Fig 2: ADAM8 expression in human atherosclerotic lesions. (a) Microarray expression profiling of carotid atherosclerotic plaque macrophages and lung, liver and spleen macrophages (n = 4 patients per tissue, nonparametric Mann-Whitney U test). (b) Relative expression of ADAM8 mRNA in early, advanced stable and unstable human plaques. Values have been corrected for GAPDH expression (n = 5–6) and ADAM8 expression levels in early plaques were normalized to 1 (one way analysis of variance followed by Dunn’s multiple comparison test). (c,d) Immunohistochemical stainings for ADAM8 in human atherosclerotic lesions. (c) Representative images of a shoulder region (scale bar, 400 µm), a region rich in foamy macrophages (scale bar, 50 µm) and a negative control (d, scale bar, 200 µm) are shown.

Fig 3: Effect of ADAM8 inhibition in an orthotopic pancreatic cancer model(A) Pancreas morphology after orthotopic injection of Panc1_A8, Panc1_ctrl, or Panc1_A8 cells followed by BK-1361 injection (10 µg/g daily, n=12 per group). (B) Tumour load [in g] 12 days after implantation (n=12); Tukey’s linear contrast test was used. **, 0.001, ***, p<0.0001 (Student’s t-test) (C) Representative ADAM8 IHC/Periodic Acid Schiffs (PAS) staining of pancreas tumours from mice injected with Panc1_A8, Panc1_ctrl compared to implanted Panc1_A8/BK-1361 treated. Significant invasion of Panc1_A8 cells (arrows, upper panel left); Panc1_ctrl and Panc1_A8/BK-1361 injected tumours show less invasion from implantation site. Lower panel, ADAM8 staining; infiltration and loss of ductal architecture, increase in ADAM8 in Panc1_ctrl implanted tumours, and delineated between implantation site and surrounding pancreas tissue in Panc1_A8/BK-1361 treated mice. Lower panel, left, enlarged image shows invasion of ADAM8-positive tumour cells; enlarged images (boxed in upper panel); box1: upregulation of ADAM8 in cell mass of Panc1_ctrl cells; box 2: enlarged view of the border between implantation site and surrounding ductal tissue; box 3: signs of intratumoural necrosis in tumours treated with BK-1361 (bar, 100 µm). (D) pERK1/2 staining of tumours. Only for Panc1_A8 (top), tumour cells stain positive for pERK1/2 (bar, 100 µm). (E) Morphology of spleen and diaphragm from mice injected with either Panc1_A8, Panc1_ctrl, or Panc1_A8/BK-1361. In PAS stains (lower panel), infiltration of tumour cells in the diaphragm is only seen in Panc1_A8. Scale bar, 100 µm. (F) Quantification of infiltration areas in peritoneum, diaphragm and spleen. Values are in % of total tissue area ±SEM (n=12). (G) ADAM8+ micrometastases in livers from Panc1_A8 injected mice; in contrast, metastases were mostly absent in livers from Panc1_ctrl and Panc1_A8/BK-1361 treated mice (bar, 200 µm); relative frequency of liver metastases in mice injected with Panc1_A8, Panc1_ctrl, and Panc1_A8/BK-1361, respectively (n=12). (H) Pancreas histology (PAS, ADAM8 IHC) after orthotopic implantation of AsPC-1_shCtrl and AsPC-1_shA8/2 cells. Scale bar in I, left upper panel, 100 µm. ADAM8+ cells in pancreas injected with AsPC-1_shCtrl cells, weaker staining with AsPC-1_shA8 cells. Infiltration areas in pancreata from 6 mice/group were quantified. Mean values ±SEM. **, p<0.01 (Student’s t-test).

Fig 4: Extracellular ADAM8 processing and inhibition of ADAM8 activity(A) ADAM8 processing: (1) autocatalytic prodomain (red) removal; (2) for active ADAM8; after membrane transport, ADAM8 cleaves membrane proteins (3a); alternatively, removal of a soluble MP (blue) domain (3b) leads to ECM cleavage and formation of remnant ADAM8. A putative interaction site (magenta) located in the disintegrin domain (DI, green). (B) Homology modelling of ADAM8 disintegrin/cysteine-rich (DC) domain based on homology to pdb file 2ao741 within the integrin-binding loop region of ADAM15. The amino acid motif “KD” is potentially accessible to peptidomimetics such as BK-1361 (see table 1). (C) FRET analysis of ADAM8 monomers in the presence of BK-1362 (control peptide “CP”) and BK-1361 (500 nM, respectively) in Panc1 cells. Fluorescence lifetime (in nanoseconds “ns”) and FRET efficiency are calculated from >12 cells in 3 independent experiments. ANOVA was used as statistical test; data are presented as mean ± SEM; p-value *, p<0.05 (Student’s t-test). (D) left panel, complex of pro-ADAM8 (100 ng/lane) at 0 and 24 hours analysed by native gel electrophoresis and immunoblotting. With CP, pro-ADAM8 forms complexes (dimers and trimers, arrowheads 2 and 3); with BK-1361, only monomers of pro-ADMA8 are detected (arrowhead 1); right panel, activation of recombinant pro-ADAM8 in vitro in the presence of CP and BK1361. Bar graph: Autocatalytic activation of pro-ADAM8 ± BK-1361 (200 nM) in vitro. Pro-ADAM8 (100 ng) incubated for indicated times; Fluorescence activity using CD23 fluorogenic peptide monitored over 5 days in triplicates. “FU”, fluorescence units. (E) Effect of BK-1361 and control peptides (see table 1) on ADAM8 MP domain removal in ADAM8-BiPro transfected COS7 cells after 12 hours. Anti-BiPro antibody detects pro (triangle), mature (circle), and remnant (diamond) ADAM8 in cell lysates (WCL); soluble ADAM8 activity from processed ADAM8 is detected in cell supernatants (SN) by CD 23 peptide cleavage. (F) Test of BK-1361 for membrane-bound CD23 in WCL and soluble CD 23 (sCD23) in SNs. Cells were incubated with CP or BK-1361 (500 nM) for 12h. (G) Dose-dependent inhibition of CD23 shedding determined by ELISA (n=5) with mean values ± SD; IC50 value for BK-1361: 182 ± 23 nM, whereas CP shows no significant inhibition of CD23 shedding.

Fig 5: (A) Correlation analyses of ADAM8+ neutrophil counts in post-capillary venules. Each data point is the average of 3 independent post-capillary venules of an area > 2000 µm2 in the core tumor tissue (n = 50). Pearson correlation revealed r = -0.463 with p = 0.0006. (B) Neutrophil density diagram from our PDAC patient cohort split into survival time less than and greater than 18 months, p = 0.0128. (C) Neutrophil counts in peripheral blood of PDAC patients from the same cohort, p = 0.2797. (D) Neutrophil-to-lymphocyte ratio in the PDAC patient cohort, p = 0.5143. Note that only the ADAM8+ neutrophil counts in post-capillary venules are significantly correlated with PDAC patient survival.