Sorting and culturing of blood and lymphatic endothelial cell lines

Human dermal microvascular endothelial cells (HDMECs), derived from human foreskin, were purchased from Promocell (no. C-12260). This cell line is generated in strict compliance with ethical and legal standards (http://www.promocell.com/products/ethical-standards/) and has been published previously in numerous articles (http://www.promocell.com/knowledge-base/search-for-information/) [9].

Separation of podoplanin-positive LECs from BECs was performed by using magnetic beads (Dynal) coated with anti-podoplanin antibody, as described previously [5]. Cells were grown in endothelial basal medium (EBM-2) supplemented with 5% fetal calf serum (FCS) and EGM-2-MV SingleQuots (CC-4147; Lonza) in an incubator at 37°C and 5% CO2. For all experiments, LECs and BECs were used below passage 6.

Immunofluorescence stainings

For cell surface immunofluorescence, cells were grown on Lab-Tek II Chamber Slides (Nunc) and fixed with 1% PFA in PBS. For tissue immunohistochemistry, human dermal biopsies were embedded in OCT medium, frozen, cut into 5μm sections at -20°C and used immediately, or stored at -20 to -80°C. The study was approved by the local ethics committee (Proposal no. 1228/2014). For immunofluorescence stainings, slides were thawed, dried at room temperature for 10 min and fixed in ice-cold acetone or in PBS/ 1% PFA for 20 min. Blocking of sections was performed for 30 min at RT in PBS/ 10% goat or donkey serum, depending on the applied antibodies (S1 Table). Slides were washed briefly in PBS, and primary antibody was added for 1 hr at RT or overnight at 4°C, depending on the antibody. Cells were incubated with scFv antibodies diluted 1:50 in PBS/ 2% BSA and, as controls, with secondary antibody alone or with unspecific scFv. Slides were washed 3 times for 5 min in PBS and the secondary antibody dilution was applied (FITC-labeled anti-flag tag antibody or fluorescence labeled secondary antibodies diluted in PBS/ 2% serum for blocking). Nuclei were counterstained with DAPI and slides were mounted with Geltol. Pictures were taken with a VANOX AHBT3 fluorescence microscope (Olympus) an inverted live cell microscope (AxioVert 200M, Zeiss), or a laser scanning microscope (LSM 5 Exciter, Zeiss).

scFv phage display library

The phage antibody library ETH-2, a non-immunised single chain antibody fragment (scFv) library of over 5 x 10⁸ clones [29], was used for selections on BECs and LECs. ETH-2 phages display human recombinant scFv antibodies, in which one human germline gene segment for the heavy and two germline gene segments for the light chains [32] were used together with randomized positions in both complement determining region 3 (CDR3) loops [33]. The library was cloned into restriction sites NcoI and NotI of phagemid vector pDN332, carrying an ampicillin resistance, a lacZ promoter, a pelB leader sequence for secretion of scFv fragments into culture supernatants, a His-tag and a Flag-tag applicable for detection.

Selection of phages on BECs and LECs

Six rounds of selection were performed on BECs and LECs with the ETH-2 scFv library, essentially according to recommended protocol [29], with changes due to panning procedure on cells. Selections were carried out using detached cells rather than confluent monolayers, as panning on these yielded mainly unspecific phage antibodies (own communication). Briefly, BECs and LECs nearly confluently grown in T75 cell culture flasks were washed with PBS and mildely detached with 5mM EDTA/ PBS at 37°C. Cells were harvested after 15–30 min and centrifuged at 1500 rpm for 3 min. Cell pellets were weighed and resuspended in 1 ml EGM-2 MV/ 1% BSA. The solution was split into two 500μl portions containing approximately 1 x 10⁵ cells. To each tube, 200 μl of ETH-2 library (~ 10¹² pfu/ml) or VCS-M13 helper phage (~ 10¹² pfu/ml) as background control were added (S2 Fig), respectively, and tubes were incubated overnight at 4°C under rotation. On day 2, cells were centrifuged at 1.500 rpm for 3 min and resuspended in 1 ml EGM2-MV/ 1% BSA. Cell suspensions were transferred to new 1.5ml tubes and unbound and weak bound phages were removed by washing the cells four times with EGM2-MV/ 1% BSA, finally resuspending them in 400 μl PBS. Phage-carrying cells were directly used for re-infection of 7.5 ml exponentially growing TG1 E. coli culture. After 30 min at 37°C, 25 μl of infected TG1 bacteria were used for determination of phage output titer. Serial 1:100 dilutions (10^–5, 10^–7, 10^–9, 10^–11) were prepared in 2xTY medium, and 10 μl (titer = colonies x 1.000) of each dilution were plated on 2xTY-Amp-Glu plates, or on 2xTY-Kana-Glu plates for helper phage infected bacteria. To amplify the whole phagemid DNA pool, remaining infected TG1 were centrifuged at 3.300 g for 10 min, pellets were resuspended in 0.5 ml 2xTY, spread on 500 cm² 2xTY-Amp-Glu agar culture dishes and incubated at 30°C overnight. On day 3, TG1 bacteria grown on the large agar plates were gently loosened by adding 5–10 ml 2xTY/ 10% glycerol with a glass spreader. The homogenous bacterial suspensions were aspirated and stored at—80°C, or immediately used for phage particle preparation. Colonies of plates with dilution series were counted and phage titers were calculated, yielding the output titer/ml (1–5 x 10¹² pfu/ml per round). Titers of input and output phage were corrected for different volumes and expressed as the total amount of cfu. Phage recovery was calculated as the ratio of output/input phage after one round of selection. Enrichment factors were calculated as output versus input ratios as previously described [17] (Table 1).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Phage recoveries and enrichment factors during selection of ETH2 phage library on BECs and LECs in suspension.

https://doi.org/10.1371/journal.pone.0127169.t001

Phage rescue from each selection round was carried out as described [29]. In brief, 50 ml of 2xTY-Amp-Glu medium were inoculated with 10–20μl bacterial suspension to yield an OD600 = 0.05–0.1, and grown at 37°C and 225 rpm until OD600 = 0.4–0.5 (~ 6 x 10⁹ Bacteria). 15 ml of this culture were infected with helper phage VCS-M13 in a ratio of at least 20:1 (phage: bacteria) for 30–40 min at 37°C in a water bath. Typically, 100 μl VCS-M13 (~ 10¹² pfu/ml) per 15 ml culture were used. The infected bacteria were centrifuged at 3.300 x g for 10 min at RT, resuspended in 100 ml 2xTY-Amp-Kana and incubated in a shaker at 30°C overnight. 100 ml of overnight culture were transferred to 250 ml buckets and centrifuged at 3.300 x g for 10 min at 4°C. The phage supernatant was transferred to new tube and ¼ volume of 20% polyethylene glycol (PEG)/ 2.5% NaCl was added for at least 1hr on ice. The mixture was placed on ice for 40 min and then centrifuged for 15 min at 10.800 x g at 4°C. Pellets were resuspended in 120 ml Aqua bidest/ 20% PEG/ NaCl (i.e. 24 ml) for a second precipitation step. After centrifugation, the supernatant was discarded and the pellet was resuspended in 3ml PBS/ 10% glycerol. The solution was rotated for 20 min at 37°C and centrifuged at 13.000 rpm for 2 min to remove any residual bacterial cell debris. Finally, phage solutions were transferred to fresh microcentrifuge tubes in 0.8 ml portions, which were stored at -80°C or used for the next panning round. For each subsequent panning step, ~10¹² amplified phage particles of the previous enrichment round were used. A total of six consecutive panning rounds was performed on BECs and LECs by repeating this selection procedure.

Phagemid preparation, DNA insert and fingerprint analyses and sequencing

Individual bacterial clones containing plasmids or phage replicative forms were resuspended in 2xTY/ 15% glycerol in 2ml plastic vials at -70°C for long term storage. 5ml 2xTY-media containing the appropriate antibiotic were inoculated with single bacterial colonies and incubated at 37°C in a shaker overnight. 1.5 ml of the overnight culture was centrifuged for 2 min at 3.000 x g to harvest bacteria. Plasmid or phagemid DNA was extracted using the Wizard Plus SV Miniprep DNA Purification System (Promega) or Wizard SV 96 Plasmid DNA Purification System (Promega) according to manufacturer´s instructions. For large scale DNA purification (up to 500 μg), endotoxin-free transfection-grade plasmid DNA EndoFree Plasmid Maxi Kit (Qiagen) was used following the manufacturer´s handbook. Clone diversity was determined by fingerprinting with the frequent-cutting enzyme BstNI (NEB, New England Biolabs) according to manufacturer’s instructions. DNA fragments were resolved on agarose gels run in TAE buffer. High throughput sequencing of single phagemid DNA was performed at Eurofins MWG Operon using primer 5´-TACTACGCAGACTCCGTGAAG-3´. Inserts were translated into correct amino acid sequences and phagemid clones were sorted according to frequency of respective CDR3 sequences. Homology trees of the CDR3 regions of variable heavy (VH) and light (VL) sequences were generated using clc main workbench (www.clcbio.com).

Phage or scFv antibody preparation for ELISA screening

The versatile TG1 semisuppressor strain can be used for both, production of phage particles bearing pIII-fused scFvs by superinfection with helper phage and amber-suppression, or production of soluble scFvs by incomplete suppression of the amber stop codon [31]. Supernatants from clones of the 5^th and 6^th rounds of selection were screened by ELISA in phage and scFv format. Individual phage-infected TG1 colonies were grown in 96 deep-well plates (Nunc) in 150 μl 2xTY/ ampicillin/ 0.1% glucose. After 3 hrs, phage production was induced by addition of 25 μl 2xTY/ ampicillin/ 0.1% glucose, containing 1x10⁹ pfu helper phage VCS-M13. After infection for 30 min at 37°C, bacteria were pelleted and supernatants discarded. Infected bacteria were resuspended in 200 μl 2xTY/ ampicillin/ kanamycin and were grown overnight at 30°C. Bacteria were spun down at 1.800 x g for 10 min at 4°C, and 100 μl of the phage-containing supernatants were used for subsequent ELISA or FACS analyses. For scFv production, single colonies of phage-infected TG1 were grown in 5 ml 2xTY/ ampicillin/ 0.1% glucose, and scFv protein production was induced with 1mM IPTG for 16–24 hrs at 30°C. Bacteria were pelleted at 1.800 x g for 10 min and supernatants were immediately used in whole-cell ELISA. scFv antibodies were purified from bacterial culture supernatants by affinity chromatography using Protein AG UltraLink Resin (Pierce) according to manufacturer´s recommendations. The final antibody concentrations were either evaluated from OD_280nm (Nanodrop), by SDS-PAGE followed by coomassie staining, or by densitometric evaluation of Western blots probed with anti-His tag antibody.

Phage amount and whole cell ELISA

For phage amount ELISA, wells of flat bottom Nunc-Immuno 96 MicroWell MaxiSorp Plates (Nunc; 436110) were coated with 500 ng (in 100μl) rabbit anti-fd bacteriophage antibody (Sigma; B7786) diluted in carbonate buffer (15mM carbonate, 35mM biscarbonate)/ pH9.6 or PBS/ pH7.4 overnight at 4°C. On the next day, wells were washed twice with 200μl TBST and blocked with 200 μl 5% MTBS for 30 min at RT. Screening of specific binding to cell surface antigens was performed by whole cell ELISA with phage antibodies or scFv fragments. BECs, LECs and additional cell lines were grown in 96-well plates to confluency. Cells were washed twice with PBS and fixed with PBS/ 1% PFA for 20 min at 4°C. Wells were washed 3 times with 200μl PBS and blocked with 200μl 2% MPBS for 30 min at RT. 100 μl of phage or scFv containing supernatants were added to wells in triplicates and incubated for 2 hrs at room temperature. Wells were washed 3 times with 200 μl TBST, and 100 μl of HRP conjugated anti-M13 monoclonal antibody (Amersham; 27-9421-01) diluted 1:5000 in 0.5% MTBS was added per well. After incubation for 30 min at RT wells were washed three times with 200 μl PBS and incubated with 1:1000 diluted HRP-conjugated anti-M13 polyclonal antibody (Amersham Pharmacia) in 2% MTBS. Final washing was performed 3 times with 200 μl TBST and once with 200 μl TBS and inverted plates were blotted on a clean paper towel to remove any remaining washing buffer. The assay was developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA (Sigma-Aldrich). Color reaction was stopped by adding of TMB stop reagent (Sigma-Aldrich), and color absorbance was immediately read at 450 nm in an ELISA plate reader (Bio-Tek).

FACS analysis with phage antibodies

Binding of phage particles or phage-displayed scFvs to HDMECs, BECs or LECs was determined by flow cytometry. Cells were plated at a cell density of 1 x 10⁵ cells/ cm² in small culture flasks. After 72 hours, cells were gently detached from the culture dish by EDTA, washed twice with PBS and kept on ice. For each FACS analysis sample, aliquots of 5x10⁵ cells were blocked with 2% MPBS/ 0.05% NaN₃ for 30 min on ice, and approximately 5 x 10¹² phage particles were blocked with 2% MPBS for 1 h at room temperature. For each determination, cells were resuspended in 100 μl FACS buffer (1% FCS, 0.01M EDTA/ PBS) mixed with blocked phage particles in micronic tubes, and incubated on ice for 1 hr. Cells were washed twice with 500 μl 2% MPBS, incubated with rabbit anti-M13 phage antibody (S1 Table) diluted 1:500 in 2% MPBS for 30 min on ice, washed twice with 2% MPBS and incubated with FITC-labeled goat anti-rabbit antibody diluted 1:1000 in 2% MPBS for 1 hr on ice. In case of scFv staining, a directly Alexa Fluor 488 conjugated anti-His-tag antibody, diluted 1:200 in FACS buffer, was used. Samples were washed twice with PBS, resuspended in 500 μl PBS and analyzed on a FACScalibur device (Becton Dickinson) using software FCS Express 2.0.

Immunoprecipitation

BECs (5x10⁶) were washed twice with ice cold PBS and lysed with 500μl lysis buffer (TBS pH7.4/ 1% NP40/ complete protease inhibitor cocktail) for 10 min on ice. Cell lysates were centrifuged for 5 min, 14.000 rpm at 4°C and cleared supernatant was transferred into a new tube. For protein deglycosylation, 30 μl of BEC lysate containing 30 μg protein (1 μg / μl) was incubated with 750 units of PNGase F (NEB, New England Biolabs) for 3 hrs at 37°C. For immunoprecipitations, one half of the lysate was incubated with 5 μg scFv, and the other without (negative control) at 4°C overnight under rotation. To circumvent failure of specific antigen enrichment (internal communication), we chose Protein A/G UltraLink Resin (Pierce) with high ability to capture VH3 segment containing scFvs [34]. Immune complexes were captured with 25 μl of Protein A/G Resin and incubated for 1hr at 4°C under rotation. Samples were centrifuged for 3 min, 2.000 rpm at 4°C and the pellets were resuspended four times with lysis buffer, each time performing transfer to a fresh tube. The pelleted resins were incubated at 95°C for 5 min with 2 x SDS sample buffer and either used for subsequent SDS-PAGE and Western blot analysis, or were sent to mass spectrometry facility for further processing.

SDS-PAGE and Western Blotting analyses

Proteins were denatured at 95°C for 5 min, and 20 μl of protein lysates were separated per slot on a 12% bisacrylamide/ acrylamide gel. Proteins were transferred overnight to nitrocellulose membranes (BA83, poresize 0.2 μm, Whatman Protran). Membranes was dried between 20 min and overnight and blocked with TBST/ 5% BSA. Membranes were washed three times for 10 min in TBST at RT and incubated with indicated antibodies in TBST/ 5%BSA for 1 hr at RT, or overnight at 4°C (depending on the applied antibody) (S1 Table). After incubation with the first antibody, membranes were washed three times for 10 min in TBST and incubated with HRP-conjugated secondary antibodies for 1 hr. Membranes were washed five times for 10 min in TBST and chemoluminescent signals were developed with ECL plus (GE) reagent. Lumi-Imager F1 (Boehringer, Mannheim) system was used to detect chemoluminescent signals, exposure times varying between 5 sec and 10 min, depending on the signal intensity.

NanoLC ESI MSMS

Samples were subjected to LC MS/MS analysis at the APAF (www.proteome.org.au) according to standard sample preparation for mass spectrometry. Briefly, proteins were released from the resin by incubation in 300 μl of 0.05% SDS/ 0.5M triethylammonium bicarbonate (TEAB) at 95°C for 15 min. The resin was centrifuged and the supernatant was collected. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and methyl methane thiosulfonate (MMTS) were added to reduce and alkylate the proteins. Subsequent trypsin digestion was performed overnight at 37°C. Cation-exchange Macro-Prep High S Support columns (Bio-Rad) were used to clean the samples. Digested peptides were separated by nano-LC using the Tempo nanoLC system (Applied Biosystems, CA, USA). The sample (~1/3 of the original amount) was preconcentrated on a peptide trap (Michrome peptide Captrap) and desalted with H₂O/ 2% CH₃CN/ 0.1% formic acid at 8 μl / min. The peptide trap was then connected with the analytical column containing C18 RP silica (SGE ProteCol C18, 300A, 3μm, 150μm x 10cm). Peptides were eluted from the column using a linear solvent gradient of H₂O:CH₃CN (0.1% formic acid) from 90:10 to 0:100% (v/v) at 500 nl / min over a period of 50 min. The LC eluent was subjected to positive ion nanoflow electrospray analysis on an Applied Biosystems QSTAR Elite mass spectrometer. The QSTAR was operated in an information dependant acquisition mode (IDA). In the IDA mode, a TOFMS survey scan was acquired (m/z 400–1600, 0.5 sec), with the three largest multiply charged ions (counts >25) in the survey scan sequentially subjected to MS/MS analysis. MS/MS spectra were acquired in dynamic exit mode with a maximum accumulation time of 2 seconds. The LC-MS/MS data were analyzed using Mascot Server (http://www.matrixscience.com) to search peptide alignments to ‘Homo sapiens’ entries in the NCBInr and SwissProt protein databases.

Recombinant pFUSE antibody engineering, eukaryotic cell expression and purification

scFv-mFc fusion antibodies were generated by PCR-amplification of a 732 bp portion of scFvs using 5’-TACGGCAGCCGCTGGATTGTTATTACTC-3’ as forward, and 5’-TTATTAGATCT-CGCGCCTAGGACGGTCA-3’ as reverse primers. After restriction digest with NcoI and BglII, scFv fragments were inserted in correct reading frame between the IL2 secretion signal and the murine Fc sequence of pFUSE-mIgG2B-Fc2 expression vector (Invivogen) (S5C Fig). Ligation products were transfected into competent TG1 E.coli, and plasmids were prepared from single clones (Qiagen). One day before transfection, cultured HEK293 cells were split and seeded at a density of 3x10⁶ cells per 10 cm cell culture dish in 10 ml antibiotic free medium (DMEM/ 10% FCS). Transfection of HEK293 cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer´s instructions. Cell culture supernatants were collected 72 hrs after transfection and antibodies were purified with proteinAG UltraLink Resin (Pierce), yielding scFv-Fc antibody concentrations of 0.2–2 mg/ml. Supernatants containing Fc alone (from vector alone transfectants) was similarly processed and used as negative control.

Recombinant CD146 ELISA

Recombinant human CD146 comprising the extracellular Met¹-Gly⁵⁵⁹ portion of the protein precursor (NP_006491.2) fused to a C-terminal poly-Histidine tag was purchased commercially (Sino Biological Inc., Nr. 10115-H08H). ELISA plates were coated with CD146 or BSA as control antigen at concentrations of 100, 10 and 1 ng / ml in triplicates overnight at 4°C and were blocked with TBST / 5% BSA for 2hrs on the following day. Commercially available antibodies, scFv fragments and scFv-Fc fusion antibodies were added serially diluted to respective wells and incubated at RT overnight, followed by HRP-conjugated detection antibodies. ELISA plates were washed and developed as described above. In Western blotting, cell lysates were electrophoresed in 12% SDS-PAGE and transferred to nitrocellulose. Blots were probed with scFv, scFv-Fc and commercially available antibodies (S1 Table).

Scratch wounding assay

For migration assay, confluent BEC monolayers grown in 24-well plates (n = 3) were starved overnight. Thereafter, an artificial wound was created using a 200 μl pipette tip. Non-adherent cells were washed away and BECs were treated with or without 10 μg/ml of purified scFv-Fc B6-11 or Fc fragment only in EBM-2 MV / 0.5% FCS for 48 hrs. Wound closure was monitored with an inverted live cell microscope (AxioVert 200M, Zeiss) in an incubation chamber by taking pictures of the same section (2 sections per scratch) every hour over the whole time period. Wound area was measured using AxioVision 4.7 and calculated as amount of wound closure (in %) from timepoint t = 0. The experiment was performed twice.

Statistical data analysis

Statistical analyses were performed using Graph Pad Prism Software. Gap closure was analysed by one-way Anova followed by pairwise comparison to the control groups using paired t-test considering a P-value ≤ 0.05 as significant.

Establishment of scFv phage selections on primary BECs and LECs

Primary human BECs and LECs were retrieved from initially mixed HDMEC cultures (S1 Fig). Successful separation of BECs and LECs was tightly controlled by immunofluorescence staining of endothelial marker molecules CD31 and podoplanin (PDPN) (Fig 1) and by established PCR control (not shown), as described previously [8]. Cells were used only up to six passages, as long-term culturing leads to marker destabilization [8]. The semi-synthetic antibody phage library ETH-2 [29] comprising a variety of 5 x 10⁸ phage clones was used for panning on BECs and LECs. Selection parameters were determined empirically: First, selections were performed with cell suspensions, as panning on cells grown in monolayers predominantly yielded plastic and serum protein binders [16]. Second, as epitope internalization might occur at higher temperatures, we panned at 4°C. Third, as cell-bound phages are hardly recovered by pH-shift [16,35] (own unpublished results), phages were not eluted chemically from the cells, but cell-bound phages were directly added to logarithmically growing TG1 E. coli. In parallel, selections were performed with identical numbers of VCS-M13 wildtype phage (S2 Fig) to determine background-binding of phage particles carrying no antibodies. Trypan blue cell-staining showed that the cells had not been lysed during phage incubation and washing procedure (not shown), indicating a high chance to select scFvs against cell surface proteins.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Purity control of HDMEC, BEC and LEC cultures used for in vitro cell panning experiments.

Light microscopy and double-immunofluorescence stainings of HDMECs that were separated into BECs and LECs by sorting with magnetic beads coated with rabbit anti-podoplanin (PDPN) antibody. A: HDMECs contain subcultures of BECs and LECs. B: BECs are CD31+ / PDPN-. C: LECs are CD31+ / PDPN+. Nuclei were counterstained with DAPI. Size bars: 50μm.

https://doi.org/10.1371/journal.pone.0127169.g001

Characterization and binding specificities of polyclonal phage antibody eluates

Established enrichment steps were performed six times on separate BEC and LEC suspensions. Cell-specific phage antibody recoveries and enrichment factors were calculated for each panning round (Table 1). Phage recoveries and enrichment factors of ETH-2 selections showed an exponential increase on BECs and LECs, while they remained at constant low levels when applying wildtype phages (Table 1, Fig 2A). Phage antibody enrichment was not enhanced by increased cell numbers or by performing more than six selection rounds (not shown). Concomitantly, a FACS analysis of polyclonal phage pools confirmed binding of cell-enriched phage antibodies (Fig 2B, black-lined graphs), but not of VCS-M13 wildtype phages (Fig 2B, grey-filled graphs), to BECs (upper row) and LECs (lower row). Next, phagemid DNA derived from randomly picked single clones of all selection rounds was analyzed by BstNI-digest. While the restriction pattern differed widely during panning rounds #1 - #3 and restriction-resistant clones were detected (Fig 2C, upper row), rounds #4 - #6 mainly consisted of intact scFv clones (Fig 2C, lower row). These data showed that six selection rounds had resulted in enrichment of phage antibodies specifically binding to BECs and LECs, relative to the original antibody library complexity.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Enrichment of phage antibodies that bind to BECs and LECs with successive biopanning rounds.

A: Numbers of recovered phage particles after each panning round on BECs and LECs. For each selection round, the phage output/input titer ratios are given on the y-axis, showing an increase on BECs (red line) and LECs (green line) when adding polyclonal ETH-2 phage. In contrast, control wild-type VCS-M13 (grey and black lines) phage did not shift significantly on either cell population. B: Enrichment of BEC- and LEC-binding phage antibodies as seen by FACS-analysis of phage antibody pools. Shown are representative histograms of BECs and LECs stained with phage antibodies after respective panning rounds (black line) and WT phage as negative control (grey-filled graphs). The number of cells (counts: Y-axis) is given as function of the fluorescence intensity of phage antibody staining of the cells (FL1-H: X-axis). Percentage of BECs and LECs shifted by phage binding is depicted in the graphics. In all experiments, cells were incubated with phage antibody pools, and cell-binding was detected by anti-M13 antibody and FITC-conjugated anti-rabbit antibody. Grey-filled graphs show WT phage binding as negative control. C: BstNI DNA fingerprint analysis of single scFv clones. Representative gel images of respective panning rounds (#1 - #6) performed on BECs and LECs are shown. Over the course of panning rounds, the number of intact clones is increasing. Phagemid DNA isolated from single clones was digested with BstNI and analyzed in agarose gel electrophoresis. M: 1 kbp molecular weight marker.

https://doi.org/10.1371/journal.pone.0127169.g002

scFv antibody sequencing and bioinformatic analysis

Phagemid DNA of 994 individual single antibodies derived from selection rounds #5 and #6 was sequenced, and corresponding amino acid sequences were determined, yielding 557 intact antibody clones (i.e. 56%). From these, 166 diverse antibodies were derived, as exemplified by their CDR3 region sequences (S2 Table). A more detailed sequence statistics revealed that the majority of the clones was unique (Table 2) and contained CDR3 insert lengths of 6 amino acids (S3 Table). Further, a homology alignment of VH and VL CDR3 inserts indicated a broad diversity of the sequences, excluding biased clone enrichment (S3 Fig). In conclusion, the scFv sequence analysis indicated a highly diverse, unbiased clone abundance in phage selection rounds #5 and #6, out of which we isolated 166 diverese phage antibodies.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. Frequency of diverse scFv antibody sequences among 557 intact clones.

https://doi.org/10.1371/journal.pone.0127169.t002

Single phage antibodies specifically bind to BECs and LECs

All 166 intact phage antibodies were produced in large scale and screened for binding to BECs and LECs by whole-cell ELISA (Fig 3A). Presence of identical cell amounts in different wells was controlled by light microscopy, using confluent monolayers (Fig 1). In parallel, supernatants of the same antibodies were applied in a phage amount ELISA to normalize the phage antibody binding signals on cells (Fig 3B). Overall, 86 clones (53%) reacted with ECs by binding at least 3-fold over the background signal produced by WT phages. The other clones reacted weaker towards the cells, probably due to off-rates higher than the threshold set by the screening ELISA, or by recognizing underrepresented antigens. Importantly, though with different affinities, nearly all clones bound to both cell lines, BECs and LECs (Fig 3A), indicating a strong overlap of surface markers between these two lineages. Although the enrichment curve and FACS analysis had shown a clear increase of LEC-binders (Fig 2A and 2B), no exclusive LEC-specific antibodies were identified, and also depletion selection and competitive elution were not successful to derive LEC-specific clones (not shown). Twelve phage antibodies showing eminent reactivity in cell ELISA were picked for detailed characterization. Cell binding specificity was assessed by applying dilution series of selected phage antibodies (Fig 3C), and FACS analysis of the three strongest binders reconfirmed specific EC-reactivity (Fig 3D). Hence, the selection had yielded a panel of high-affinity BEC- and LEC-binding phage antibodies.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Binding assessment of single phage antibodies to BECs and LECs in cell ELISA and FACs.

A: Representative whole cell ELISA of phage antibodies prepared from 166 unique scFv clones derived from panning rounds #5 and #6 on BECs (red) and LECs (green). Values represent mean ± SD (n = 3). Negative controls were: WT-phage, 2xTY medium without phage, 2^nd antibody only. Phage antibody binding was visualized via peroxidase-conjugated anti-M13 phage antibody. OD value represents absorbance at 450nm. B: Phage amount ELISA of single phage antibodies. To allow for normalization of cell ELISA signal intensities, phage antibody concentrations were determined in a second ELISA format, where anti-fd bacteriophage antibody (S1 Table) was coated, and phage containing supernatants were added. Bound phage particles were detected by peroxidase-conjugated anti-M13 phage antibody (S1 Table). C: Effect of decreasing numbers of phage antibody concentrations on binding to BECs and LECs by cell-ELISA. Phage antibody dilution series in cell ELISA assessing specific binding to BECs and LECs. Control: same dilutions of WT phage. D: Binding of three selected phage antibodies to BECs and LECs analyzed by flow cytometry. Shown are histograms of BECs and LECs stained with the three phage antibodies (black lines). Grey-filled graphs show VCS-M13 wildtype phage binding as negative control. Cell binding was detected by sequential incubation with anti-M13 and FITC-conjugated anti-rabbit antibodies. The percentage of BECs and LECs bound to phages was evaluated by setting a marker (M1) indicated by the blue bar and is depicted in the graph.

https://doi.org/10.1371/journal.pone.0127169.g003

Soluble expression of scFvs and phage-independent binding to BECs and LECs

According to high binding affinity and antibody sequence abundance, eight antibodies were chosen for further analysis. Soluble scFv fragments were produced in E. coli non-suppressor strain (Fig 4A) and purified from culture supernatants, which resulted in 0.2–0.5 mg / ml culture soluble scFv antibody. Binding of this scFv antibody panel to BECs, LECs and human dermal fibroblasts (HDFs, see S1 Materials and Methods) as control cell line was re-examined in cell ELISA. The background was low, and when diluted 1:10, signals were in the range of commercial anti-PDPN and anti-CD31 antibodies (Fig 4B). All scFvs retained binding capacity towards BECs and LECs, with similar reactivities as the corresponding phage antibodies (Fig 3C). Immunoprobing of BEC and LEC lysates with scFv antibodies under reducing and non-reducing condition did not yield specific signals (not shown), indicating that binding of scFvs required native antigen. When tested in immunofluorescence on HDMECs, all eight scFv antibodies revealed a membrane-associated staining pattern (Fig 4C), as would be expected from the whole cell-selection procedure. A more detailed inspection by co-staining against podoplanin revealed that one antibody (B5-105) reacted equally to both, BECs and LECs, four antibodies stained BECs in combination with subpopulations of LECs (B5-83, L5-80, L6-05, L6-73), while three scFv antibodies showed selective BEC-labeling (B6-11, B6-112, B6-117). In accordance with previous cell ELISA screenings (Fig 3A), there was no LEC-specific staining. These data proved that soluble, phage-derived scFv antibodies retained binding capacity to BECs and LECs, out of which we identified three BEC-specific antibodies.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. scFv antibody production and binding to BECs and LECs in cell ELISA and immunofluorescence.

A: SDS-PAGE analysis of scFv antibody expression. Eight single scFv clones were grown in E. coli TG1 and scFv production was induced by IPTG. scFvs were purified from culture supernatants by using protein A sepharose and elution with glycine buffer at pH 2.2. Samples were captured and adjusted to PBS on Zeba spin desalting columns (Thermo Scientific). Proteins were separated on a 10% SDS-polyacrylamide gel and stained with Coomassie blue for molecular weight detection. Typical purification results of eight clones are shown with Mr in the range of 30 kDa. B: Whole cell ELISA with soluble scFv antibodies prepared from eight phage clone cultures. BECs, LECs and human dermal fibroblasts (HDF) cultivated in 96-wells were incubated with scFv fragments purified from bacterial culture supernatants. Comparison with anti-podoplanin and anti-CD31 antibodies as positive control. C: scFv antibodies show different labeling patterns on HDMECs in immunofluorescence. Representative double immunofluorescence images showing staining of eight different monoclonal scFv antibody clones on HDMECs. As seen from co-immunofluorescent staining against podoplanin (green), B5-83, B5-105, L5-80, L6-05 and L6-73 showed overlapping staining for BECs and LECs, while B6-11, B6-112 and B6-117 revealed specific BEC staining. Nuclei were counterstained with DAPI. Size bars: 50μm.

https://doi.org/10.1371/journal.pone.0127169.g004

Antigen retrieval for clone scFv B6-11 identifies CD146/MCAM

Showing highest specificity for BECs versus LECs (Fig 4B and 4C), we aimed to identify the antigen bound by scFv B6-11 antibody. Therefore, BEC lysates were used for immunoprecipitations with soluble scFv B6-11. Although no obvious band was detected in SDS-PAGE analysis of immune complexes (not shown), immunoprecipitates were subjected to trypsin digest and subsequent LC-MS/MS analysis. MS/MS spectra of the sample were searched in the Mascot database, which identified hits in the scFv B6-11 list that were clearly distinct from controls (S4 Fig). Nine peptides of the main hit showed exact identity with MUC18 surface glycoprotein (NP_006491), also known as CD146 / MCAM (Fig 5A). CD146 is a cell adhesion molecule belonging to the Ig super-family proteins [36]. Its mucin-type 71 kDa protein core is highly glycosylated, which brings the total molecular mass to 113–130 kDa, depending on the cell type. To test whether scFv B6-11 was specific for CD146, we followed several strategies: Immune complexes captured from BEC lysates with scFv B6-11 were probed with commercial anti-CD146 antibody, revealing a band at approximately 120 kDa (Fig 5B) that corresponds to the predicted molecular weight of CD146. Further, treatment of BEC lysates before and after addition of scFv B6-11 with PNGase F, which removes oligosaccharides from N-linked glycoproteins, led to a down-shift of the detected band to 95 kDa (Fig 5B), indicating that scFv B6-11 bound to CD146 independent of post-translational glycosyl-modification. As scFv B6-11 did not detect CD146 in immunoblotting (not shown), direct binding of scFv B6-11 was analyzed using an immobilized recombinant protein representing the Met¹-Gly⁵⁵⁹ portion of human CD146 (see Materials and Methods) in ELISA. scFv B6-11 as well as the commercial anti-CD146 antibody, but not control scFv B6-117, bound concentration-dependent this domain compared to control antigen (Fig 5C). These data suggested that scFv B6-11 bound to CD146 in native condition.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. LC-MS/MS identification of CD146 binding to scFv B6-11, and antigen confirmation by immunoprecipitation and ELISA.

A: Alignment of LC-MS/MS identified peptides (S4 Fig) with the sequence of MCAM/CD146MUC18. The eluates from scFv B6-11 immunoprecipitation were subjected to trypsin digestion (see Materials and Methods section) and subsequently analyzed by LC-MS/MS. B: Immunoprecipitation of CD146 by soluble scFv B6-11 from BEC lysates. Immune complexes were tested by Western blot in reducing conditions using commercial anti-CD146 antibody. Lane 1: input BEC lysate, lanes 2–5: immunoprecipitates with scFv B6-11, lanes 2 and 4: under addition of PNGase F. Treatment of BEC lysates with PNGase prior or after addition of scFv B6-11 had no influence on co-immunoprecipitation capacity of scFv B6-11, showing that scFv B6-11 binding to CD146 is glycosylation-independent. C: scFv B6-11 binds to immobilized extracellular domain of recombinant human CD146 in ELISA. An irrelevant antigen (BSA), a non-binding scFv and uncoated wells served as controls. scFv binding was detected with peroxidase-conjugated anti-His tag antibody (S1 Table), and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. D: CD146 expression of different cell lines as shown by immunoprobing with anti-CD146 antibody. CD146 is expressed in BECs, in A375, CRL1676, HTB71 melanoma cells, but not in primary LECs and HEK293 cells. The same blot was probed with anti-tubulin antibody for control of equal protein loads. E: scFv B6-11 stains cell lines expressing CD146 with similar intensity as commercial anti-CD146 antibody in ELISA. Negative controls were a non-binding scFv and 2^nd antibody only. F: Similar to commercial anti-CD146 antibody, scFv B6-11 stains BECs (upper lane, red) but not LECs (lower lane, green) in immunofluorescence. Size bars: 50μm. G: scFv B6-11 stains blood, but not lymphatic vessels in human skin. Double immunofluorescence staining of skin sample with Cy3-labeled scFv-B6-11 or anti-CD146 (red) and anti-PDPN (green) antibodies. Blood (BV) and lymphatic (LV) vessels are indicated by lines. Nuclei were counterstained with DAPI. Size bars: 50μm.

https://doi.org/10.1371/journal.pone.0127169.g005

scFv B6-11 labels native CD146/MCAM on BECs and blood vessels

CD146 was initially identified in melanoma and later also on endothelial cells [37,38]. Screening a panel of cell lines with commercial anti-CD146 antibody in immunoblotting showed that CD146 was highly expressed in BECs and in melanoma cell lines A375, CRL1676 and HTB71, but not in LECs and HEK293 cells (Fig 5D). Application of scFv B6-11 besides the commercial anti-CD146 antibody on these cell lines in ELISA revealed comparable reactivity (Fig 5E), suggesting that both antibodies recognized the same antigen. Further, when applied in immunofluorescent stainings, scFv B6-11 separately labeled BECs, but not LECs (Fig 5F, left panel), consistently with differential labeling of BECs versus LECs with anti-CD146 antibody (Fig 5F, right panel). Moreover, scFv B6-11 proved discriminatory labeling of blood versus lymphatic vessels in human skin cryosections (Fig 5G, left panel), similar to CD146 that did not colocalize with podoplanin-positive lymphatic capillaries (Fig 5G, right panel). These results indicated that phage derived scFv B6-11 recognized a conformational epitope of CD146 present on BECs and melanoma cells, and showed that B6-11 specifically labeled dermal blood versus lymphatic vasculature.

scFv-FcB6-11 fusion antibody retains specificity for native CD146

We next examined whether the three scFvs B6-11, B6-112 and B6-117 retained BEC-specificity as scFv-Fc fusion antibodies (S5A Fig) expressed in eukaryotic HEK-293 cells. By subcloning into the eukaryotic expression vector pFUSE mIgG2B-Fc2 [39] (S5B Fig), scFv regions (S5C Fig) were fused to a murine IgG Fc region. Immunoblotting of cell culture supernatants with anti-mouseFc-IgG antibody showed that scFv-Fc fusion antibodies migrated as monomers of approximately 80 kDa under reducing condition (R) (Fig 6A, lanes 2–4), while they behaved as dimers of 160 kDa under non-reducing condition (NR) (Fig 6A, lanes 5–7), confirming that they were expressed and secreted correctly. As control, Fc portion only was also expressed and purified (Fig 6A, first lane). Again, immunoprecipitates of BEC lysates with scFv-Fc B6-11, but not those treated with Fc only, contained CD146 (Fig 6B), confirming that the fusion antibody retained its capability to bind CD146. As scFv-Fc B6-11 did not detect CD146 in immunoblotting (not shown), it was re-examined in ELISA format, showing that scFv-Fc B6-11 specifically bound to recombinant CD146, but not to control protein (Fig 6C). Further, scFv-Fc B6-11 bound to BECs and melanoma cell lines A375, CRL-1676 and HTB71 harbouring CD146, but not to LECs and HEK-293 cells, in cell ELISA (Fig 6D), and signal intensities correlated with CD146 levels seen in WB (Fig 5D). In immunofluorescence, scFv-Fc B6-11 showed discriminatory labeling of PDPN-negative BECs in HDMECs (Fig 6E, left panel), similar to the commercial anti-CD146 antibody (Fig 6E, right panel). Further, on A375, CRL-1676 and HTB71 melanoma cells, scFv-Fc B6-11 co-stained with an antibody against melanoma surface antigen S-100 (S6A Fig) and with commercial anti-CD146 antibody (S6B Fig). These data confirmed that scFv-Fc antibody B6-11 recognized an antigenic site of CD146, which is common to BECs and melanoma cells.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. Production of scFv-Fc B6-11 fusion antibody and reconfirmation of binding to CD146.

A: Western Blot analysis showing molecular size of scFv-Fc fusion antibodies B6-11, B6-112 and B6-117. The scFv-region of the phagemids was cloned into the pFUSE-mIgG2B-vector (see S5 Fig). Constructs were transfected into HEK293-cells. Cell culture supernatants were loaded on reducing (R) and non-reducing (NR) 10% SDS-PAGE, and nitrocellulose blots were probed with peroxidase-labeled anti-mouseFc antibodies. scFv-Fc fusion antibodies are secreted as approximate 140 kDa dimers, as seen under non-reducing condition (-DTT). Control: Fc only protein, produced from pFUSE vector without scFv insert. B: Immunoprecipitation with scFv-Fc B6-11 reconfirms CD146-binding. G1S1 lysates were incubated with scFv-Fc B6-11 and Fc only as control. Blots of immunoprecipitates were probed with anti-CD146 and anti-mouseFc antibodies. C: scFv-Fc B6-11 binds to recombinant CD146 in ELISA. scFv-Fc B6-11, commercial anti-CD146 antibody and Fc only were used on respective dilutions of recombinant human CD146 or BSA as control antigen coated on 96-well ELISA plates. Absorbance was measured at 450nm. D: scFv-Fc B6-11 fusion antibody binds to cells expressing CD146 in ELISA. Purified scFv-Fc B6-11 (light grey bars), commercial anti-CD146 antibody (dark grey bars) and Fc only (black bars) were added to monolayers of respective cell lines. Bound antibodies were detected using anti-Fc and HRP-conjugated anti-rabbit antibodies, and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. E: On HDMECs, scFv-Fc B6-11 fusion antibody shows the same reactivity as a commercial anti-CD146 antibody. F: scFv-Fc fusion antibodies (red) reveal diverse membraneous labling patterns in co-immunofluorescent stainings with anti-CD31 antibody (green). Nuclei were counterstained with DAPI. Size bars: 50μm.

https://doi.org/10.1371/journal.pone.0127169.g006

scFv-Fc fusion antibodies label BEC membranes

To compare the specificities of scFv-Fc fusion antibodies B6-11, B6-112 and B6-117, co-stainings against endothelial marker CD31were performed on HDMECs. Although there was some variation in the staining intensity, each antibody, but not Fc portion only, re-confirmed the membrane-associated labeling pattern (Fig 6F). A more detailed comparative inspection denoted an intense membrane staining by B6-11 (S7A Fig), while B6-112 showed labeling of the membranous linings (S7B Fig) and B6-117 revealed a more heterogeneous staining pattern (S7C Fig). These subtle differences suggested that the three antibodies recognized diverse cellular epitopes. In conclusion, the data showed that scFv-Fc fusion antibodies, as their scFv parent antibodies, retained reactivity towards antigens presented by BECs in native condition.

scFv-Fc fusion antibodies label blood vasculature in different organs

Next, the binding profile of scFv-Fc fusion antibodies was characterized against a panel of human tissues. As seen by co-localization with endothelial marker CD31 (Fig 7A), but not with lymphatic marker podoplanin (Fig 7B), scFv-Fc B6-11, B6-112 and B6-117 fusion antibodies specifically stained blood vessel structures of human skin sections. Staining of blood vasculature of kidney specimens was seen by scFv-Fc B6-11, B6-112 and B6-117 fusion antibodies (S8A–S8C Fig), but not with Fc portion only (S8D Fig). Moreover, scFv B6-11 showed specific blood vessel labeling on human colon and lung cryosections (S8E and S8F Fig). These data proved that scFv-Fc fusion antibodies were highly useful as specific labels of blood capillaries from different organs.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. Fusion antibodies scFv-Fc B6-11, B6-112 and B6-117 stain blood vessels in the dermis.

A: Representative images of double immunofluorescence stainings of frozen human skin sections with scFv-Fc fusion antibodies (red) in combination with anti-CD31 (green) show overlapping staining, but B: not with anti-PDPN antibody (green) as control. Nuclei were counter-stained with DAPI. Size bars: 50μm.

https://doi.org/10.1371/journal.pone.0127169.g007

scFv-Fc B6-11 interferes with gap closure of immortalized BECs

Finally, considering the putative functional role of CD146 for endothelial cell migration [38], we analyzed whether binding of scFv-Fc B6-11 to CD146 present on BECs showed implications in vitro. In a scratch wounding assay, addition of scFv-Fc B6-11 to cell culture medium lead to a significantly decreased gap closure of a BEC monolayer (Fig 8A, upper panel, and Fig 8B, red line), when compared to addition of Fc portion (Fig 8A, middle panel and Fig 8B, grey line) or PBS only as controls (Fig 8A, lower panel, and Fig 8B, black line), suggesting that scFv-Fc B6-11 might exert an inhibitory influence on the migratory capabilities of BECs via binding to CD146.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 8. scFv-Fc B6-11 fusion antibody inhibits motility of BECs in a scratch wound assay.

A: Migration analysis of BECs by scratch wound assay in the presence of scFv-Fc B6-11, Fc fragment or PBS as controls. Shown are representative images of wounds created in BEC monolayers (n = 3 in each group) at indicated time points. Size bars: 500μm. B: Reoccupation of the gap by BECs after t = 24 hrs and t = 48 hrs versus t = 0 hrs was measured using AxioVision 4.7 software. Areas repopulated by BECs and % of gap area newly covered by BECs were calculated at each timepoint. Assay was performed twice and values are means ± SD. Gap closure was analysed by one-way Anova (P < 0.05) followed by pairwise Student´s t-testing. Shown are significant differences between scFv-Fc versus Fc control, and scFv-Fc versus PBS control, respectively (P-values: * < 0.05, ** < 0.005, ***< 0.0005).

https://doi.org/10.1371/journal.pone.0127169.g008

In conclusion, we show the feasibility of phage antibody enrichment on BECs versus LECs, yielding 166 unique phage antibodies (S9A Fig). Out of these, we isolated three antibodies, B6-11, B6-112 and B6-117, that exclusively target blood vasculature, and by using IP and LC-MS, one antibody specificity was identified as MCAM/CD146 (S9B Fig) revealing an antigenic link between blood endothelial and melanoma cells.