Materials and reagents
The human anti-Paxillin antibody was purchased from BD (cat. Num. 610,052), the anti-ZO-1 from Zymed (cat. Num. 617,300), the anti-FN1 and anti-GAPDH from Sigma (cat. Num. F-3648, cat. Num. G-8795), anti-Actin from Santa Cruz (cat. Num sc-1616), anti-VE-Cadherin from ABcam (ab33168), anti-Nidogen-1 from R&D (cat. Num. MAB2570–100), anti-pSTAT3 and anti-Stat3 from Cell Signaling (cat. Num. 9131, 9132), anti-CD31 from BD Pharmigen (cat. Num. 550,274), anti-phospho Histone 3 from Millipore (cat. Num. 06–570). Fluorescently labeled secondary antibodies and anti-Phalloidin-568 were purchased from Invitrogen, DAPI was obtained from Sigma. FLLL31 was purchased from Sigma (cat. Num. F9057). RhNID1 (cat. Num.: 2570-ND-050) and rhBGN1 (cat. Num. 2667-CM-050) were purchased from R&D, Minneapolis, MN, USA.
Primary human umbilical vein endothelial cells (HUVECs; PromoCell, Heidelberg, Germany) and primary human dermal microvascular endothelial cells (HDMECs; PromoCell, Heidelberg, Germany) were expanded in endothelial growth medium (EGM) and supplements (Lonza, Basel, Switzerland) on 1.5% gelatine-coated plates. The cancer cell lines SK-BR-3, MCF7, PC-3 and H460 were cultured in DMEM with 10% FBS (Sigma Aldrich, Buchs, Switzerland).
To prepare endothelial cell-conditioned medium (EC-CM), confluent HUVEC monolayers were detached, and the cells were plated onto new cell culture dishes of the same diameter in EGM. To prepare conditioned medium from confluent HUVEC 9/10 of the total cell number was seeded for expansion. To obtain conditioned medium from subconfluent HUVEC 1/10 of the total cell number was plated. After 24 h of culturing, medium was changed with conditioning medium (M199 medium (Sigma Aldrich, Buchs, Switzerland) containing 2% FBS supplemented with 1% penicillin, 0.1% glutamine, 4 μg/ml bovine pituitary extract (Thermo Fisher, Waltham, MA, USA) and 8 U/ml heparin). After a conditioning time of 24 h the supernatant was collected, cell debris were removed and the conditioned medium was stored at − 80 °C. For the experiments the cancer cell lines were expanded for 24 h, then the expansion medium was removed and the cancer cells exposed to the HUVEC conditioned medium for 4 days.
Immunofluorescence microscopy analysis
Cells, plated and treated on glass coverslips, were washed and fixed in 4% PFA for 20 min at RT, permeabilized with 0.5% NP40 in PBS for 5 min, washed and blocked with 3% BSA in PBS-Triton for 1 h at RT. The cells were then incubated with the primary antibody over night at 4 °C. Fluorescently labeled secondary antibodies were added together with DAPI 1 μg/ml for 1 h in the dark. Cells were washed 5 times, mounted and imaged on a fluorescent microscope (Leica DMI4000).
Endothelial cells were transfected with 4 siRNAs for each target (siGENOME Human SMARTpool, Dharmacon Lafayette, Colorado, USA) with Lipofectamine 3000 (Invitrogen, Thermo Fisher Waltham, MA, USA) according to the manufacturer’s instructions. The siRNA sequences are as follows: NID1: GGGCGAACCUGCUAUGAUA, GAAGGUUUAUUAUCGAGAA, UAACCUGGAUCGAAUAGAA, and CCUUCAUAACUGCGACAUA; BGN: GGAGAACAGUGGCUUUGAA, UGAAUGAACUCCACCUAGA, CCAAAGAGAUCUCCCCUGA, and GAACAACGACAUCUCCGAG; HSPG2: GCGCUGCGAUGGUGACUUU, CAACACACACCACGAGCUA, GAGCUAUGUGAAUGCAAU, and ACGGUGGGAAGUUGCGAUA; CLU: GAUAAAGACUCUCAUAGAA, GAAAGAGGAUGCCCUAAAU, GGAAGUAAGUACGUCAAUA, and GUAGAAGUCUCCAGGAAGA; C1QTNF5: GCGAAUCCAUUGCCUCUUU, UGAACGAGCAGGGACAUUA, GGGCCAGCCUGCAGUUUGA, and UGACUACAUUGGCAUCUAU; CYR61: GGGCAGACCCUGUGAAUAU, GGCCAGAAAUGUAUUGUUC, GGUCAAAGUUACCGGGCAG, and GCAGCAAGACCAAGAAAUC; TIE1: GGGAAGCCUCCUACCCUUA, GAAGUUCUGUGCAAAUUGG, CAACAUGGCCUCAGAACUG, and UCGAAACUGUGACGAUGAA; VWF: GGACAGAUCAUGACACUGA, GGAAGACCCUGUGGACUUU, GAAGAGGCCUGCACUCAGU, and GGUCACAUCUUCACAUUCA. After 72 h, the transfection medium was changed, and a second transfection was performed. Twenty-four hours after the second siRNA transfection, the cells were used for experiments.
Migration and transendothelial migration assay
A total of 50′000 SK-BR-3 cells were plated into Boyden-Chamber inserts with 8 μm pore size (BD Falcon, Corning Tewksbury MA, USA) in 200 μl M199 (Sigma Aldrich, Buchs, Switzerland) 2% FBS medium; the bottom chamber was filled with 800 μl of M199 20% FBS medium. After a migration time of 18 h, the cells were fixed with 4% paraformaldehyde in PBS. Cells that had not crossed the membrane were removed, and cells on the bottom of the insert membrane were stained with DAPI (1 μg/ml), visualized with a Leica DMI4000 fluorescence microscope and quantified using ImageJ software. The trans-endothelial migration assay was performed as previously described . Briefly, a total of 15′000 CellTrace CFSE (Invitrogen, Thermo Fisher Waltham, MA, USA)-labelled SK-BR-3 cells were seeded onto a confluent HUVEC monolayer and incubated over 48 h in 5% CO2 at 37 °C without a serum gradient. After removal of non-migrated cells, cells that crossed the HUVEC monolayer were fixed with 4% paraformaldehyde, visualized and quantified as described above. Immunofluorescence and immunoblotting were performed as previously described . All experimental conditions were tested and analysed in three replicates and each condition was tested in biological triplicates.
RNA isolation and RT-PCR
Total RNA was prepared with Tri-Reagent (Sigma Aldrich, Buchs, Switzerland) and reverse-transcribed with ImProm-II™ reverse transcriptase (Promega, Madison, Wisconsin, USA). Transcripts were quantified using SYBR-green PCR Master Mix in a StepOnePlus PCR system (Applied Biosystems, Foster City, California, USA). Real-time PCR reactions were performed in triplicate, and fold-induction was calculated using the comparative Ct method (ΔΔCt) normalized to ribosomal protein L19 expression. The following primers (sequence 5′-3′) were used: human nid1: TCTACGTCACCACAAATGGCA; human hspg2: GTGTGGTGTTCATCAAGGAGC; human nid2: GAAACGCAGTATGTGGACTATGA; human cyr61: GGTCAAAGTTACCGGGCAGT; human vwf: CCGATGCAGCCTTTTCGGA; human clu: CCAATCAGGGAAGTAAGTACGTC; human c1qtnf5: AACGAGCAGGGACATTACGAC; human tie1: AAGCAGACAGACGTGATCTGG; human bgn: CAGTGGCTTTGAACCTGGAG; and human rpl: GATGCCGGAAAAACACCTTG.
Cells were lysed with 0.5 M Tris-Hcl ph 6.8, 10% SDS, glycerol and the lysates boiled for 5 min. Lysates were normalized for equal amount of protein and loaded onto SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Whatman Protran). Blots were sequentially incubated with 5% milk, the primary antibody (1:1000) overnight, and the HRP-labeled secondary-antibody for 1 h at room temperature. Signals were revealed with Upti-Light™ chemiluminescence reagent (Uptima) and detected with X-Ray films.
Stable isotope labelling by amino acid in cell culture (SILAC) of HUVEC
HUVECs were cultured in custom-made EGM-2 without arginine and lysine (Lonza), with the addition of SILAC amino acids (13C6 L-arginine and 2H4 L-Lysine for the “SILAC medium” or 13C6 15N4 L-arginine and 13C6 15N2 L-Lysine for the “SILAC heavy”) (Cambridge Isotope Laboratories) until more than 97% of SILAC amino acids were incorporated into proteins.
Labelled “heavy” and “medium” cells were split either sub-confluent or confluent (ratio of sub-confluent to confluent being 1:20) for 3 days (until the confluent cells had reached 100% confluency), washed in PBS with Ca2+ and Mg2+, and incubated in EBM-2™ medium (Lonza, Basel, Switzerland) for 4 h. The supernatants were collected and spun at 4 °C (300 g for 10 min, followed by 2000 x g for 10 min and 10,000 x g for 30 min). The cells were counted and the “heavy” supernatant coming from the confluent cells was pooled together with the “medium” supernatant coming from the sub-confluent cells (forward experiment) and vice versa (reverse experiment), adapting the volume to the corresponding cell number. Proteins were extracted using Strataclean resin (Agilent Technologies) as previously described , dissolved in 4x sample buffer (NuPAGE LDS loading buffer, Life Technologies) supplemented with 0.1 M DTT and separated on a 4–12% NuPAGE Novex Bis-Tris gel (Life Technologies). Each gel lane was cut in seven slices and proteins were in-gel digested with trypsin. Peptides were loaded onto Empore-C18 StageTips and eluted with 80% ACN and 0.5% acetic acid.
Mass spectrometry (MS) analysis of secretome
Digested peptides were separated by nanoliquid chromatography (Easy nLC, Thermo Fisher Scientific) coupled on line to a linear trap quadrupole (LTQ)-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray ion source (Nanospray Flex Ion Source, Thermo Fisher Scientific). Peptides were loaded onto a 20 cm fused silica emitter (New Objective) packed with C18-AQ, 1.9 μm resin (Dr Maisch GmbH) and eluted with 5–25% solvent (80% ACN, 0.5% acetic acid) over 90 min (200 nl/min). Full scan MS spectra were acquired in the Orbitrap analyzer with a resolution of 120,000 at 400 Th, and a target value of 106 charges. The 10 most intense ions were selected for high collision dissociation fragmentation with a target value of 40,000 charges and acquired in the Orbitrap with resolution of 15,000 at m/z 400 Th. Data were acquired with Xcalibur software (Thermo Fisher Scientific).
MS data quantification and analysis
The relative quantification based on SILAC labelling was performed by processing the RAW MS files with MaxQuant version 18.104.22.168 . Proteins and peptides were identified using the Andromeda search engine  against the human UniProt database (release-2012 01, 88,847 entries). To search for precursor and fragment ions, an initial maximal mass deviation of 7 ppm and 20 ppm, respectively, was required. Trypsin with full enzyme specificity and only peptides with a minimum length of 7 amino acids were selected. A maximum of two missed cleavages was allowed. Carbamidomethylation (Cys) was set as fixed modification, while Oxidation (Met) and N-acetylation as variable modifications. For protein and peptide identification, we required a maximum false discovery rate (FDR) of 1%. The “requantify” option was enabled.
Data analysis and normalization
Statistical analysis and annotation of the MS data were performed using the Perseus module of MaxQuant version 22.214.171.124. The reverse and contaminant hits from the MaxQuant output files were excluded from the analysis. Only proteins identified with at least 1 unique peptide and quantified with a minimum of two ratio counts were considered for the analysis.
For each experiment (forward and reverse), the SILAC ratios (sub-confluent/confluent cells) were transformed using the binary logarithm (log2) and normalised by subtracting the median value. Proteins were considered up or down-regulated if the SILAC ratio was higher than one standard deviation from the mean of the calculated ratios in both replicate experiments. Protein annotations were added based on the Uniprot ID of each entry (GO and KEGG categories).
Human phospho-kinase antibody array
The protein lysates from 10′000’000 cells were analysed in a sandwich immunoassay with membrane-bound capture antibodies and biotinylated phospho-specific detection antibodies on the human phospho-kinase antibody array (ARY003B, R&D System, Minneapolis, MN, USA) according to the manufacturer’s instructions. The spot signals were quantified using ImageJ software.