J Hepatobiliary Pancreat Sci (2011) 18:821–828 DOI 10.1007/s00534-011-0395-4

ORIGINAL ARTICLE

The chemotactic interaction between CCL21 and its receptor, CCR7, facilitates the progression of pancreatic cancer via induction of angiogenesis and lymphangiogenesis Bin Zhao • Kai Cui • Chang-Liang Wang • Ai-Liang Wang • Bo Zhang • Wu-Yuan Zhou Wen-Hua Zhao • Sheng Li

•

Published online: 19 May 2011 Ó Japanese Society of Hepato-Biliary-Pancreatic Surgery and Springer 2011

Abstract Background In this study, we report the influence of CCL21 and its receptor, CCR7, on the progression of pancreatic cancer and illuminates the correlation between the CCL21/CCR7 axis and the angiogenesis and lymphangiogenesis of pancreatic adenocarcinoma (PAC). Methods A total of 30 patients with pancreatic cancer was involved in the current study. The expression of CCL21 and CCR7 in cancerous tissues, paracancerous tissues and normal pancreas were investigated using realtime PCR, Western blot and immunohistochemistry, respectively. In addition, we assessed microvessel density (MVD) and microlymphatic vessel density (MLVD) in tumor tissues using immunohistochemistry. Results Compared to paracancerous tissues and normal pancreas, CCL21 expression in cancerous tissues was detected at a significantly low level. In contrast, the CCR7

expression was considerably higher in cancerous tissues than in normal pancreas and paracancerous tissues. Additionally, a significant correlation between the expression pattern of the CCL21/CCR7 axis and clinicopathological features, such as lymph node metastasis, was identified. Furthermore, we found that CCL21 expression was significantly associated with MVD but not significantly associated with MLVD, while CCR7 expression was significantly associated with MLVD but not significantly associated with MVD. Conclusions The chemotactic interaction between CCR7 and its ligand, CCL21, may be a critical event during progression in pancreatic cancer, and its underlying mechanism may be induction of angiogenesis and lymphangiogenesis regulated by this chemotactic interaction. Keywords Chemokines  Chemokine receptors  Angiogenesis  Lymphangiogenesis  Pancreatic neoplasms

B. Zhao and K. Cui are co-first authors.

Electronic supplementary material The online version of this article (doi:10.1007/s00534-011-0395-4) contains supplementary material, which is available to authorized users. B. Zhao Shandong University, Ji’nan 250011, China K. Cui  C.-L. Wang  B. Zhang  W.-Y. Zhou  S. Li (&) Shandong Tumor Hospital, Ji’nan 250117, Shandong, China e-mail: [email protected] A.-L. Wang Affiliated Hospital of Jining Medical College, Ji’ning 272111, China W.-H. Zhao (&) Shandong Qianfoshan Hospital, Ji’nan 250014, Shandong, China e-mail: [email protected]

Introduction Pancreatic cancer is a devastating disease. Surgical resection of pancreatic adenocarcinoma remains the cornerstone of treatment but is only available for a small minority of patients. Even after surgical intervention, the prognosis remains poor, since median and 5-year survival after resection are 7.5 months and 8%, respectively [1]. A recent meta-analysis suggests that gemcitabine-based chemotherapy seems to improve overall survival of patients with advanced and metastatic disease to some extent [2]. However, even with this treatment the prognosis continues to be poor. The predilection of metastases for specific organs may depend on a variety of factors [3, 4]. The most recent

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theory proposes that chemoattractants, produced by stromal or immune cells, lead invasive cancer cells to the tissue of their potential secondary growth [5]. Chemokines, a group of homologous, yet functionally divergent proteins, directly mediate leukocyte migration and activation and play a role in regulating angiogenesis [6]. Chemokines also function in maintaining immune homeostasis and secondary lymphoid organ architecture [7]. Several chemokines are known to have antitumor activity. Of those chemokines, the role of CCL21, and also its receptor, CCR7, in the progression of several kinds of cancers were intensively studied recently. CCR7 is highly expressed in human breast cancer cells, malignant breast tumors, and metastases; and ligand binding to CCR7 induces chemotactic and invasive responses, including actin polymerization and pseudopodia formation [8]. Several other tumor types express CCR7, and expression correlates with lymph node metastases and poor prognosis in gastric carcinoma [9], squamous cell carcinoma of the head and neck [10], non-small cell lung cancer [11], thyroid carcinomas [12], esophageal cancer [13], cervical cancer [14], tonsillar cancer [15], colorectal adenocarcinomas [16], oral and oropharyngeal squamous cell carcinoma [17], and prostate cancer [18]. Melanoma cells express CCR7 and migrate to CCL21 in vitro [19]. Although Nakata et al. [20] have reported chemokine receptor CCR7 expression correlates with lymph node metastasis in pancreatic cancer, there have been few reports describing their functions in pancreatic cancer. Based on previous findings, we hypothesized that the CCL21/CCR7 axis may play an important role in progression and metastasis of pancreatic carcinoma. Therefore, in this study, we have evaluated the expression of the CCL21/CCR7 axis and its relationship with tumor stage and grade in 30 patients with pancreatic cancer. We also analyzed the intratumoral and peritumoral microvessel density (MVD) and microlymphatic vessel density (MLVD) in pancreatic cancer, and the relationship with expression of CCL21/CCR7. These data were then tested for their significance for tumor progression. Our data suggest that CCL21/CCR7 mediates potent responses in the progression and metastasis of pancreatic cancer.

Materials and methods Tissue samples Tissue samples were obtained from 30 patients who underwent macroscopically curative resection at Shandong Tumor Hospital between 2005 and 2007. Samples of the pancreatic tumor, paracancerous tissues (paracancerous tissues is defined as tissue within 2 cm of the edge of the

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tumor tissue) and normal pancreas were immediately frozen in liquid nitrogen or formalin-fixed after surgery and then embedded in paraffin. Sections from each case were stained with hematoxylin and eosin (H&E) for histological examination according to the tumor–node–metastasis (TNM) classification system. Tissue was collected based on the protocol approved by the Ethics Committee of the Medical Faculty of Shandong Tumor Hospital. All patients had complete clinical and pathologic data. The patients were comprised of 17 men and 13 women with a median age of 57.2 years (ranging from 35 to 78 years). No patients received preoperative chemotherapy or radiotherapy. Among the 30 patients, 17 well-differentiated and 13 poorly differentiated cases were identified. The study included 12 Union for International Cancer Control (UICC) stage I–II patients and 18 who were stage III–IV. Immunohistochemical staining Immunohistochemistry was performed on the tissue sections using the treptavidin–peroxidase immunohistochemical staining methods. We used 4-lm-thick sections of representative blocks with antibodies against the following: CCL21 (dilution 1:200), CCR7 (dilution 1:200), VEGFR-3 (dilution 1:250), and CD34 (dilution 1:80) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Briefly, the sections were deparaffinized and rehydrated. After blocking of endogenous peroxidase with methanol containing 0.3% H2O2, the sections were autoclaved at 121°C for 10 min in citrate buffer (10 mmol/L sodium citrate, pH 6) for antigen retrieval. After blocking with normal goat serum, the sections were reacted overnight with appropriately diluted primary antibodies. The sections were then reacted sequentially with biotin-conjugated anti-mouse immunoglobulin G antibodies (Vector Laboratories, Burlingame, CA, USA) and Vectastain Elite ABC reagent (Vector Laboratories). Diaminobenzidine was used as the chromogen, and the nuclei were counterstained with hematoxylin. A breast cancer sample was used as positive control, and PBS as negative control. The sections were read by two double-blinded pathologists according to staining intensity and the proportion of positive tumor cells. The upper quartile was used as the cut-off point. For evaluation of MVD, microvessels were detected by morphologic observation and immunohistochemical labeling with the endothelial marker CD34. All independent CD34-positive vessel structures were counted, irrespective of the presence of an identifiable lumen. For assessment of MVD, we used the following method. Firstly, the area with the most intense vascularization was selected at low magnification. Then, average MVD was analyzed by selecting 5 randomized fields per tumor at a magnification of 4009. All blood vessels with lumens larger than 8 red cells or

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with muscular layers were not counted. The number of CD34-positive vessel structures in 5 high power fields was recorded and the average value was taken as the MVD for each case. The results of immunostaining were assessed by two pathologists who were blinded to the clinicopathological findings. Meanwhile, VEGFR-3 was used for MLDV evaluation, and the process was the same as MVD assay described above. RT-PCR Real-time PCR was carried out according to the manufacturer’s instructions (TaKaRa, Dalian, China). In brief, RNA extraction from frozen human specimens was performed using the acid guanidinium thiocyanate method. RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water (0.1% DEPC was added to water overnight and then autoclaved for 20 min to destroy DEPC). The prepared RNA (1 lg) was mixed with reverse transcription reagents at a total volume of 20 lL and incubated for 30 min at 42°C to produce first-strand cDNA. A total of 1 lL cDNA was used for PCR amplification (Invitrogen, Carlsbad, CA). The primer sequences were as follows: CCR7 forward: 50 -CAGCCTTCCTGTGTGGTT-30 CCR7 reverse: 50 -AGGAACCAGGCTTTAAAGT-30 SLC forward: 50 -GGTTCTGGCCTTTGGCATC-30 SLC reverse: 50 -AGGCAACAGTCCTGAGCCC-30 b-Actin forward: 50 -CCCAAGGCCAACCGCGAG AAGAT-30 b-Actin reverse: 50 -GTCCCGGCCAGCCAGGTC CAG-30 . Following the manufacturer’s instructions, reverse transcription was performed at 42°C in the presence of 5 units AMV reverse transcriptase and 1 lg RNA for 60 min. AMV RT inactivation and RNA/cDNA/primer denaturation were performed at 95°C for 5 min to activate modified Taq polymerase followed by 40 cycles at 95°C for 10 min, 95°C for 10 s and 56°C for 1 min and 1 cycle at 72°C for 35 s. The PCR product was separated by 2% agarose gel electrophoresis. The gels were viewed using UV transillumination and photographed by a Kodak 120 gel imaging system. Western blot analysis Frozen samples were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (complete, Mini, Roche Diagnostics Corp., Indianapolis, IN). Protein concentration was determined by using a bicinchoninic acid (BCA) protein assay kit (Pierce-BioLynx, Brockville, Ontario, Canada). Following boiling for 2 min

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in SDS sample buffer, 20 lg of total protein and molecular weight standard (Precision Plus Protein All Blue Standards, Bio-Rad Laboratories, Hercules, CA) were simultaneously subjected to 10% SDS-PAGE and the separated protein bands were transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in TBS containing 1% Tween 20 for 1 h and then incubated overnight at 4°C with anti–SLC (mouse; 1:400 dilution; Roche Diagnostics) and anti-CCR7 antibodies (mouse; 1:1,000 dilution; Stressgen, Ann Arbor, MI). The membranes were immersed in secondary antibody (goat antimouse or anti-rabbit IgG-horseradish peroxidase; 1:5,000 dilution; Cedarlane Laboratories Ltd., Hornby, ON, Canada) for 1 h, incubated with Supersignal (Pierce-BioLynx), and exposed to X-ray film for detection of antibody-bound proteins. As a loading control, the membranes were immunoblotted for b-actin (mouse, 1:20,000 dilution; Cedarlane Laboratories). Statistical analysis The data were analyzed using the SPSS software program (version 13.0 for Windows; SPSS Inc., Chicago, IL). Comparison among different groups was performed by Chi-square test, Fisher’s exact probabilities, one-way ANOVA with Bonferroni t test and Spearman rank correlation coefficient. P \ 0.05 was considered statistically significant. After the step of determining statistical power of 0.8, we calculated that the minimum required sample size is thirty cases in our study.

Results Expression of CCL21 and CCR7 in pancreatic cancer At first, the expression of CCL21 and CCR7 in primary tumor, paracancerous tissues and normal pancreas were compared using various methods including real-time PCR and immunohistochemistry. To further determine the expression and location of CCL21 and CCR7, immunohistochemical assay was performed (Fig. 1). In keeping with above results, CCL21 expression was low in pancreatic tumor tissues, but high in other tissues. The immunostaining results of CCR7 are shown in Table 1; 76.7 and 66.7% of positive expression was detected in cancerous tissues and paracancerous tissues, respectively. This is statistically significant compared with normal tissues. As shown in Table 2, expression of the CCL21 gene in tumor tissues was found to be low at the mRNA level, but in paracancerous tissues and normal pancreas, the CCL21 mRNA level was found to be fairly high (Fig. 2a). At the

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Fig. 1 Immunohistochemical staining of CCL21 in cancerous tissues (a), paracancerous tissues (b) and normal pancreas (c). Immunohistochemical staining of CCR7 in cancerous tissues (d), paracancerous tissues (e), normal pancreas (f) (9100)

Relationship between CCL21, CCR7 and clinicopathological factors of pancreatic cancer To define the role of CCL21 and CCR7 in the progression of pancreatic cancer, we analyzed the association of expression profile of CCL21 and CCR7 with tumor grade and stage. It was revealed that an increase in or induction of expression of CCR7 and the cytoplasmic localization of CCR7 was significantly associated with tumor grade and stage (Table 3). In addition, CCR7 expression was also correlated with lymph node status in pancreatic cancer patients (P = 0.01). As for CCL21 expression, no pronounced correlation was observed with progression in pancreatic cancer patients. Lymphatic and blood vessels in pancreatic cancer Fig. 2 a CCL21 mRNA expression. b CCR7 mRNA expression

protein level, CCL21 showed the same trend as at the mRNA level (Fig. 3). As for CCR7, the opposite was observed not only at the mRNA level (Fig. 2b), but also at the protein level (Fig. 3). Compared with normal pancreas, the expression of CCR7 in tissues originating from other locations was significantly increased (P \ 0.05) as shown in Table 2. Similar results were observed using 2% agarose gel electrophoresis after real-time PCR (Fig. 2a, b).

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Table 4 lists the degree of angiogenesis and lymphogenesis, and the relationships between these variables and clinical outcomes were determined. Patients with high intratumoral MVD had a significantly lower degree of differentiation and higher class of TNM staging than those with low intratumoral MVD (P \ 0.05). However, no obvious relationship was found between lymph node metastasis and blood vessel formation (P [ 0.05). In addition, it was revealed that MLVD status was also related to the tumor stage and grade. Formation of lymphatic vessels was significantly increased in patients with higher

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Table 1 The positive expression of CCL21 and CCR7 determined by immunohistochemical staining Group

CCL21 expression

CCR7 expression

Negative

Positive (%)

Cancerous tissues

25

5 (16.7)

Paracancerous tissues

17 7

Normal pancreas

P

Negative

13 (43.3)

0.049

10

20 (66.7)

0.567

23 (76.7)

0.000

21

9 (30.0)

0.000

7

Positive (%)

P

23 (76.7)

Table 2 The expression of CCL21 mRNA and CCR7 mRNA by RT-PCR Group

CCL21 mRNA CCL21/b-actin

CCR7 mRNA P

CCR7/b-actin

P

Cancerous tissues

0.257 ± 0.173

0.793 ± 0.291

Paracancerous tissues

0.428 ± 0.247

0.003 0.698 ± 0.324

Normal pancreas

0.695 ± 0.302* 0.000 0.233 ± 0.197* 0.000

0.237

* P \ 0.001 compared with paracancerous tissue values by one-way ANOVA and Bonferroni t test

Fig. 4 CD34 is positive in a microvascular endothelial cell of pancreatic cancer (9100)

Fig. 3 CCL21 and CCR7 expression

TNM staging and lymph node metastasis, but there was no statistically significant change in patients with a lower degree of differentiation (Figs. 4, 5).

associated with angiogenesis of PAC, andthat a higher expression of CCR7 protein is significantly associated with lymphangiogenesis of pancreatic cancer. The MVD in patients with positive expression of CCL21 was increased but no effects were revealed on CCR7 expression. As for MLVD, by contrast, there was a statistically significant increase in the mumber of patients with CCR7 expression, but not CCL21 expression.

Discussion Analysis of association between CCL21, CCR7 and MVD, MLVD As shown in Table 5, the CCL21 protein was negative in 25 samples, and among these samples the MVD was higher than that in positive samples of CCL21 protein (P = 0.012). Also there was no significant correlation between the expression of CCL21 protein with MLVD of PAC (P [ 0.05). There were 23 samples in which the CCR7 protein was positive, and among these samples the MLVD was higher than that in the group with negative CCR7 protein (P = 0.004). These results indicate that a lower expression of CCL21 protein is significantly

Chemokines and their receptors have been suggested to play a key role in regulating the metastatic destination of tumor cells [21]. Chemokine molecules constitute a superfamily of inducible, secreted, proinflammatory proteins [22–25] involved in a variety of immune responses, acting primarily as chemoattractants and activators of specific types of leukocytes [26, 27], which mediate their effects by binding to G-protein-coupled receptors [28]. It is becoming increasingly evident that chemokines play an integral role in the initiation of a specific immune response [29]. One such chemokine, CCL21, which is a CC chemokine found on high endothelial venules and within the T

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Table 3 Correlation between CCL21, CCR7 and clinicopathological factors of pancreatic cancer Factors

n

CCR7 expression

CCL21 expression

Negative

Positive (%)

Histology

P

Negative

Positive (%)

0.018

0.510

Well-differentiated

17

6

11 (64.7)

13

4 (23.5)

Moderately/poorly differentiated

13

1

12 (92.2)

12

1 (7.8)

I–II

12

6

6 (50.0)

9

3 (33.3)

III–IV

18

1

17 (94.2)

16

2 (11.1)

TNM stage

0.011

Lymph node metastasis Negative

0.617

0.001

Positive

n

0.617

12

7

5 (41.7)

11

1 (8.3)

18

0

18 (100.0)

14

4 (22.2)

Table 4 Correlation between MVD, MLVD and clinicopathological factors of pancreatic cancer MVD

P

P

Table 5 Correlation between CCL21, CCR7 and MVD, MLVD n

MVD

P

0.012

MLVD

P

MLVD P CCL21

Histology

0.000

0.194

Negative

25

63 ± 7.1

Well-differentiated

17 54 ± 4.3

5 ± 5.9

Positive

5

54 ± 6.8

Moderately/poorly differentiated

13 65 ± 6.5

8 ± 6.4

CCR7 Negative

7

52 ± 4.7

Positive

23

54 ± 4.2

TNM stage

0.000

0.017

I–II

12 52 ± 7.4

4 ± 2.9

III–IV

18 63 ± 7.3

8 ± 4.9

Lymph node metastasis

0.456

0.009

Negative

12 52 ± 7.4

4 ± 2.9

Positive

18 54 ± 6.9

9 ± 5.7

Fig. 5 Immunohistochemical staining of VEGF-R3 in micro-lymph vessel endothelial cell of pancreatic cancer (9100)

cell zones of both spleen and lymph nodes [30–32], is selective in its recruitment of naive T cells and dendritic cells (DCs) [33]. In the lymph nodes, CCL21 is believed to play an important role in the initiation of an immune

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8 ± 5.4

0.266

6 ± 3.2 0.292

6 ± 5.7

0.004

12 ± 4.1

response by colocalizing naive T cells with DC-presenting antigen [34, 35]. The receptor for this ligand, CCR7, is expressed on all naive T cells, some memory T cells, B cells, and mature dendritic cells and plays a central role in lymphocyte trafficking and homing to lymph nodes [36, 37]. Recent studies have shown that CCL21/CCR7 signaling is involved in regulating the progression of several types of cancer [38, 39]. However, there are few reports of correlation of the CCL21/CCR7 axis with clinical features in pancreatic cancer. In our study, we have evaluated the expression of CCL21/CCR7 and revealed a critical relationship between them and tumor stage and grade in pancreatic cancer. Our data show that, in pancreatic carcinoma, CCL21 expression in tumor tissues is decreased. However, in paracancerous tissues, normal pancreas and lymph nodes, CCL21 was detected at a fairly high level. In contrast, CCR7 expression showed the opposite trend. 76.7% of pancreatic carcinoma samples expressed CCR7 compared with 30% in normal samples. We also found significant differences in the following clinicopathological features and prognosis among CCL21/CCR7-positive and CCL21/ CCR7-negative samples: (a) lymph node metastasis; (b) tumor TNM staging; and (c) degree of tumor differentiation. To elucidate the underlying association between the mode of CCL21/CCR7 expression and clinicopathological

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features, we assessed MVD and MLVD in tumor tissues. We found that CCL21 expression was significantly associated with the formation of blood vessels, but there was no obvious link with lymphatic microvessel density. As for CCR7, higher levels of expression were detected in patients with higher MLVD but lower MVD. Angiogenesis and lymphangiogenesis, the formation of new blood vessels and lymphatic vessels, are required for many pathological processes, including tumor growth and metastasis as well as physiological organ/tissue maintenance. In general, the molecular mechanisms that control carcinoma progression and metastasis are related to alterations in various oncogenes, tumor suppressor genes, metastasis suppressor genes, and growth factors and their receptors, including Src, Ras, p16, KiSS-1, Nm23, FasL, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and interleukin (IL)-6 [40–42]. These abnormalities affect the downstream signal transduction pathways involved in the control of cell growth and other malignant properties, such as tumor staging and degree of tumor differentiation. Interestingly, one of the most recently recognized events involves the interaction between chemokines and their receptors. Several studies found that the chemokine receptor was highly expressed in breast and ovarian carcinomas, and the interaction between the receptor and its ligand resulted in chemotaxis or directed migration of tumor cells from their primary site via the circulation to preferential sites of metastasis [43– 45]. These studies strongly supported our hypothesis. The interaction between CCR7 and CCL21 may play crucial roles in the metastasis and progression of pancreatic cancer by effects on the formation of new blood vessels and lymphatic vessels. In conclusion, the chemotactic interaction between CCR7 and its ligand, CCL21, may be a critical event during progression in pancreatic cancer, and the potent mechanism for that may be the induction by cancer cells of angiogenesis and lymphangiogenesis. This hypothesis was supported by the findings that the clinicopathological features are significantly correlated with the expression of CCR7 in pancreatic cancer tissues, but not CCL21. Our data also suggest that the CCL21/CCR7 axis could be associated with formation of lymphatic vessels and blood vessels induced by pancreatic cancer cells. Additional work is under way to determine pathways responsible for tumor cell chemokine and/or chemokine receptor-associated angiogenesis and lymphangiogenesis. Controlling such a poor prognostic feature would be likely to enable more successful loco-regional tumor control and improve survival in patients with pancreatic cancer. Acknowledgments This work was financially supported by Grants No. 30571712 and No. 30810403081 from the National Natural

827 Science Foundation of China and Grant No. 2007GG20002022 from the Department of Science and Technology of Shandong Province of China. We thank Wei-xia Zhong, Dian-bin Mu and Yin-ping Yuan for their excellent technical support in evaluating the results of immunohistochemical staining. Conflicts of interest peting interests.

All authors declare that they have no com-

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