with low expression of FAP (10 mo vs 33 mo, P = 0.0085) (Figure 4). In univariate analyses, the following parameters were significantly associated with overall survival in PDAC: patient age (P = 0.013), tumor size (P = 0.008), fibrotic focus (P = 0.023) and perineural invasion (P = 0.009). The multivariate survival analysis further revealed that high expression of FAP was a predictor of overall survival (P = 0.017; relative risk, 2.513) independent of patient age (P = 0.029), tumor size (P = 0.011), fibrotic focus (P = 0.037) or perineural invasion (P = 0.020).

Table 2 Correlation of fibroblast activation protein expression with clinicopathological features of pancreatic ductal adenocarcinoma patients n (%).
ItemsCategorynFAP negativeFAP positiveP value
Age (yr) ≤ 606319 (30.2)44 (69.8)0.000
> 607113 (18.3)58 (81.7)
GenderMale9222 (23.9)70 (76.1)0.990
Female4210 (23.8)32 (76.2)
Location of tumorHead9727 (27.8)70 (72.2)0.082
Body/tail375 (13.5)32 (86.5)
Size of tumor (cm)1 ≤ 2.01710 (58.8)7 (41.2)0.000
> 2.011221 (18.8)91 (81.2)
Histological gradeG131 (33.3)2 (66.7)0.889
G211627 (23.3)89 (76.7)
G3154 (26.7)11 (73.3)
Fibrotic focusAbsent137 (53.8)6 (46.2)0.003
Present477 (14.9)40 (85.1)
Perineural invasionAbsent2511 (44.0)14 (56.0)0.009
Present10921 (19.3)88 (80.7)
pT1T1105 (50.0)5 (50.0)0.170
T2438 (18.6)35 (81.4)
T34612 (26.1)34 (73.9)
T4326 (18.8)26 (81.2)
pN1Absent8421 (25.0)63 (75.0)0.725
Present4510 (22.2)35 (77.8)
pTNM1I327 (21.9)25 (78.1)0.895
II308 (26.7)22 (73.3)
III339 (27.3)24 (72.7)
IV347 (20.6)27 (79.4)
Figure 4
Figure 4 Kaplan-Meier survival curves of pancreatic ductal adenocarcinoma patients. The prognosis was significantly worse in the fibroblast activation protein (FAP)-positive group compared with the FAP-negative group (P = 0.0085).
DISCUSSION

In this study, we confirm and extend previous findings that FAP expression is significantly elevated in PDAC tissues. FAP overexpression was positively correlated with patient age, tumor size, fibrotic focus, perineural invasion and poor survival, implying its involvement in PDAC development. Moreover, the protein product of FAP was found in all four pancreatic cancer cell lines and in PDAC tumor cells. To our knowledge, this was a novel finding of interest in the current study.

Solid tumors are a composite of cancer cells, endothelial cells, inflammatory cells, and fibroblasts. Although the relevance of fibroblasts in cancer progression is increasingly being recognized, little is known about their origin. Activated pancreatic stellate cells (myofibroblast-like cells), connective tissue-type fibroblasts, and mesenchymal cells were originally identified as the source of the fibrosis in chronic pancreatitis[9], and are now thought to be responsible for the dense stroma associated with pancreatic adenocarcinoma[9,28]. Cancer cells were surrounded by extremely dense collagen bundles associated with abundant reactive stromal fibroblasts, which were closely related to tumor prognosis[29,30]. The presence of a fibrotic focus correlated positively with disease progression, greater tumor size, the presence of lymph node metastases, and poor outcome in breast[31-33] and pancreatic[29,34] cancers. We found that the fibrotic focus was positive in 78.3% (49/60) of the PDAC tissues examined. Nevertheless, statistical analysis supported no correlation between the fibrotic focus and the prognosis of PDAC, which might be due to the fact that the majority of patients in our study were at an advanced stage, when a fibrotic focus would be common; the high short-term mortality rate of PDAC patients following surgery may also contribute to this result.

FAP expression has been described to be present predominantly in the tumor stroma of epithelial malignancies, and its presence has been associated with epithelial cancer invasion, tumor angiogenesis, and subsequent growth and metastasis[15-19,23]. FAP staining of the epithelial neoplasm has also been seen in some of studies, mainly located in the cytoplasm and the cell membrane in carcinoma cells of the stomach[20], colorectum[18,21], breast[22], ovaries[35] and uterine cervix[24], and increased expression of FAP was also associated with histological grade, invasion and metastatic progression. Cohen et al[15] reported that FAP expression was seen mainly in the fibroblasts immediately adjacent to PDAC tissues, but was extremely rare in the carcinoma cells. In contrast, we found in the present study that FAP was highly expressed in the carcinoma cells (76.1%) as well as in the fibroblasts (73.1%) of PDAC tissues. The results of the Western blotting assay also demonstrated the presence of FAP protein expression in four human pancreatic cancer cell lines. Our findings suggested that PDAC cells might directly contribute to stroma desmoplasia through an autocrine mechanism of the FAP protein. We further analyzed the correlation of FAP expression in PDAC cells with patient survival. As we expected, FAP expression in the tumor cells was correlated with a shorter patient survival and served as an independent prognostic indicator for PDAC. These data suggest that FAP acts through an autocrine and/or paracrine mechanism in human pancreatic cancer, and may be a potential new therapeutic target in the treatment of PDAC.

As noted, we obtained different results concerning FAP expression in PDAC and its relation to desmoplasia from those reported previously[15]. We used a commercial primary antibody, and a non-biotinylated secondary antibody, so there would not be a false impression that FAP was localized to carcinoma cells. Although we do not know the reasons for this difference in expression, it may be related to use of a different source of FAP antibody. According to a recent study, the presence of collagen structures radially aligned with tumor cells was suggested to promote tumor progression and invasion[36]. The difference in FAP expression may reflect distinct biological features of these types of cancer.

To conclude, we found that PDAC cells, as well as CAFs, can also express FAP, which is closely associated with desmoplasia in PDAC. This suggests that PDAC cells may be directly responsible for the desmoplastic reaction through an autocrine mechanism of FAP production, and may partly explain why desmoplasia is particularly conspicuous in PDAC compared with the other carcinomas. This suggests that carcinoma cells may play a direct role, through an autocrine and/or paracrine mechanism, in desmoplasia in PDAC development and progression. Further research is needed to investigate the mechanism and some studies are already in progress. Our findings also provide evidence for ongoing clinical investigations with FAP as a therapeutic target for PDAC.

COMMENTS
Background

Fibroblast activation protein (FAP) is expressed on reactive fibroblasts during embryonic development, in healing wounds, in chronic inflammation, in liver cirrhosis, and in cancer-associated fibroblasts (CAFs) of many human carcinomas.

Research frontiers

FAP can enhance stromal cell proliferation and invasiveness, affect cell apoptosis, and is closely correlated with poor prognosis of a variety of tumors. Stromal cells expressing FAP may be involved in tumor immune system suppression. FAP expression has been found to be present predominantly in the tumor stroma of epithelial malignancies, and its presence has been associated with cancer invasion, tumor angiogenesis, and subsequent growth and metastasis.

Innovations and breakthroughs

FAP was expressed in both stromal fibroblast cells and carcinoma cells. All four pancreatic cancer cell lines expressed FAP protein at different levels. Higher FAP expression in carcinoma cells was associated with tumor size, fibrotic focus, perineural invasion and worse clinical outcome.

Applications

FAP is highly expressed in carcinoma cells and fibroblasts in pancreatic adenocarcinoma tissues, and its expression is associated with desmoplasia and worse prognosis. This suggests that carcinoma cells may play a direct role, through an autocrine and/or paracrine mechanism, in desmoplasia in pancreatic ductal adenocarcinoma (PDAC) development and progression, and FAP may serve as a potential therapeutic target in PDAC.

Terminology

FAP, or seprase, a cell surface glycoprotein belonging to the serine protease family, is a 170 kDa dimer that is catalytically active and has dipeptidase and gelatinase activities; CAF, is a predominant stromal cell type, which actively communicates with and stimulates tumor cells, thereby contributing to tumor development and progression.

Peer review

This is an interesting work.

Footnotes

Peer reviewer: Andrada Seicean, MD, PhD, Third Medical Clinic Cluj Napoca, University of Medicine and Pharmacy Cluj Napoca, Romania, 15, Closca Street, Cluj-Napoca 400039, Romania

S- Editor Tian L L- Editor Cant MR E- Editor Li JY

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