path4181-sup-0001-TableS1.pdfPDF document189KClinical and histological features of primary prostatic adenocarcinoma patients investigated in the present study. All patients were clinically staged according to the WHO TNM/AJCC 2010. HG = high grade; B = bilateral; D = diffuse; F = focal; NE = not evaluated.
path4181-sup-0002-TableS2.pdfPDF document174KSurface Plasmon Resonance (SPR) analysis of FGFs/PTX3 interaction. Recombinant PTX3 (20 µg/ml in 10 mM sodium acetate, pH 2.4) was allowed to react with a Fc2 flow cell of a CM4 sensor chip previously activated with a mixture of 0.2 M EDC and 0.5 M NHS (flow rate 10 µl/min). These experimental conditions allowed the immobilization of 1020 resonance units (RU) corresponding to 22 fmoles/mm2 of PTX3. After ligand immobilization, matrix neutralization was performed with 1.0 mM ethanolamine pH 8.5 (flow rate 10 µl/minute). Activated/deactivated Fc1 flow cell was used as a reference cell and for blank subtraction. Increasing concentrations of recombinant FGFs (ranging between 20–400 nM) were injected over the PTX3-coated sensorchip and the response was recorded as a function of time. Injection lasted for 4 min (flow rate 30 µl/min) to allow FGF association to immobilized PTX3 and was followed by 10 min of dissociation. Scatchard plot analysis of steady-state SPR data were used to calculate the affinity (mean ± S.E.M) of FGFs/PTX3 interaction (KD). After each run, the sensorchip was regenerated by injection of HBS containing 10 mM glycine pH 2.0. FGFs assayed for PTX3 binding were: FGF1, FGF2, FGF4, FGF6, FGF7, FGF8b, FGF9, FGF10, FGF17 (PeproTech) and FGF3 (R&D Systems). n.d., no significant binding.
path4181-sup-0003-FigureS1.pdfPDF document159KEffect of PTX3 on DHT and FGF2-dependent FGFR1 phosphorylation and signalling. Serum-starved TRAMP-C2 cells were stimulated with 10 nM DHT (A) or 0.55 nM FGF2 (B) in the absence or in the presence of 120 nM PTX3, 300 nM neutralizing anti-FGFR1 single chain RR-C2 scFv antibody, or 10 nM FGFR1 tyrosine kinase inhibitor PD173074. After 15 min, cells extracts were probed with anti-pFGFR1 (Tyr 766), anti-pAKT and anti-pERK1/2 antibodies. Uniform loading of the gel was assessed by probing the membrane with anti-FGFR1 and/or anti-α-tubulin antibodies.
path4181-sup-0004-FigureS2.pdfPDF document184KEffect of PTX3 on LNCaP human prostate tumor cell proliferation. Serum-starved LNCaP cells were counted 48 h after stimulation with 10 nM DHT, 0.4 nM FGF8b or 0.5 nM FGF2 in the absence (black bars) or in the presence of 66 nM PTX3 (dashed bars), 300 nM of 2 neutralizing anti-FGFR1 single chain RR-C2 scFv antibody (RR-C2 scFv, open bars) or of irrelevant single chain antibody (a-scFv, grey bars). Under these experimental conditions, DHT, FGF2 and FGF8b induce a 2.0-, 1.8- and 1.7-fold increase in cell proliferation, respectively, when compared to non-stimulated cells. Data, expressed as percentage of cell proliferation measured in the absence of any antagonist, are the mean ± S.D. of 3 determinations. No effect was exerted by PTX3 or the two antibodies on the basal proliferation of serum-starved cells grown in the absence of any mitogenic stimulus (data not shown). (*, P < 0.05 or better, Student's t-test).
path4181-sup-0005-FigureS3.pdfPDF document187KhPTX3 overexpression in TRAMP-C2 cells. A) Naïve TRAMP-C2 cells, stable mock_TRAMP-C2 and hPTX3_TRAMP-C2 transfectants were analyzed for human and murine PTX3 expression by RT-PCR. Also, their conditioned media were assessed for the levels of PTX3 monomer in a Western blot (WB) with a polyclonal anti-PTX3 antibody that recognizes both murine and human proteins. B) Cells grown in complete cell culture medium were analyzed for FGF2, FGF8b and VEGF-A expression by RT-PCR.
path4181-sup-0006-FigureS4.pdfPDF document170KhPTX3 overexpression inhibits TRAMP-C2 tumor growth in immunodeficient athymic nude mice. Athymic nude male mice were injected s.c. with 5x106 mock_TRAMP-C2 (image) or hPTX3_TRAMP-C2 (image) cells in 350 µl of PBS. A) Tumor take represents the percentage of tumors larger than 100 mm3 versus the total number of injected grafts for each cell type (n) at the indicated time after challenge. B) Tumor volume (mean ± S.E.M) was measured with callipers up to 100 days after challenge. *, P < 0.001, Student's t-test.
path4181-sup-0007-FigureS5.pdfPDF document162K Nterm-hPTX3 and sCterm-hPTX3 overexpression in TRAMP-C2 cells. A) Stable mock_TRAMP-C2, hPTX3_TRAMP-C2, Nterm-hPTX3_TRAMP-C2 and sCterm-hPTX3_TRAMP-C2 transfectants were analyzed for transgene expression by RT-PCR. The schematic position of the primers utilized for analysis are shown (s = leader secretion sequence; F1 = hPTX3 forward, R1 = hPTX3 reverse, R2 = hPTX3 reverse. B) C57BL/6 male mice were injected s.c. with 5x106 EGFP_TRAMP-C2, sCterm-hPTX3_TRAMP-C2 or Nterm-hPTX3_TRAMP-C2 cells in 350 µl of PBS and tumor volume was measured with callipers. a Number of tumors larger than 100 mm3 versus the total number of injected grafts at the indicated time after challenge. b Time to reach a tumor volume ≥ 200 mm3. *, P < 0.001, Student's t-test. 3
path4181-sup-0008-FigureS6.pdfPDF document93KPTX3 is down-regulated in human prostate cancer. Data mining was performed on the cancer microarray database Oncomine 4.0 (Oncomine DB at Oncomine boxed plot of PTX3 expression levels between human normal prostate gland and prostate cancer in multiple datasets from ref. 1 (a), ref. 2 (b), ref. 3 (c), ref. 4 (d), and ref. 5 (e). 1. Luo JH, Yu YP, Cieply K, Lin F, Deflavia P, Dhir R, et al. Gene expression analysis of prostate cancers. Mol Carcinog 2002;33:25–35. 2. LaTulippe E, Satagopan J, Smith A, Scher H, Scardino P, Reuter V, et al. Comprehensive gene expression analysis of prostate cancer reveals distinct transcriptional programs associated with metastatic disease. Cancer Res 2002;62:4499–506. 3. Welsh JB, Sapinoso LM, Su AI, Kern SG, Wang-Rodriguez J, Moskaluk CA, et al. Analysis of gene expression identifies candidate markers and pharmacological targets in prostate cancer. Cancer Res 2001;61:5974–8. 4. Wallace TA, Prueitt RL, Yi M, Howe TM, Gillespie JW, Yfantis HG, et al. Tumor immunobiological differences in prostate cancer between African-American and European-American men. Cancer Res 2008;68:927–36. 5. Singh D, Febbo PG, Ross K, Jackson DG, Manola J, Ladd C, et al. Gene expression correlates of clinical prostate cancer behavior. Cancer Cell 2002;1:203–9.
path4181-sup-0009-FigureS7.pdfPDF document137KPTX3 immunoreactivity in the stroma of invasive human prostate adenocarcinoma. Sub-serial sections obtained from prostatic adenocarcinoma case#20 were stained for PTX3 (brown in A-D), neurofilament (NF) (blue in B), D2-40 (blue in C), and Factor VIII (blue in D). A low power view of the invasive adenocarcinoma area shows PTX3 immunoreactivity in the extracellular space surrounding vessels and intra-prostatic nerves (A). These findings were confirmed by double immunohistochemistry of anti-PTX3 coupled with NF for nerve fibres (B), D2-40 for lymphatic endothelium (C) and Factor VIII for blood vessel endothelium (D). PTX3 immunoreactivity surrounding vascular spaces and intra-prostatic nerves in invasive tumor areas was observed in 30/33 (91 %) and 25/33 (76 %) of prostate adenocarcinoma biopsies, respectively. Original magnification = 100x (A; scale bar 200 µm), 200x (C and D, scale bar = 100 µm), 600x (B; scale bar = 33 µm). 4
path4181-sup-0010-FigureS8.pdfPDF document205KFRS2α activation in human prostate adenocarcinoma. Sub-serial sections obtained from normal prostate and two prostatic adenocarcinoma (PAC) cases were immunostained for phosphorylated FRS2αpFRS2αand double immunostained for pFRS2 p63 as labelled. In normal prostate gland, pFRS2αis weakly present on luminal cells and completely absent on p63+ basal cells. In PACs, FRS2α phosphorylation is strongly induced on neoplastic cells whereas a weak immunoreactivity is observed in luminal cells of normal prostate gland (highlighted by asterisks). Original magnification = 400x for normal prostate (scale bar = 50 µm) and 200x for PACs (scale bar = 100 µm).
path4181-sup-0011-AppendixS1.pdfPDF document354KSupplementary Materials And Methods

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