B7-H1 up-regulated expression in human pancreatic carcinoma tissue associates with tumor progression

Abstract

Purpose

Aberrant tumor cell B7-H1 expression, a member of B7 family that can predominantly stimulate interleukin 10 (IL-10) products, contributed to the tumor immune evasion and tumor progression. This study was designed to investigate the expression of B7-H1 and IL-10 in normal pancreas tissues and pancreatic carcinoma samples, and to evaluate clinical significance of B7-H1 expression in pancreatic carcinoma.

Methods

First, the B7-H1 and IL-10 expression in 40 pancreatic carcinoma samples and 8 healthy pancreas specimens using reverse transcription-PCR (RT-PCR) and western-blotting was detected. Localization of B7-H1 and IL-10 was confirmed by immunohistochemical (IHC) staining. Next, the association between B7-H1 expression and tumor differentiation and tumor stage was analyzed. Finally, the correlation between tumor-associated B7-H1 and IL-10 was evaluated.

Results

Pancreatic carcinoma samples demonstrated the up-regulated expression of B7-H1 and IL-10 at mRNA and protein level compared with normal pancreas tissues. IHC staining revealed that B7-H1 and IL-10 was almost localized in tumor cells. Analysis of relationship between B7-H1 and tumor clinicopathological characteristics showed that B7-H1 expression was significantly associated with poor tumor differentiation (P < 0.01) and advanced tumor stage (P < 0.01). Meanwhile, tumor-associated B7-H1 expression was also correlated with IL-10 products (P < 0.01, R ² = 0.6985, mRNA level; P < 0.01, R ² = 0.7236, protein level) in tumor cells.

Conclusions

Our findings for the first time demonstrated up-regulated B7-H1 expression in human pancreatic carcinoma tissues, which might play a role in tumor progression and invasiveness. This expression seemed to be related to the ability of B7-H1 to promoting IL-10 secretion.

Introduction

Pancreatic carcinoma is the major leading cause of cancer death worldwide. The lethal nature of pancreatic carcinoma stems from its propensity to rapidly disseminate to the lymphatic system and distant organs (Sarkar et al. 2006). However, the mechanism of pancreatic carcinoma development and its etiology have not been completely elucidated so far.

B7-H1 is a cell-surface glycoprotein belonging to the B7 family of costimulatory molecules (Dong et al. 1999), and an overwhelming number of studies supported the role of B7-H1 as a negative regulator of T-cell responses (Carter et al. 2002). Aberrant tumor cell B7-H1 expression has been described in a number of human malignancies including lung carcinomas, ovarian carcinomas, breast carcinomas, colon carcinoma, melanoma, renal cell carcinoma, glioblastoma, and squamous cell carcinoma of the head and neck (Konishi et al. 2004; Dong et al. 2002; Wintterle et al. 2003; Thompson et al. 2004; Strome et al. 2003). Tumor-associated B7-H1 has been shown to inhibit antitumoral T-cell immunity by interacting with T-cell programmed death 1(PD-1) or a putative non-PD-1 receptor to induce tumor-specific T-cell apoptosis or by impairing cytokine production and the cytotoxicity of activated T cells (Dong et al. 2002; Wintterle et al. 2003; Curiel et al. 2003; Iwai et al. 2002). These findings suggest a potential role for B7-H1 in tumor immune evasion and tumor progression. However, to our knowledge, expression of B7-H1 and its clinical significance in pancreatic carcinoma have not been demonstrated so far.

Ligation of B7-H1 to T cells can result in the preferential production of IL-10 (Dong et al. 1999; Tamura et al. 2001). IL-10 molecule is a multifunctional cytokine and has strong immunosuppressive effects through the inhibition of T helper1-type cytokines, including interferon-γ and interleukin-2 (Moore et al. 2001; Akdis and Blaser 2001). It has also been widely speculated that IL-10 could favor the development of tumors through immuno-suppressive mechanisms, including the modulation of antigen presenting cell and especially dendritic cells functions, and the development of Treg-cell activity (Vicari and Trinchieri 2004; Erdman et al. 2003). However, whether IL-10 production by tumor cells is relevant to tumor-associated B7-H1 expression is currently unclear.

To gain a better insight into the mechanism of pancreatic carcinoma progression, in this study, we detected the expression of B7-H1 in pancreatic carcinoma specimens, and then analyzed the association between B7-H1 expression and clinicopathological characteristics of tumor. Finally, to explore the possible mechanism underlying this association we also investigated the correlation between B7-H1 and IL-10 in tumor cells.

Materials and methods

Patients and tissue samples

Pancreatic carcinoma specimens (histopathologically confirmed primary pancreatic duct adenocarcinomas) were surgically removed from 40 pancreatic carcinoma patients at First Affiliated Hospital, Zhejiang University School of Medicine, China, between 2002 and 2005. Patient clinicopathological characteristics were showed in Table 1. Surgically resected pancreatic cancer specimens were stored at −80°C immediately after surgical removal and for protein and RNA extraction. Formalin-fixed and paraffin-embedded sections were used for routine histopathological diagnosis and immunohistochemical staining. Eight healthy pancreas tissues obtained through an organ donor program were used as control in this study. The study was approved by the local ethics committee and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. All patients in the study gave informed consent.

Table 1.

Association between B7-H1 expression and clinicopathological characteristics of 40 pancreatic carcinoma specimens

	B7-H1				IL-10
	Western-blot	RT-PCR	IHC (%)	P-value	Western-blot	RT-PCR	IHC (%)	P-value
Sex
Male (n = 19)	0.521 ± 0.200	0.527 ± 0.198	59	P > 0.05	0.566 ± 0.187	0.567 ± 0.184	62	P > 0.05
Female (n = 21)	0.532 ± 0.218	0.531 ± 0.213	51		0.548 ± 0.196	0.570 ± 0.183	59
Differentiation
Well (n = 6)	0.234 ± 0.105	0.251 ± 0.118	38	P < 0.01	0.277 ± 0.095	0.316 ± 0.107	42	P < 0.01
Moderate (n = 20)/poor (n = 14)	0.580 ± 0.155	0.570 ± 0.163	72		0.600 ± 0.148	0.611 ± 0.141	79
TNM Stage
I (n = 8)	0.239 ± 0.096	0.237 ± 0.090	40	P < 0.01	0.297 ± 0.102	0.335 ± 0.118	38	P < 0.01
II–IV (n = 32)	0.603 ± 0.138	0.611 ± 0.097	70		0.597 ± 0.172	0.607 ± 0.162	83
Site
Head (n = 33)	0.525 ± 0.153	0.520 ± 0.117	53	P > 0.05	0.550 ± 0.166	0.559 ± 0.147	61	P > 0.05
Body-tail (n = 7)	0.563 ± 0.165	0.566 ± 0.168	57		0.585 ± 0.180	0.614 ± 0.186	60
Size
<2 cm (n = 6)	0.223 ± 0.104	0.240 ± 0.101	52	P > 0.05	0.273 ± 0.097	0.320 ± 0.130	57	P > 0.05
>2 cm (n = 34)	0.587 ± 0.172	0.579 ± 0.171	58		0.607 ± 0.155	0.612 ± 0.155	64

Data were expressed as mean ± SD

Reverse transcription-PCR (RT-PCR) for IL-10 and B7-H1 mRNA expression

The tumor tissues total RNA was extracted using TRIzol reagent (Gibco BRL, Life Technologies Ltd, Paisley, UK) according to the manufacturer’s instruction. The RNA concentration was quantitated by measuring absorbance at 260 nm and was reversely transcripted to cDNA in a 20-μL final volume containing 1 μM random hexanucleotide primers, 1 μM dNTP, and 200 U Moloney murine leukemia virus reverse transcriptase (Fermantas, Hanover, German). The cDNA samples were then subjected to PCR analysis using the following primers: B7-H1, sense 5′-GCCGACTACAAGCGAATTAC-3′, antisense 5′-TCTCAGTGTGCTGGTCACAT-3′; IL-10, sense 5′-GAACCAAGACCCAGACAT-3′ antisense 5′-ATTCTTCACCTGCTCCAC-3′; ß-actin, sense 5′-CGGGAAATCGTGCGTGAC-3′ antisense 5′-TAGAAGCATTTGCGGTGG-3′. PCR were performed in a total volume of 25 μL in PCR buffer in the presence of 0.2 mM dNTP, 1 μM of each primer, and 1 U of Taq DNA polymerase. Conditions used for the PCR amplification were as follows: B7-H1, 94°C 4 min; 35 cycles: 94°C 1 m, 59°C 1 m, 72°C 1 m; and 72°C 10 m. IL-10, 94°C 4 min; 35 cycles: 94°C 1 m, 56°C 1 m, 72°C 1 m; and 72°C 10 m. ß-actin was used as internal control and coamplified. After 35 cycles of amplification, the PCR products were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. All RT-PCR reagents were from Invitrogen (Carlsbad, CA, USA).

Western-blotting for IL-10 and B7-H1 protein expression

The primary antibodies used were as follows. Monoclonal anti-human B7-H1 antibody (Clone130002) and monoclonal anti-human IL-10 antibody (Clone23738) were purchased from R&D systems (Mineapolis, MN, USA). Primary antibodies were diluted 1:500 in 5% bovine serum albumin (BSA) with TBS-0.1% Tween 20.

Protein samples were solubilized and boiled in SDS sample buffer for 2 min and then separated by 12% SDS-PAGE gel. Next, the separated proteins were transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). Following incubation in blocking buffer (TBS with 5% BSA and 0.1% Tween 20) for 2 h at room temperature, the membranes were probed for 1 h at 4°C with anti-B7-H1 antibodies or anti-IL-10 antibodies and then probed overnight at 4°C. Next day, after probing for 2 h at 4°C with primary antibodies again, the membranes were washed and probed with a horseradish peroxidase-linked secondary antibody (Dako Corp, Carpinteria, CA, USA) (1:2,000) for 2 h at room temperature. After washing four times with TBS-Tween, the blot was developed with enhanced chemiluminescence substrate (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and imaged using a Storm 860 PhosphorImager.

Immunohistochemical (IHC) staining for location of B7-H1 and IL-10 in pancreatic carcinoma tissues

Paraffin-embedded tissue sections of 4 μm were dried for 40 min at 58°C before deparaffinization in xylene and rehydration by sequential incubation in EtOH/water solutions. The sections were treated with 3% hydrogen peroxide in methanol for 15 min at room temperature, in darkness, to quench endogenous peroxidase activity. After rinsing in water and PBS, sections were blocked with serum (diluted 1:20 in PBS) from a non-immunized animal for 30 min to reduce non-specific binding. Subsequently, the sections were incubated with a primary monoclonal antibody against B7-H1 (Clone130002, R&D systems, diluted 1:50 in PBS) or IL-10 (Clone23738, R&D systems, diluted 1:150 in PBS) in a moist chamber at 4°C overnight. After washing in PBS, biotinylated antimouse immunoglobulin (Dako Corp) at a dilution of 1:200 was added for 45 min, followed by a washing step and incubation with ABC reagent (strept ABC complex/HRP; Dako Corp.) for 45 min. The peroxidase reaction was developed with 0.02% 3,3′-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, USA) in PBS containing 0.06% hydrogen peroxide for 1 min. Finally, sections were rinsed with water and counterstained with Harris’ hematoxylin. Immunoreactivity in more than 10% cells per field was defined as positive.

Statistical analysis

Statistical analysis was done using SPSS software (Version 11.5.1, SPSS Inc., Chicago, IL, USA). Comparisons between two groups and the association between B7-H1 or IL-10 levels and clinicopathological variables were analyzed statistically using Student’s paired t test. Spearman correlation coefficient was used to determine the association between B7-H1 expression and IL-10 levels. Values of P < 0.05 were considered to indicate statistical significance. All tests used were two sided.

Results

B7-H1 and IL-10 up-regulated expression by pancreatic carcinoma at mRNA level

Previous study has revealed that the expression of human B7-H1 mRNA was detected in a variety of non-lymphoid parenchymal organs, including heart, skeletal muscles, placenta, lung, thymus, spleens, kidney and liver by Northern blot (Dong et al. 1999). In our study, B7-H1 mRNA expression in pancreatic carcinoma and normal tissue was tested by RT-PCR. Results demonstrated that in normal pancreas tissue low level of B7-H1 mRNA was detectable (Fig. 1). In contrast, B7-H1 mRNA expression was markedly increased in pancreatic carcinoma tissues (0.518 ± 0.197) compared with normal tissue (0.136 ± 0.019) (Fig. 1, P < 0.05). IL-10 mRNA expression pattern was similar to B7-H1 mRNA expression profile that pancreatic carcinoma showed higher IL-10 mRNA level (0.568 ± 0.194) than that of normal tissue (0.152 ± 0.027) (Fig. 1, P < 0.01). This suggests that the expression of B7-H1 and IL-10 in pancreatic carcinoma is pretranscriptionally controlled.

Fig. 1.

Identification of B7-H1 and IL-10mRNA transcripts in pancreatic carcinoma and normal pancreas by RT-PCR. Top B7-H1 mRNA was detected at higher level in pancreatic carcinoma tissue (lane 1, 2, 3) compared with normal pancreas tissue (lane 4, 5, 6), P < 0.05. Bottom IL-10mRNA was detected at higher level in pancreatic carcinoma tissue (lane 1, 2, 3) compared with normal pancreas tissue (lane 4, 5, 6), P < 0.01

B7-H1 and IL-10 up-regulated expression by pancreatic carcinoma at protein level

We next investigated whether a similar increase in B7-H1 expression in pancreatic carcinoma could be detected at the protein level. Western-blotting analysis showed that all tumor samples were observed intensely at B7-H1 protein band (Fig. 2). In contrast, normal pancreas tissue demonstrated minimal quantities of B7-H1 protein (Fig. 2). There was a statistically significant difference in B7-H1 protein expression between pancreatic carcinoma (0.532 ± 0.181) and normal pancreatic tissue (0.125 ± 0.049) (P < 0.01). Meanwhile, IL-10 protein expression was also analyzed in pancreatic carcinoma and normal tissue. Consistent with previous reports (Bellone et al. 1999), we also detected the increasing IL-10 protein expression in pancreatic carcinoma tissue (0.556 ± 0.206) compared with normal pancreatic tissue (0.174 ± 0.041) (Fig. 2, P < 0.01).

Fig. 2.

Detection of B7-H1 and IL-10 protein expression in pancreatic carcinoma specimens and normal pancreas specimens by western-blotting. Increased expression of B7-H1 in pancreatic carcinoma tissues (top lane 1, 2, 3) was observed compared with normal pancreas tissues (top lane 4, 5, 6). IL-10 expression was also significantly increasing in pancreatic carcinoma tissue (middle lane 1, 2, 3) compared with normal pancreatic tissue (middle lane 4, 5, 6)

B7-H1 and IL-10 localization in pancreatic carcinoma samples

IHC results confirmed the western-blotting data. Using IHC staining, B7-H1 and IL-10 protein expression was analyzed in all tumor specimens and normal tissue. IHC staining showed that more than 50% of the tumor cells expressed B7-H1 in the pancreatic carcinoma specimens. B7-H1-positive cells were evenly scattered throughout the specimens, B7-H1 was located primarily in the cytoplasm of the tumor cells (Fig. 3b). In normal pancreatic tissues, we found positive staining in islets cells (Fig. 3c), but no B7-H1 expression was found in other type of pancreatic cells. IL-10 localization in pancreatic carcinoma cells was also detected by IHC staining. Consistent with the previous reports, intense staining for IL-10 (about 60%) was identified in pancreatic carcinoma specimens. Meanwhile, IHC indicated that IL-10 was almost expressed in the cytoplasm of tumor cells (Fig. 3e). Interestingly, staining for IL-10 was also only found in islets cells in normal pancreas tissue (Fig. 3f). Representative IHC staining was showed in Fig. 3.

Fig. 3.

IHC analysis of B7-H1 and IL-10 expression in paraflin-embedded sections of pancreatic carcinoma and normal pancreas. Magnification of a–f ×400. Pancreatic carcinoma tissue showed high intense staining for B7-H1 (b arrow) and IL-10 (e arrow). In normal pancreas tissue, only islets cells showed positively staining for B7-H1 (c arrow) and IL-10 (f arrow). B7-H1 and IL-10 immunoreactive cells were localized in the cytoplasm. Negative control, in which the primary B7-H1 (a) and IL-10 (d) detecting Ab was replaced by 3% BSA/PBS serum, showed complete lack of immunoreactivity

B7-H1 or IL-10 expression associated with tumor differentiation and stage

To define the clinical role of B7-H1 in pancreatic carcinoma progression, we analyzed the relationship between B7-H1 expression and clinicopathological features of pancreatic carcinoma. 40 pancreatic carcinomas were classified according to the International Union Against Cancer tumor node metastasis (TNM) classification for pancreatic cancer (Table 1). The media expression levels of B7-H1 for stage I was significantly reduced compared with stage II, stage III, and stage IV. B7-H1 expression between stage II and stage III and stage IV were not significantly different. Moderately and poorly differentiated tumor demonstrated the higher levels of expression of B7-H1 than well-differentiated tumor. Univariate analysis showed that B7-H1 expression was significantly associated with tumor stage (P < 0.01) and tumor grade (P < 0.01). No correlation was observed between B7-H1 expression and either sex, site, or size of tumor. The relationship of IL-10 expression with tumor differentiation and stage was similar to the association between B7-H1 and clinicopathological features. Detailed results are presented in Table 1.

Over-expression B7-H1 correlated with elevated IL-10 level in pancreatic carcinoma tissues

To explore the possible mechanism underlying the association between B7-H1 and tumor progression, we further analyzed whether B7-H1 expression would be correlated with IL-10 levels. As shown in Fig. 4, at mRNA level, results indicated a positive correlation between B7-H1 expression and IL-10 products (P < 0.01, R ² = 0.6985, Fig. 4a). A positive correlation was also seen between B7-H1 expression and IL-10 products at protein level (P < 0.01, R ² = 0.7236, Fig. 4b).

Fig. 4.

Correlation of B7-H1 expression with IL-10 level in pancreatic carcinoma. a The B7-H1 expression was significantly correlated with IL-10 level at mRNA level in pancreatic carcinoma samples (P < 0.01, R ² = 0.6985). b The B7-H1 expression was significantly correlated with IL-10 level at protein level in pancreatic carcinoma samples (P < 0.01, R ² = 0.7236)

Discussion

Our study for the first time provides evidence for up-regulated expression of B7-H1 in pancreatic carcinoma tissue. More importantly, we found the association between B7-H1 up-regulated expression and poorly differentiated pancreatic carcinomas and advanced tumor stage. Meanwhile, elevated IL-10 product was correlated with B7-H1 expression, which may be responsible for clinical significance of B7-H1expression.

Cancer cells can grow by escaping from the attack of immune cells, thus, disrupting the host immune system, which is progressively suppressed as a result of tumor progression and metastasis (Zou 2005). Inadequate, inappropriate, or inhibitory T-cell costimulatory pathway signaling have all been shown to restrict a host’s ability to generate productive immune responses against cancer. Recent studies identify a new inhibitory B7-H1 pathway, which negatively regulates T-cell immune responses by selectively costimulating a putative T-cell subset that secretes IL-10 and increasing programmed cell death of activated T cells (Dong et al. 1999) Aberrant tumor cell B7-H1 expression has been described in a number of human malignancies and implicated as a negative regulator of antitumoral T cell-mediated immunity. Herein, we first reported that B7-H1 up-regulated expression in pancreatic carcinoma and IHC staining demonstrated increased B7-H1 was almost located at pancreatic carcinoma cells. Additionally, B7-H1 transcripts and protein expression were also found in normal human pancreatic tissue by RT-PCR and western-blot. IHC demonstrated B7-H1 positive staining was in normal islets cells. Keir et al. (2006) reported the expression of B7-H1 in mouse normal islets cells, which could protect islets cells against attack by autoreactive T cells. We found the similar phenomenon that human islets cells have the B7-H1 positive expression in normal pancreas tissue. Meanwhile, new observation showed that islets cells demonstrated concomitant expression of IL-10; this implicated IL-10 might particulate in the protective function of B7-H1 in islets cells. But functional significance of B7-H1 expression in human islets required further investigation.

Tumor-associated B7-H1 has recently garnered much attention as a potential inhibitor of host antitumoral immunity. B7-H1 expressed by tumor cells was shown to enhance apoptosis of activated tumor-specific T cells and to inhibit CD4+ and CD8+ T cell activation in vitro and lead to growth of immunogenic tumor in vivo (Dong et al. 2002). Up-regulating B7-H1 expression on Myeloid dendritic cells (MDC) impaired MDC-mediated T-cells activation in ovarian cancer patients, and blockade of MDC-B7-H1 could improve T-cells antitumor immunity (Curiel et al. 2003). In addition, mice succumb to tumors transfected with B7-H1 even after adoptive T-cell immunotherapy, whereas blockade of PD-1/B7-H1 inhibits tumorigenesis in vivo (Strome et al. 2003; Iwai et al. 2002). Clinical significance of B7-H1 has been reported to exhibit a strong association with the aggressiveness of a renal cell carcinoma and patient cancer-specific survival (Thompson et al. 2004). In present study, similar results were obtained that B7-H1 expression was associated with poorly differentiated pancreatic cancer cells and advanced tumor stage; this implicated that B7-H1 expression in human pancreatic carcinoma tissues might play a role in tumor progression and invasiveness. The mechanisms regulating B7-H1 expression on tumor cells are still unclear. Inflammatory mediators are implicated by up-regulation of B7-H1 expression on the surface of several tumor cell lines after exposure to IFN-γ. Moreover, B7-H1 expression is more frequent in freshly isolated cancer tissue specimens than in cultured tumor cell lines, and the expression of B7-H1 on tumor-related dendritic cells may be up-regulated by tumor environmental factors (IL-10 or VEGF) of ovarian cancer. These observations suggest that the cytokine microenvironment induces the expression of B7-H1 on tumor cells.

Next, we investigated the possible mechanisms underlying the association of B7-H1expression with tumor aggressiveness. IL-10 plays an important role in the oncogenetic and metastatic ability of neoplasms. In patients suffering from pancreatic carcinoma, elevated IL-10 levels affected systemic immunity in favor of a T helper2-like phenotype and were correlated with poor survival (Bellone et al. 1999). Ligation of cognate receptor(s) on T cells by B7-H1 was reported to predominantly stimulate IL-10 production. Furthermore, we have previously reported that B7-H1 up-regulated expression in peripheral blood monocytes was correlated with higher serum IL-10 levels in chronic hepatitis B virus infection patients (Geng et al. 2006). Trabattoni et al. (2003) showed the coexistence of B7-H1 up-regulated expression and intracellular increased IL-10 production in CD14⁺ cells of HIV infection, which could be responsible for T-lymphocyte unresponsiveness and loss of protective immunity. Thus, in present study, we also analyzed the relationship between B7-H1 and IL-10. Data demonstrated that B7-H1 expression was statistically significantly correlated with IL-10 level in pancreatic carcinoma. This suggest a potential mechanism that tumor B7-H1 contributed to pancreatic carcinoma aggressiveness by promoting excessive IL-10 secretion, which can lead to strong immunosuppressive effects through the inhibition of T helper1-type cytokines, including interferon-γ and interleukin-2. Curiel et al. demonstrated that myeloid dendritic cells (MDC) in ovarian carcinoma expressed B7-H1; blockade of B7-H1 could enhance MDC-mediated T activation and was accompanied by down-regulation of IL-10 in MDC. However, PD-1Ig did not decrease IL-10 expression in MDC. Thus, we surmised that ligation of additional receptors other than PD-1 by B7-H1 regulate the IL-10 production in pancreatic carcinoma cells.

In summary, our findings for the first time suggest the potential role of B7-H1 in fostering cancer cell growth and metastasis of pancreatic carcinoma. This implicates a new mechanism by which pancreatic carcinoma escapes from immune surveillance and tumor progression. Recognition of this new mechanism of tumor evasion will necessitate a new approach to the design of B7-H1-based immunotherapy.

Abbreviations

BSA

Bovine serum albumin

IL-10

Interleukin-10

IHC

Immunohistochemical

MDC

Myeloid dendritic cells

PD-1

Programmed death 1

RT-PCR

Reverse transcription-PCR

TNM

Tumor node metastasis