Enzyme-linked immunosorbent assay for BAFF and APRIL

Serum levels of BAFF and APRIL in all subjects were assayed using an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA for BAFF; BioVendor, Candler, NC, USA for APRIL) following the manufacturer's recommendations.

Immunohistochemistry of PDAC specimens

Pancreatic tissues were fixed in formalin. Sections (3 µm thick) were cut from each block and adjacent sections were stained with standard hematoxylin and eosin (H&E) and immunohistochemical staining techniques. Paraffin-embedded samples were dewaxed and rehydrated, and then antigens were retrieved by autoclaving for 1 min at 125°C in EDTA buffer (pH 9.0). Endogenous peroxidase activity was inactivated by incubation with methanol containing 1% hydrogen peroxidase for 20 min. The sections were then incubated in 1% blocking serum for 30 min to reduce nonspecific reactions. For immunohistochemistry, the sections were incubated with the relevant primary antibody (Table S1) at 4°C overnight. The tissue sections were treated with peroxidase-labeled secondary antibody (Histofine Simplestain Max PO; Nichirei, Tokyo, Japan) for 1 h at room temperature, and incubated with Simple Stain DAB Solution (Nichirei). Photomicrographs were taken using a Nikon Microphot FXA with a Nikon Digital Camera DXM 1200 (Nikon, Tokyo, Japan).

For the immunofluorescence staining, mounted and formalin-fixed sections were used. Sections were incubated with the relevant primary antibody (Table S1) at 4°C overnight. After washing with phosphate-buffered saline, the sections were incubated with DyLight488-conjugated donkey anti-goat antibody for BAFF or BAFF-R, and with DyLight549-conjugated donkey anti-mouse antibody for CD20. The nuclei of the sections were stained with 4,6-diamidino-2-phenylindole (Wako, Osaka, Japan). Slides were imaged using a Zeiss Axioskop 2 Plus fluorescence microscope and captured by a Zeiss AxioCam HRc digital camera (Zeiss, Tokyo, Japan) and saved to a computer. The photographs stained for BAFF, BAFF-R and CD20 were merged to evaluate their locations in the cells.

Cells and culture conditions

Four pancreatic cancer cell lines (PANC-1, BxPC-3, AsPC-1 and MIA PaCa-2 cells), which were initially generated from patients with PDAC, and Ramos cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The PANC-1 and MIA PaCa-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin. BxPC-3, AsPC-1 and Ramos cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin. Microphotographs were obtained after various treatments on an inverted microscope (Olympus IX70, Olympus, Tokyo, Japan) equipped with an Olympus DP12 digital camera. After being serum starved for 24 h, cells were incubated with recombinant BAFF (Reliatech, Braunschweig, Germany) and recombinant TGF-β (R&D Systems) for 48 h. For neutralizing the BAFF, goat anti-BAFF-R antibody (R&D Systems) was used, and goat IgG antibody (R&D Systems) was used as a control antibody.

RNA extraction, cDNA synthesis and real-time RT-PCR

The RNA was reverse transcribed using RT-PCR kits (Applied Biosystems, Foster City, CA, USA) with an oligo d(T)₁₆ primer under standard conditions. Real-time PCR amplification was performed using a LightCycler 480 (Roche, Basel, Switzerland) and 2 µL of purified cDNA product, 0.5 µL of sense primer (10 pmol/ µL), 0.5 µL of antisense primer (10 pmol/ µl), 1 µL of LightCycler Fast Start DNA Master SYBR Green I (Roche), and 0.8 µL of MgCl₂ (25 mmol/L) (experimental conditions and sequences of the primers used to amplify human genes are indicated in Table S2). Commercial glyceraldehyde phosphate dehydrogenase (GAPDH) primer sets and β-actin primer sets (Roche) were used for PCR amplification under the conditions recommended by the manufacturer. GAPDH served as an internal reference gene, and the relative change was calculated by relative quantification, applying the formula 2^−ΔΔCt. Reaction products were separated on 2% agarose gels.

Western blotting

For Western blotting, 20 µg of protein was applied to the lanes of 4% to 12% Bis-Tris Gels (Life Technologies), then blotted onto Immobilon-P membranes (Millipore, Bedford, MA, USA), and incubated with the relevant primary antibody (Table S1). Appropriate species-specific conjugated secondary antibody kits were commercially obtained (GE Healthcare, Tokyo, Japan). Proteins were detected using the ECL prime Kit or the ECL Kit (GE Healthcare) with an ImageQuant LAS 4000 system (GE Healthcare).

Wound healing/scratch test

PANC-1 cells were seeded into 6-well plates and incubated for 24 h under a serum-starved condition. After confirming that a complete monolayer had formed, the monolayers were wounded by scratching lines with a plastic tip. The wells were then washed once to remove any debris, and observed and photographed under the microscope. Thereafter, the plates were incubated at 37°C under 5% CO₂ for 24 h with the recombinant human BAFF (Reliatech), after which the cells were observed and photographed. Cells were visualized with an Olympus Model IX70 inverted microscope (Olympus) using a 4× objective. Images were captured with an Olympus DP12 a digital camera (Olympus). The distance that the cells had migrated was measured on the photomicrographs. The percent wounded area filled was calculated as follows: {(mean wounded breadth – mean remained breadth)/mean wounded breadth} × 100 (%) [19].

Invasion assay

To investigate cell invasion, a 96-well cell invasion assay kit (Cultrex, Trevigen, Gaithersburg, MD, USA) was used according to the manufacturer's instructions.

Establishment of human BAFF-R transfectant cell clones

Cell clones overexpressing BAFF-R were developed using a plasmid, which could express the BAFF-R gene and the G418-resistant gene (pBCMGS-BAFF-R) [20]. PANC-1 cells were transfected with pBCMGS-BAFF-R using Lipofectamin 2000 (Life Technologies), and the transfected cell clones were selected by incubation with G418 (1000 µg/mL, Life Technologies). Finally, four cell clones that over-expressed BAFF-R were established.

Flow cytometric analysis

Flow cytometric analysis was performed for the stained PANC-1 cells and human BAFF-R transfected cell clones. BAFF-R was detected with primary antibody specific for the BAFF-R (Table S1) followed by an additional incubation with Alexa 488-conjugated secondary antibody for goat IgG (Abcam, Tokyo, Japan). Those stained cells were examined using a FACScalibur (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed with FlowJo software (TreeStar Corporation, Ashland, OR, USA).

Expression of BAFF and BAFF-R in human PDAC tissue

Immunohistochemistry experiments were performed to determine whether BAFF is expressed in pancreatic tissue. Pancreatic tissue from surgical species obtained from patients with PDAC was stained for BAFF and BAFF-R (Fig. 2 and ​and3).3). Tumor-infiltrating immune cells surrounding PDAC cells were found to remarkably express BAFF and BAFF-R (Fig. 2). Additional staining of CD20 (a marker of B lymphocytes), CD3 (T cell receptor, a marker of T lymphocytes) and CD68 (a marker of monocytes/macrophages) was performed to identify their types of infiltrating cells expressing BAFF or BAFF-R. The distribution of BAFF- and BAFF-R-positive cells appeared consistent with the distribution of B lymphocytes, rather than that of T lymphocytes or monocyte/macrophages (Fig. 2). Recently, it has been shown that B lymphocytes can secrete BAFF [7], [8], [16]. Double immunofluorescence staining with BAFF and CD20, CD3, CD68 (Fig. 3A), and staining with BAFF-R and CD20, CD3, CD68 (Fig. 3B) was performed. The distribution of BAFF (green) and CD20 (red) was merged and appeared yellow (Fig. 3A). Moreover, the distribution of BAFF-R (green) and CD20 (red) was also merged and appeared yellow (Fig. 3B). However, the distribution of BAFF and BAFF-R were not co-localized with CD3 or CD68. These results indicate that B lymphocytes, which express high levels of BAFF and BAFF-R, are in the infiltrate and proliferate in the tissue surrounding the PDAC.

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Figure 2

Immunohistochemistry of BAFF and BAFF receptor in human PDAC tissue.

(A) Staining with hematoxylin and eosin (H&E) and immunohistochemistry of BAFF, BAFF-R, CD3, CD20 and CD68 in PDAC and surrounding tissues are shown. First Ab (-) indicates staining control without first antibody. The B lymphocytes in the tissue surrounding PDAC had a remarkably high level of BAFF expression. Magnification as indicated above each photomicrograph. Scale bars represent 200 µm under low magnification (x40), and 50 µm under high magnification (x200 and ×400).

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Figure 3

Immunofluorescence of BAFF and BAFF receptor in human PDAC tissue.

(A) Immunofluorescence staining with BAFF (green), and CD20, CD3, CD68 (red) are shown. The merged image is indicated as “merge”. Expressions of BAFF and CD20 were co-localized (yellow); however, BAFF and CD3 or CD68 were not co-localized. (B) Immunofluorescence staining with BAFF-R (green) and CD20, CD3, and CD68 (red) are shown. Expression of BAFF-R and CD20 were co-localized (yellow); however, BAFF-R and CD3 or CD68 were not co-localized. First Ab (-) indicates staining control without the first antibody.

Expression of BAFF receptors in human PDAC tissues and human PDAC cell lines

In order to investigate the expression of BAFF receptors in PDAC, immunohistochemistry was performed on PDAC tissues obtained from patients as well as on human PDAC cell lines. BAFF-R was expressed in PDAC cells, which had positive signals of MIB-1 and CA19-9 (Fig. 4A, left column). However, other BAFF receptors (TACI and BCMA) were not expressed in the PDAC cells (Fig. 4A, right column). The human PDAC cell lines, PANC-1, BxPC-3, AsPC-1, and MIA PaCa-2, were used for the in vitro studies. Qualitative RT-PCR for the gene expression of BAFF-R, TACI, and BCMA revealed only expression of BAFF-R (Fig. 4B). Ramos cells, known as a tissue that expresses BAFF-R, TACI and BCMA, were used as a positive control [21]. Moreover, Western blotting was performed to evaluate the protein expression of these BAFF receptors (Fig. 4C). Expression of BAFF-R was confirmed in all of the PDAC cell lines, but expression of TACI and BCMA could not be detected. Taken together, these results showed that increased levels of BAFF resulted from the infiltrating and proliferating B lymphocytes surrounding PDAC tissues; these increased levels of BAFF can stimulate PDAC through BAFF-R signaling. To identify the role of BAFF in PDAC, an in vitro assay was performed using the PANC-1 PDAC cell line, its cloned transfectant of BAFF-R, and recombinant human BAFF for further analysis.

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Figure 4

Expression of BAFF receptors in human PDAC tissue and human PDAC cell lines.

(A) Staining with hematoxylin and eosin (H&E) and immunohistochemistry of MIB-1, CA19-9, BAFF-R, TACI and BCMA in PDAC are shown. First Ab (-) indicates staining control without the first antibody. Magnification is as indicated above each photomicrograph. Scale bars represent 200 µm under low magnification (x40), and 50 µm under high magnification (x400). (B) Qualitative RT-PCR analysis of BAFF receptors in PDAC cell lines. GAPDH is indicated as a housekeeping gene. RNA isolated from Ramos cell was used as a positive control. N.C. is a negative control without sample. (C) Western blot analysis of BAFF receptors in PDAC cell lines. β-actin is indicated as a housekeeping protein. Ramos cell was used as a positive control. N.C. is a negative control without sample.

BAFF induces EMT in a PDAC cell line

In the in vitro assay, the cell morphology of PANC-1 was observed to change after addition of recombinant human BAFF to the culture medium (Fig. 5A). The cells appeared to adopt a more fibroblast-like (spindle type) morphology and showed reduced cell-cell contact. These morphologic changes were similar to changes seen with TGF-β treatment. From this result, it was hypothesized that increased BAFF might induce alterations in genes related to the epithelial-mesenchymal transition (EMT) in PDAC cells. PANC-1 is isolated from undifferentiated PDAC tissue [22] and is positive for the epithelial marker E-cadherin and the mesenchymal protein vimentin. Thus, PANC-1 was used as a model of a PDAC cell that still has an epithelial potential. Analysis for altered expression of representative genes related to EMT was performed by adding human recombinant BAFF to the culture medium. A significant downregulation of E-cadherin mRNA, as well as significant upregulation of vimentin and Snail mRNAs, were observed in a dose-dependent manner with BAFF (Fig. 5B). GAPDH mRNA was used as a housekeeping gene for the quantitative PCR, and it was confirmed that the levels of GAPDH mRNA were not altered by treatment with BAFF (Fig. S1). Western blotting results for these molecules were similar to the results of real-time RT-PCR (Fig. 5C). The BAFF-induced alterations in PDAC cells were similar to alterations seen during EMT. Increased BAFF in PDAC could promote gene alterations associated with EMT through a decrease in E-cadherin and an increase in vimentin via the Snail signaling pathways.

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Figure 5

BAFF induces changes associated with epithelial-mesenchymal transition (EMT) in a PDAC cell line.

(A) The cellular morphology of PANC-1 cells changed after the addition of BAFF to the supernatant. TGF-β was used as a positive control. Similar to TGF-β, addition of BAFF caused the morphology of PANC-1 cells to become spindle-like. (B) Analysis of real-time RT-PCR for EMT markers (E-cadherin, vimentin, and Snail) are indicated. These genes were modulated in a dose-dependent manner with BAFF. Data are shown as means ± SE of four separate experiments (*p<0.05 vs. BAFF 0 ng/mL). (C) Western blotting analysis of EMT markers indicates that the expression of each protein was modulated by the addition of BAFF. β-actin is shown as a housekeeping protein.

Treatment with human recombinant BAFF and motility and invasion of PDAC cells

In conjunction with morphologic changes, it was suggested that BAFF induces EMT in PDAC. These results prompted study of the other effects of BAFF, such as motility and invasion. PANC-1 cells were incubated with human recombinant BAFF, and changes in motility were evaluated with the wound healing/scratch test. Treatment with BAFF increased the motility of PANC-1 cells and resulted in accelerated wound closure (p<0.01) (Fig. 6A and 6B). Moreover, treatment with human recombinant BAFF significantly improved the ability of cells to invade the extracellular matrix in vitro (Fig. 6C).

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Figure 6

Treatment with human recombinant BAFF increases motility and invasion of PDAC cells.

(A) The wound healing/scratch test indicated that the motility of PANC-1 cells was enhanced by 24 h of incubation with BAFF. (B) The percent wounded area filled in the wound healing/scratch test is shown as a graph (*p<0.01 vs. BAFF 0 ng/mL). (C) The invasion assay shows enhancement of invasion with BAFF in PANC-1 cells (*p<0.05 vs. BAFF 0 ng/mL). (D) The wound healing/scratch test with neutralizing anti-BAFF-R antibody indicated that the motility of PANC-1 cells with BAFF was inhibited by the antibody. (E) The percent wounded area filled in the wound healing/scratch test is shown as a graph. Data are shown as means ± SE of six separate experiments (*p<0.05, **p<0.01).

Additional assays were performed using anti-human BAFF-R antibody, which can inhibit the binding of BAFF to BAFF-R [23]. With addition of human recombinant BAFF to the supernatant of the cultured cells, the percent wounded area filled increased significantly (15.1 ± 2.3 vs. 27.2 ± 2.3; p<0.01). With addition of both human recombinant BAFF and the anti-human BAFF-R antibody, wound closure was diminished significantly compared to the addition of control goat antibody (27.2 ± 2.3 vs. 20.9 ± 2.4; p<0.05) (Fig. 6D and 6E). These data suggest that BAFF stimulation caused increased motility and invasion of PANC-1 cells.

We thank Dr. Takeshi Imamura (Department of Molecular Medicine for Pathogenesis, Ehime University School of Medicine, Ehime, Japan), Dr. Aristidis Moustakas (Ludwig Institute for Cancer Research, Uppsala University, Sweden) and Dr. Gorgoulis Vasillis (Department of Histology and Embryology, School of Medicine, University of Athens, Athens, Greece) for valuable advice, and also Dr. Shuichiro Shigematsu, Mr. Kenji Tanimoto, Ms. Shiyi Chen, Ms. Satomi Yamanaka, Ms. Sakiko Sugawara, Ms. Chie Takeichi and Ms. Sakiko Inoh (in our department) for valuable technical assistance.