PDAC (P48Cre+:LSLKrasG12D+:INK4Alox/lox) mice were crossed with SPARC+/+ or SPARC−/− mice to produce WT PDAC or SPARC-null PDAC (knockout [KO] PDAC) mice. For tumor analyses, mice were sacrificed once becoming moribund, with at least six mice per group. Tumors were preserved in formalin or snap frozen using liquid nitrogen. Animal experiments were performed at the University of Texas Southwestern Medical Center at Dallas in compliance with the Animal Welfare Act, the Public Health Service Policy, and the U.S. Government Principles Regarding the Care and Use of Animals.
Antibodies to the following proteins were used for indirect immunofluorescent microscopy: α-SMA (NeoMarkers), NG2 (AB5320; Millipore), desmin (Ab907; Millipore), vinculin (V4139; Sigma-Aldrich), MECA32, SPARC (R&D Systems), total TGF-β (SC146; Santa Cruz Biotechnology, Inc.), and endoglin (MJ7/18). For function-blocking assays, a pan–TGF-β–neutralizing antibody was purchased from R&D Systems (1D11), an αVβ6-blocking antibody was a gift from D. Sheppard (University of California, San Francisco, San Francisco, CA), and an αV integrin–blocking antibody was purchased from BioLegend (RMV-7). The hybridomas that produce mAb293 and mAb303 were grown in our laboratory and purified by protein A chromatography. For Western blots, ALK5 (SC-398; Santa Cruz Biotechnology, Inc.), TβRII (SC-220; Santa Cruz Biotechnology, Inc.), endoglin (clone MJ7/18), αV integrin (AB1930; Millipore), FAK (3285; Cell Signaling Technology), phospho-SMAD2 (AB3849 serine 465/467; Millipore), and total SMAD2 (tSMAD2; 3107; Cell Signaling Technology) were used. For solid-phase binding assays, endoglin (MJ7/18) and SPARC (mAb 236) were used. For immunoprecipitations, SPARC (mAb303), ALK5, TβRII (SC-220), and endoglin (MJ7/18) were used. The hybridomas MECA32 and MJ7/18, developed by E.C. Butcher (Stanford University, Palo Alto, CA), were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development, and maintained by The University of Iowa.
Primary pericyte isolation, cell culture, and transfections
Mouse pancreata from 4-wk-old SPARC+/+ and SPARC−/− mice were minced and then subjected to digestion with 1% collagenase type 1, DME, 10 mM Hepes, 1% fetal bovine serum, and PBS at 37°C until a single-cell suspension was obtained. Cell suspensions were centrifuged at low speed to pellet large debris, resuspended in wash buffer, and passed through a 70-µm cell strainer. The resulting cell suspension was then incubated with sheep anti–rabbit IgG-conjugated magnetic Dynabeads (Invitrogen) and rabbit anti-NG2 IgG (Millipore) at 4°C. Dynabeads were preincubated with anti-NG2 IgG overnight at 4°C on a nutator and then washed three times in wash buffer to remove NaN2. Bead-bound cells were separated from unbound cells using a cell separation magnet (IMagnet; BD). Primary pericytes were maintained in 10% fetal bovine serum–supplemented DME and used between passage 1 and 7 for experiments. 10T1/2 cells were used before 10 passages and maintained in 10% fetal bovine serum–supplemented DME. Primary pericytes were transfected using Lipofectamine 2000 (Invitrogen), whereas 10T1/2 cells were transfected using Fugene (Roche). For shRNA knockdown of SPARC, endoglin, and TGF-β1, shRNA expression plasmids were purchased from Sigma-Aldrich (MISSION). 2 µg plasmid DNA was mixed with 3 µl transfection reagent and used to transfect 150,000 cells. Cells were used 48 h after transfection. For negative control transfections, a nontargeting shRNA expression plasmid was used (MISSION).
Cord formation assay
10,000 bEnd.3 endothelial cells were plated onto matrigel-coated 3-well chamber slides (BD) in the presence or absence of either 10,000 SPARC+/+ or SPARC−/− pericytes and allowed to self-assemble into cords for 17 h in DME supplemented with 0.75% fetal bovine serum at 37°C before visualization by fluorescent microscopy. Before use in the assays, bEnd.3 cells and pericytes were stained with either the red fluorochrome PKH26 or the green fluorochrome PKH67, respectively (Sigma-Aldrich). Experiments were performed three times and in triplicate. Images were taken at a 4× magnification, with five images taken per well. The peripheral zone of matrigel was avoided during image acquisition so to avoid cord artifacts associated with changes in surface elevation. Cord lengths and widths were calculated using NIS-Elements software (Nikon). For cord width measurements, widths were taken halfway into the length of each cord.
Transwell inserts with 8-µm pores were used for migration assays. Inserts were placed in 24-well tissue-culture plates for the duration of experiments. The bottom sides of the insert membranes were coated with 10 µl of 1-µg/µl fibronectin for 1 h at 37°C. Inserts were then used immediately for experiments (Sigma-Aldrich). 7,000 or 5,000 primary pericytes or 10T1/2 cells were added into the insert reservoir in DME in a total volume of 125 µl, whereas DME containing 0.1% fetal bovine serum was added into the tissue-culture plate well. Experimental conditions were always added to both the top and bottom of the transwell. Cells were allowed to migrate to the fibronectin-coated side of the insert membrane for 6 h. Cells on the noncoated side of the insert membrane were removed. Cells that migrated to the underside of the membrane were fixed in formalin and manually counted. Experiments were performed in triplicate and repeated two or three times as indicated in the figure legends.
TGF-β ELISA and TGF-β1 response gene expression
A TGF-β1 ELISA kit (TGF-β1 EMAX Immunoassay G7591) that detects the active form of TGF-β1 was purchased from Promega. Sample preparation for active TGF-β1 ELISA was performed as follows: primary pericytes were seeded at 150,000 cells per well in 6-well tissue-culture plates and cultured in 0.75% fetal bovine serum–supplemented DME in triplicate. Cells did not exhibit any expansion and remained subconfluent and viable for the duration of the experiment. Conditioned media and cell lysates were collected at 24, 48, 72, and 96 h after seeding. A mammalian protein extraction reagent (M-PER) cell lysis buffer supplement with protease inhibitor (Complete Mini) was used for lysate preparation (Roche). ELISA was performed according to kit instructions. Active TGF-β1 concentrations were calculated by interpolating values onto a standard curve generated with TGF-β1 accompanying the kit. For qPCR expression analyses, fold change was calculated using the ΔΔ cycle threshold method, in which WT at 0 pg/ml was the reference sample, and GAPDH was the reference gene. Sample preparation for qPCR expression analyses were as follows: serum-starved primary pericytes were seeded at 100,000 cells per well in fibronectin-coated 6-well tissue-culture plates in triplicate. Active TGF-β1 purchased from PeproTech was added to serum-starved pericytes at final concentrations of 0, 50, and 5,000 pg/ml. Cells were then incubated for 17 h at 37°C. RNA was harvested using TRIZOL reagent (Sigma-Aldrich). cDNA was synthesized using iScript (Bio-Rad Laboratories). 12.5 ng cDNA was used per 96-well PCR plate well, with each tissue-culture plate well represented in three individual PCR plate wells. The following primer sets were used for qPCR or RT-PCR: CTGF forward, 5′-AGCCTCAAACTCCAAACACC-3′, and reverse, 5′-CAACAGGGATTTGACCAC-3′; PAI-1 forward, 5′-GACACCCTCAGCATGTTCATC-3′, and reverse, 5′-AGGGTTGCACTAAACATGTCAG-3′; BIGH3 forward, 5′-TGATAAGAGGGGACGGTTTG-3′, and reverse, 5′-ATTGGTGGGAGCAAAAACAG-3′; and GAPDH forward, 5′-AGAAGGCTGGGGCTCATTTG-3′, and reverse, 5′-AGGTCGGAGTCAACGGATTTG-3′.
To assess the effect of SPARC on SPARC−/− pericyte transcription, pericytes were cultured for 72 h in the presence or absence of recombinant SPARC or BSA control. Media were replaced with fresh SPARC- or BSA-containing media every 24 h before RNA extraction.
Cells were incubated in 0.75% fetal bovine serum–supplemented DME overnight before RNA extraction and cDNA synthesis. The following primer sets were used for RT-PCR: endoglin (L-endoglin) forward, 5′-GCACTCTGGTACATCTATTCTCACACACGTGG-3′, and reverse, 5′-GGGCACTACGCCATGCTGCTGGTGG-3′; SPARC forward, 5′-CTGCGTGTGAAGAAGATCCA-3′, and reverse, 3′-TGGGACAGGTACCCATCAAT-3′; ALK5 forward, 5′-GGCGACGGCATTACAGTGTT-3′, and reverse 5′-TGTACATACAAATGGCCTGT-3′; TβRII forward, 5′-GCAAGTTTTGCGATGTGAGA-3′, and reverse, 5′-GGTATCTTCCAGAGTTGAAGC-3′; TGF-β1 forward, 5′-TTGCTTCAGCTCCCACAGAGA-3′, and reverse, 5′-TGGTTGTAGAGGGCAAGGAC-3′; αV integrin: itgav forward 5′-GGGTGATCATCTTGGCAGTT-3′, and reverse, 5′-GAACTTGGAGCGGACAGAAG-3′; β1 integrin: itgb1 forward, 5′-GTGACCCATTGCAAGGAGAAGGAC-3′, and reverse 5′-GTCATGAATTATCATTAAAAGTTT-3′; β3 integrin: itgb3 forward, 5′-CTGGTGTTTACCGATGCCAAG-3′, and reverse, 5′-TGTTGAGGCAGGTGGCATTGAAGG-3′; β6 integrin: itgb6 forward, 5′-CCGGCTGGCCAAAGAGATGT-3′, and reverse, 5′-AGTTAATGGCAAAATGTGCT-3′; RPS6: rps6 forward, 5′-AAGCTCCGCACCTTCTATGAGA-3′, and reverse, 5′-TGACTGGACTCAGACTTAGAAGTAGAAGC-3′; and β-actin: actb forward, 5′-ATATCGCTGCGCTGGTCGTC-3′, and reverse, 5′-AGGATGGCGTGAGGGAGAGC-3′.
Detection of basal SMAD2 phosphorylation
Pericytes were seeded at 100,000 cells per well of 6-well culture plates and cultured in 1.5% fetal bovine serum–supplemented DME for 17 h before being lysed in 300 µl sample buffer (62.5 mM Tris-HCl, pH 6.8, at 25°C, 2% SDS, 10% glycerol, 50 mM DTT, and 0.01% bromophenol blue). Lysates were subjected to SDS-PAGE and Western blotting for tSMAD2 and pSMAD2 (serine 465/467) immediately thereafter.
10T1/2 cells were lysed in modified radioimmunoprecipitation assay buffer (0.5% deoxycholate, 0.5% SDS, 1% Triton X-100, 10 mM sodium phosphate, pH 7.2, 150 mM sodium chloride, and protease inhibitor [Complete Mini]). Pericytes were lysed in a milder buffer containing 1% NP-40, 10 mM sodium phosphate, pH 7.2, 150 mM sodium chloride, and protease inhibitor (Complete Mini). Lysis was performed on serum-starved adherent cells after washing with chilled PBS. Cells were scraped using 1 ml modified radioimmunoprecipitation buffer. Lysates were allowed to rotate at 4°C on a nutator for 1 h and then vortexed several times before centrifuging at 13,000 rpm for 10 min to pellet any insoluble material. Lysates were then precleared with protein A/G beads (Thermo Fisher Scientific). 200 µg cellular protein in 1 ml lysis buffer was used per immunoprecipitation reaction. 1 µg of the appropriate IgG was added with 20 µl protein A/G bead slurry to each sample; each sample was then allowed to rotate overnight at 4°C on a nutator. Immunoprecipitated complexes were washed twice in lysis buffer and then boiled in sample buffer and subjected to SDS-PAGE and Western blot analysis.
Solid-phase binding assays
Wells of 96-well clear-well assay plates were coated with recombinant human SPARC, recombinant human endoglin (R&D Systems), or serum (EastCoast Bio), blocked, and incubated with recombinant endoglin or recombinant SPARC. Bound endoglin or SPARC was detected with antiendoglin (MJ7/18) or anti-SPARC (mAb 303) antibodies or detected with horseradish peroxidase–conjugated secondary IgG. Assays were developed using tetramethylbenzidine reagent (Thermo Fisher Scientific). Samples were added in triplicate, and the experiment was repeated three times.
Surface protein labeling
Primary pericytes were grown to 80% confluency and then switched to 0.75% fetal bovine serum–supplemented DME. Cells were then labeled with a cell surface protein isolation kit (Sulfo-NHS-SS-Biotin; Thermo Fisher Scientific) according to the manufacturer’s instructions. 4–10-cm dishes per pericyte genotype were used per fractionation. Fractionations were performed twice with identical results.
Epifluorescent images were taken using a microscope (Eclipse E600; Nikon) and a camera (CoolSNAP HQ; Photometrics). Images were acquired and analyzed using NIS-Elements software. For visualization of immunofluorescently stained cells, images were thresholded so as to not include a signal caused by the nonspecific binding of the fluorophore-conjugated secondary antibody alone and analyzed as JPEG 2000 files. Confocal images were taken using either a TCS-SP5 confocal microscope (Leica) or an Eclipse TE2000E confocal microscope (Nikon). Leica images were taken using the Imaging Application for Confocal SP5 software (Leica). Images were saved as Leica Image Files (.LIF) and analyzed using ImageJ software (National Institutes of Health). Contrast and brightness were adjusted equally in all channels using Photoshop (CS3 Extended; Adobe). Nikon images were taken using a camera (CoolSNAP ES) and EZ-C1 3.8 software (Photometrics). Images were saved in the native ICS/IDS format. Images were processed using NIS-Elements software. Channels were thresholded so as to not include autofluorescence from the assay medium (10% FBS in DME containing phenol red). Nikon epifluorescent and confocal objectives (plan fluorite) had the following numerical apertures: 10×, 0.3; 20×, 0.5; 40×, 0.75; and 100×, 1.3 in oil. Leica confocal images were taken using a 63× objective with a 1.4 numerical aperture in oil. Fluorescent staining was performed using cyanine (Cy3) or fluorescein isothiocyanate–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.).
Student’s t test analysis or analysis of variance was performed for all experiments.
Online supplemental material
Fig. S1 shows vinculin and phalloidin staining used to assess focal adhesion formation in primary pericytes. Fig. S2 shows exogenous SPARC blocked the anti-TGF-β–induced enhancement of SPARC−/−
pericyte migration. Fig. S3 shows that SPARC−/−
pericytes exhibit enhanced basal TGF-β activity. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201011143/DC1