Tumour-derived Interleukin 35 promotes pancreatic ductal adenocarcinoma cell extravasation and metastasis by inducing ICAM1 expression

Interleukin 35 (IL-35) is a novel member of the IL-12 family, consisting of an EBV-induced gene 3 (EBI3) subunit and a P35 subunit. IL-35 is an immune-suppressive cytokine mainly produced by regulatory T cells. However, the role of IL-35 in cancer metastasis and progression is not well understood. Here we demonstrate that IL-35 is overexpressed in human pancreatic ductal adenocarcinoma (PDAC) tissues, and that IL-35 overexpression is associated with poor prognosis in PDAC patients. IL-35 has critical roles in PDAC cell extravasation and metastasis by facilitating the adhesion to endothelial cells and transendothelial extravasation. Mechanistically, IL-35 promotes ICAM1 overexpression through a GP130-STAT1 signalling pathway, which facilitates endothelial adhesion and transendothelial migration via an ICAM1–fibrinogen–ICAM1 bridge. In an orthotopic xenograft model, IL-35 promotes spontaneous pancreatic cancer metastasis in an ICAM1-dependent manner. Together, our results indicate additional functions of IL-35 in promoting PDAC metastasis through mediating ICAM1 expression.


Supplementary Figure 1. The expression levels of cancer-related genes in PDAC tissues and the corresponding adjacent normal tissues.
A, immunohistochemistry staining for EBI3, P35, CA125 and CEA in consecutive sections of pancreatic carcinoma tissues and the adjacent normal pancreatic tissues (2-3 cm around the tumor border). B, the mRNA expression levels of 11 cancerrelated genes detected by RT-PCR in 10 PDAC tissues and the corresponding adjacent normal tissues. C, the mRNA levels of CEA, CA125, c-MYC, COX2, YAP1, BCL2 and HER2 in the pancreas tissue of patients with PDAC and duodenal carcinoma (used as normal controls). D, immunohistochemical staining with no primary antibodies was shown as a negative control. Unpaired t test was used; shown are mean ± S.D. Experiments were replicated for three times. Tests of significance are two-sided. *P < 0.05. Figure panels pairs in A and D represent images taken with different zooming options; scale bars, 100μm. Figure 2. The expression of IL-35 subunits in PDAC tissues, adjacent preneoplastic lesions and adjacent normal pancreas tissues. A, the heat map showing immunohistochemical staining of EBI3 and P35 in PDAC tumor tissues, the corresponding adjacent pre-neoplastic lesions and corresponding adjacent normal pancreas tissues; N=64. B, Wilcoxon Test was used to analyze the differential expression of EBI3 and P35. C, representative images of differential IL-35 expression in pancreatic ductal adenocarcinoma tumor tissue and the adjacent preneoplastic tissue. T, tumor; P/Pre., pre-neoplastic lesions; N, normal pancreas tissues. Tests of significance are two-sided. *P < 0.05. Figure panels pairs in C represent images taken with different zooming options; scale bars, 100μm. Figure 3. The correlation between EBI3, P35, P28 and P40 expression in specimens of PDAC patients. A, immunohistochemical analysis of EBI3, P35, P28 and P40 correlative expression in consecutive sections from human PDAC surgical samples. Scale bars, 200 μm. B, the real distribution of immunohistochemical results of EBI3, P35, P28 and P40. C-D, statistical analysis of immunohistochemical results of EBI3 and P28 expression (C), as well as that of P35 and P40 (D) in human PDAC surgical samples. E, Co-immunoprecipitation and western blot analysis of EBI3:P35, EBI3:P28 and P35:P40 protein from lysates of 4 primary PDAC cell lines. F, a total of 5 × 10 5 cells were seeded onto flask and cultured in 4 mL medium for 24 hours. ELISA assays were done to detect the levels of IL-35, IL-12 and IL-27 in supernatant of 4 primary PDAC cell lines. G, immunohistochemical analysis of EBI3, P35 and IL-12(IL-12p70) expression in consecutive sections from human PDAC surgical samples. H, the real distribution of immunohistochemical results of EBI3, P35 and IL-12. I, Wilcoxon Test used to analyze the differential IL-12 levels. Figure panels pairs in A and G represent images taken with different zooming options; scale bars, 100 μm.

Supplementary Figure 4. The expression of IL-35 ligand and receptor in PDAC cell lines.
A-B, expression levels of the two subunits of IL-35 (EBI3 and p35) and the two subunits of IL-35 receptor (gp130 and IL12rβ2) in five PDAC cell lines (Panc-1, BxPC-3, AsPC-1, MIA PaCa-2 and CFPAC-1) and one normal pancreatic ductal epithelial cell line (HPDE6c7) were detected by RT-PCR (A) and Western blot (B). CD4+CD25+ Treg (nTreg) cells were used as positive expression control; β-actin was used as a loading control. C, co-immunoprecipitation and western blot analysis of EBI3 and p35 protein from lysates of MIA PaCa-2 and CFPAC-1 cells. D, a total of 5 × 10 5 cells were cultured in 4 mL medium for 24 hours. The supernatant was subjected to ELISA assays to detect IL-35 levels. Unpaired t test was used. Tests of significance are two-sided. *P < 0.05. E, subcellular localization of endogenous EBI3 and p35 in 5 PDAC cell lines. EBI3 and p35 was co-located in the cytoplasm of the cells. Immunofluorescence images were taken by a confocal microscopy. Scale bars, 50μm. F, the membrane localization of gp130 and IL12rβ2 was detected by a microscopy after immunofluorescent staining. Scale bars in E and F, 100μm.

Supplementary Figure 5. Establishment and verification of stable cells lines overexpressing or downexpressing IL-35.
A, the schematic of construction of IL-35-overexpressing and IL-35downexpressing cell lines is presented; the EBI3-P35 fusion sequence was cloned from a commercial IL-35-expressing plasmid (InvivoGen). B-C, PANC-1 and BxPC-3 cells were stably transfected with the fused EBI3-p35 gene, while empty vectors were transfected as controls. RT-PCR (B) and Elisa analysis (C) were used to detect the production and secretory level of IL-35. D-E, MIA PaCA-2 and CFPAC-1 cells were stably transfected with both shEBI3 and shP35 sequences to knockdown IL-35 expression, while scrambled sequences were transfected as controls. RT-PCR (D) and Elisa analysis (E) were used to detect the production and secretory level of IL-35. F, verification the biological function of fused IL-35 protein and commercial recombinant IL-35. Human conventional CD4 + T cells were purified by a magnetic beads sorting kit from peripheral blood of healthy volunteers, then activated for 3 days with anti-CD3 and anti-CD28 antibodies in the presence of conditioned medium from the IL-35-overexpressing PANC-1 cells (50%) or commercial recombinant IL-35 (100ng/mL). Thereafter an EDU incorporation kit was used to detect the proliferation of the cells. Representative immunofluorescence images of the EDU incorporation assay (top). Scale bars, 50 μm. Nuclei of T cells were labelled with blue color (Hoechst33342) and EDU were labelled with green color (Apollo®488). Statistical results are presented as the average EDU incorporation rates of 5 randomed fields of 400 × magnifications. Data represent three independent experiments (mean±S.D.) (bottom). Experiments were replicated for three times. Unpaired t test was used; Shown are mean values ± SD; *P < 0.05; tests are two-sided.

Supplementary Figure 6. The roles of IL-35 in PDAC migration, invasion and proliferation
assays. Panc-1 and BxPC-3 cells stably transfected with pLV-IL35 or pLV-Vector were subjected to Transwell migration assays (A), Transwell invasion assays (B), EDU incorporation assays (C) and the CCK8 assays (D). The experiments were performed in triplicate. Experiments were replicated for three times. Unpaired t test was used; Shown are mean values ± SD; *P < 0.05; tests are two-sided. Scale bars, 100μm.

Supplementary Figure 7. IL-35 facilitates PDAC cells adhesion to endothelial monolayer in static adhesion assays. Stable cell lines of BxPC-3 and PANC1 with IL-35 down-regulated or upregulated were detected by RT-PCR (A) and Western blot assays (B)
. Then the stable cell lines were subjected to static HUVEC adhesion assays (C). The detailed procedure was shown in Method. Experiments were replicated for three times. Unpaired t test was used; Shown are mean values ± SD; *P < 0.05; tests are two-sided. Scale bars, 100μm.

Supplementary Figure 8. IL-35 enhances the PDAC -human lymphatic endothelial cells (HELC) adhesion in dynamic flow assays.
A, the FACS assays was performed to compare ICAM1 expression between HUVECs and HLECs. B, representative immunofluorescence image demonstrates the dynamic flow assays at the shear force of 1 dyne/cm2; scale bar, 100μm. C, the adhesion-shear force curves of the adhesion assays done at shear forces from 0.1 to 4 dyne cm -2 . Experiments were replicated for three times. Unpaired t test was used; Shown are mean values ± SD; *P < 0.05; tests are two-sided. Scale bars, 100μm. Figure 9. Verification of the mRNA sequencing data. A, the mRNA heat-map of 10 genes up-regulated in PANC-1 cells overexpressing IL-35; the normalized counts (RPKM) were normalized to log2 rank. B, RT-PCR assays were perormed to verify the mRNA sequencing results. Experiments were replicated for three times. Unpaired t test was used; Shown are mean values ± SD; *P < 0.05; tests are two-sided. were harvested and subjected to immunohistochemistry staining to detect the expression level IL-35 (EBI3 and p35) and ICAM1. An Immunohistochemical staining with no primary antibodies was shown as the negative control (N.C.). B, representative H.E. staining and immunohistochemistry staining of visible metastases in peritoneum, mesenterium and liver. Immunohistochemical stainings without primary antibodies was used as the negative controls (N.C.). C, representative immunohistochemistry staining for EBI3 and P35 in consecutive sections of invisible micrometastases in mice liver. D, immunofluorescence staining for the co-locolization of EBI3 and P35 in invisible micrometastases in mice liver. Red arrows, micro-metastases; Figure panels pairs represent images taken with different zooming options; scale bars, 100μm. Figure 13. IL-35-ICAM1 axis promotes PDAC adhesion and metastasis to mesothelium. A, the primary mesothelial cells was extracted from human epiploon donated by PDAC patients, then the mesothelial cells was verified by the staining of mesothlial markers: cytokeratin and Vimentin. B, the FACS assays was performed to compare ICAM1 expression between HUVEC and mesothelial cells. C-D, the indicated tumor cells were added onto HUVEC monolayers and the adhered tumor cells were determined in the absence or presence of fibrinogen. E-F, Representative images (E) and total numbers (F) of metastasis in gut and mesenterium (n = 8 for each group). Unpaired t test was used; Shown are mean values ± SD; *P < 0.05; tests are twosided. Scale bars in A and C, 100μm. Figure 14. Uncropped Western blotting scans. A, Uncropped Western blotting scans for Figure 1G. B, Uncropped Western blotting scans for Figure 3E. C, Uncropped Western blotting scans for Supplementary Figure 3E. D, Uncropped Western blotting scans for 3D. Spearman rank correlation test