Syndecan-1 promotes the angiogenic phenotype of multiple myeloma endothelial cells.
Sara Lamorte1, Simone Ferrero2, Simona Aschero2, Luigia Monitillo2, Benedetta Bussolati1, Paola Omedè2, Marco Ladetto2 and Giovanni Camussi1.
1Department of Internal Medicine, Research Center for Experimental Medicine (CeRMS) and Molecular Biotechnology Center, University of Torino, Torino, Italy and 2Division of Hematology, Department of Oncology and Experimental Medicine; University of Torino, Torino, Italy.
Running title: Syndecan-1 and multiple myeloma endothelial cells.
Address correspondence: Dr. G. Camussi, Dipartimento di Medicina Interna, Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti 14, 10126, Torino, Italy;
Phone +39-011-6336708, Fax +39-011-6631184.
E-mail:
Supplementary Materials & Methods:
Syndecan-1 siRNA and shRNA transfection
A pool of 3 target-specific 20-25nt siRNAs designed to knock down expression of syndecan-1 waspurchased from Santa Cruz Biotechnology, Inc.,Santa Cruz, CA. MMECs were seeded in a 6-well plate to reach 80% of confluence and transfected with 50 pM of siRNAs and incubated over night in a siRNA Transfection Medium, according to the manufacturer’s instructions. As control, MMECs were transfected with a scrambled siRNA (scr-siRNA), a random RNA that does not match any particular sequence (Santa Cruz). Successful RNAi was evaluated after 48 hours by FACS analysis and 72 hours by RT-PCR. For shRNA transfection, MMECs were transfected with five different expression plasmids containing a gene-specific shRNA cassette coding for syndeacan-1 shRNAtargeted to different sites in the syndecan-1 message and a puromicin-N-acetyl transferase gene located downstream of the SV40 promoter, resulting in resistance to puromycin selection (Origene, Beijing, China). MMECs were seeded in a 12-well plate to reach 50% of confluence and, then, incubated with 0.5 μg of shRNA expression plasmid DNA or, as control, with a scrambled shRNA (scr-shRNA) (Origene). After 48 hours of transfection, cells contained the plasmid were selected adding 0.5 μg/ml of puromycin in the growth media. The selection pressure was maintained for 1 week. Cells were then incubated 72 hours before harvesting for RNA and 48 hours for protein analysis to evaluate RNAi efficiency. A titration analysis was performed to assess the minimum effective concentration of both siRNA and shRNA expression plasmid.1
Flow cytometry and immunofluorescence
Cells were stained with a panel of FITC- or PE-conjugated murine antibodies to human CD31 (Miltenyi), CD14, CD45 (Caltag Laboratories, Burlingame, CA), CD154 (Serotec, Oxford, UK), CD40 (Immunotech, Coulter Company, Marseille, France), VEGF receptor 1 (VEGFR-1), VEGFR-2 and VEGFR-3 (R&D Systems, Minneapolis, MN), CD29, CD144, CD146 (Chemicon, Temecula, CA), CD105 (Biolegend San Diego, CA), CD34, CD44, CD133, CD138, CD38 (Becton Dickinson Biosciences), CD90 (DakoCytomation) and Ulex europaeus agglutinin-1 (UEA-1) (Sigma). FITC- or PE-conjugated antibodies to mouse IgG (Sigma) were used as secondary antibody in indirect assay. Secondary antibody or the corresponding fluorochrome-conjugated isotype antibody was used as the negative control. For phenotype characterization by immunofluorescence, cells were fixed in 3.5% paraformaldehyde (PFA) plus 2% sucrose and permeabilized with 0.1% N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Triton X-100 buffer (Sigma), then incubated with rabbit anti-human Von Willebrand Factor (vWF) (DakoCytomation, Denmark), mouse anti-human vimentin (Sigma) or VE-cadherin (Biolegend, San Diego, CA). For VEGFR-2 localization studies cells, fixed in 3.5% or 2% PFA and permeabilized or not with HEPES-Triton X-100, were incubated with rabbit anti-human VEGFR-2 with or without mouse anti-human syndecan-1 (Santa Cruz Biotechnology), mouse anti-human EEA-1, Rab11, GM130 or TGN38 (BD Transduction Laboratories™, Becton Dickinson Biosciences). As secondary antibodies we used Alexa Fluor® 594 goat anti-rabbit and Alexa Fluor® 488 goat anti-mouse antibodies (Invitrogen, San Diego, CA). Hoechst 33258 dye (Sigma) was added for nuclear staining. Secondary antibody alone was used as control. Markers expression were analyzed by confocal microscopy using a Zeiss LSM 5 Pascal model confocal laser scanning microscope (Carl Zeiss Int., Oberkochen, Germany) equipped with a helium/neon 543mmlaser, an argon 450 to 530mmlaser, and an EC planar NEOFluar 40x/1.3 and 63x/1.4 oil DIC objective lens; acquisition software, Zeiss LSMS, version 3.2.
Cell proliferation assay
Cells were seeded into 96-well plates at a density of 8 X 103 cells/well in RPMI with 10%, 5% or 2% FBS and, as control, complete EBM with 10% FBS. Proliferation wasevaluated after 48 hours by detection of 5-bromo-2’-deoxyuridine (BrdU) incorporation into thecellular DNA using an enzyme-linked immunosorbent assay kit (Roche Diagnostics, Basel, Switzerland), according to the manufacturer’s instructions.
Apoptosis Assay
Cells (1 X 104) were plated in 96-well plate in RPMI with 10% or 0% FBS and, as control, in complete EBM with 10% FBS for 24 hours. TUNEL assay (Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick-end labeling) was performed using the In Situ Cell Death Detection kit (Roche Diagnostics), according to the manufacturing instructions. Apoptosis was determined as the percentage of positive cells per 100 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Plate was visualized under a fluorescence microscope (Nikon Eclipse 50i) at a magnification of X200.
Apoptosis and cell cycle arrays and quantitative RT- PCR
For polymerase chain reaction (PCR) array studies, MMECs were compared with MMECs syndecan-1 siRNA and BMECs. Total RNA was extracted with mirVANATM miRNA isolation kit (Ambion,Inc) and DNase-treated (DNAse I; Sigma-Aldrich). cDNA was synthesized from 1 µg of total DNase-treated RNA using RT2 Profiler PCR array first strand kit C-02 (SABiosciences, Sweden) according to protocol. Human cell cycle (Catalog no. PAHS-020; SABiosciences) and apoptosis RT2 Profiler PCR arrays (Catalog no. PAHS-012; SABiosciences) were performed according to the manufacturer's instructions using the StepOnePlusTM Real Time instrument (Applied Biosystems, Foster City, CA) and StepOnePlusTM software 2.2. Acquired data were analyzed with PCR array data analysis template downloaded from the SuperArray Web site ( and normalized to the expression level of housekeeping control genes. Fold changes below of 4 were not considered.Quantitative reverse transcription-PCR (qRT-PCR) was used to verify data from the PCR arrays. For qRT-PCR studies, 400 ng of RNA, extracted with TRIzol reagent (Invitrogen, San Diego, CA), were reverse transcribed with the miScript Reverse Transcription Kit and cDNA was analysed to detect and quantify genes of interest using Power SYBR® Green PCR Master Mix (Applied Biosystems). All samples were run in triplicate using 4 ng of cDNA and 300 nM primers for each reaction. Specific primers (See supplementary Table 2) were designed using the Primer Express software (Applied Biosystems) and purchased from MWG-Biotech AG, Ebersberg, Germany. Negativecontrols were run in parallel and melting curve analysis was performed to confirm specificity. The relative expression of different mRNAs was determined by relative quantification: δCt = Ct target – Ct ACTB/GAPDH. Fold change in expression with respect to control was calculated for all samples.
Matrigel invasion
The invasion ability was evaluated using transwell chambers(Costar, Cambridge, MA), in which upper and lower chambers, separated by 8-μm pore-sizepolyvinylpyrrolidone-free polycarbonate filters, were coated with 100 μg/well of growth factor-reduced Matrigel (Becton Dickinson Biosciences). Chambers were loaded with not complete EBM with 10% FBS and to the lower compartment was added or not VEGF (25 ng/ml) (Immunological Sciences, Rome, It.). Cells (5 X 104 cells/well) wereseeded onto the upper compartment and incubatedfor 48 hours.Cells migrated from the upper to the underside of the filter were analysed as previously described 36. Each experiment was done in triplicate.
Adhesion Assay
MMECs and MMECs syndecan-1 shRNA were harvested with non enzymatic solution (Sigma) and plated at the density of 5x104 cells/wellin 24-well plates coated with gelatin (DIFCO Laboratories, Lawrence, KS) or growth factor-reduced Matrigel (Becton Dickinson Biosciences) in media used in the experimental assays. After 1 or 2 hours, medium was aspirated and wells were washed twice to remove non adherent cells. Adherent cells where trypsinised and counted with a Burker haemocytometer. Each experiment was done in duplicate.
In vitro angiogenesis assay
The assay was performed as previously described.35 Briefly, ECs were seeded on growth factor reduced Matrigel (Becton Dickinson Biosciences) coated wells in RPMI with 5% FBS at the density of 3.5 x 104 cells/well in 24-well plates. To evaluate the endothelial organization into capillary-like structures cells were observed after 5 or 24 hours incubation with a Nikon Eclipse TE 200 inverted microscope (objectives, 10x/0.25; Tokyo, Japan) and the experimental results were recorded. The extent of capillary-like structures was analyzed in five different fields by the Micro-image System (Cast Imaging Srl, Venice, Italy) and expressed in arbitrary units system.
In vivo angiogenesis assay
For the in vivo studies cells were subcutaneously injected into SCID mice (Charles River, Calco, Italy; n = 4/5 for each experimental condition). Briefly,2 X 106ECs were resuspended in Hanks’ balanced salt solution (200 μl) (Sigma), added to Matrigel (300μl) (Becton Dickinson Biosciences) and mixed with heparin (64 U/ml) (Sigma). Cells were injected subcutaneously into the mid-abdominal region of SCID mice. At day 7 mice were sacrificed and the plugs recovered and processed for histology, as previously described.35Vessel structures were counted only if showing a patented lumen with red globuli and/or leukocytes, using Nikon Eclipse TE 200 inverted microscope (objectives, 10x/0.25; Tokyo, Japan), analyzed by the Micro-image system (Casti Imaging) and expressed as percent area per field. The human nature of ECs was assessed by immunofluorescence staining with a rabbit anti-hHLA class I (Santa Cruz Biotechnology) and a rat anti-hCD31 antibody (Abcam, Cambridge, UK) by Zeiss LSM 5 Pascal confocal laser scanning microscope (40x/1.3 oil DIC objective lens). Animal housing, care and applications of experimental procedures complied with the Guide for the Care and Use of Laboratory Animals of the Government of Italy and are in accordance to the recommendations of the Society for Laboratory Animal Science and the Federation of European Laboratory Animal Science Associations.
In vitro endothelial cell migration
Cells (8 x 104) were plated on gelatin-coated T25 flask in cultured media and allowed to adhere overnight. The day after, cells were rested and incubated in RPMI with 1% FBS with or without VEGF (25ng/ml) (Immunological Sciences). Cell division did not start to any significant degree during the experiments. Cell migration was studied over a 14 hours period under a Nikon Diaphot inverted microscope (Melville, NY) with a X10 phase-contrast objective in an attached, hermetically sealed Plexiglas Nikon NP-2 incubator at 37° C. Cell migration was recorded using a JVC (Tokyo, Japan) 1-CCD video camera. Image analysis was performed with a MicroImage analysis system (Cast Imaging Srl) and an IBM-compatible system equipped with a video card (Targa 2000; True Vision, Santa Clara, CA) as previously described. 35 Migration of at least 30 cells was analyzed for each experimental condition.
Supplementary Reference
1. Whither RNAi? Nature cell biology 2003 Jun; 5(6): 489-490.