Methods
(Words 2069)
Materials: The OPN-R3 aptamer (APT) and OPN-R3 mutant aptamer (MuAPT) were
synthesized by Dharmaco, Lafayette, CO. APT was developed in our lab to specifically bind to
and functionally block extracellular OPN (in vitro Kd=18  0.2 nM). Mutant APT (MuAPT),
which lacks OPN binding or IgG served as negative controls. All cysteine and uracil bases are
2’-O-methylated, and the molecules have 5’ cholesterol and 3’ IDT modification. The sequences
for the OPN-R3 aptamer and mutant aptamer are as follows:
APT
5’-GCCACAGAAUGAAAAACCUCAUCGAUGUUGCA -3’
MuAPT 5’-CGGCCACAGAAUGAAUCAUCGAUGUUGCAUAGUUG-3’
Recombinant human OPN protein and TGF-1 proteins were purchased from R&D Systems,
Minneapolis, MN. The v3 integrin Ab (SC-7312) and CD44 Ab (SC-7051) were purchased
from Santa Cruz Biotechnology, Santa Cruz, CA. The v3 integrin and CD44 Abs (Santa Cruz
Biotechnology, Santa Cruz, CA); Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE) (Sigma-Aldrich,
St Louis, MO); SB431542 a TGF- β1 type I receptor inhibitor (EMD Millipore, Billerica, MA);
TGF-β1 type II receptor shRNA (Santa Cruz Biotechnology); MZF1 siRNA (Life Technologies,
Carlsbad, CA) were all obtained. The sequence of Control siRNA used for the experiments was:
UAAGGCUAUGAAGAGAUAC.
Cell culture: Human mesenchymal stem cells (MSCs), CD105/CD73/CD44 positive and
CD34/CD45/CD14/HLA-DR negative, were obtained from the Texas A&M Institute for
Regenerative Medicine Health Science Center College of Medicine (Bryan, TX) and maintained
in Minimal Essential Medium (MEM) media with 20% fetal bovine serum; a subset was GFP+.
All cells were cultured at 37°C in a humidified atmosphere of 5% CO2. Transduction with MZF1
siRNA generated MSCs with constitutive knockdown of MZF1 (MSC(dMZF1)) for use in
animal studies. After IRB approval, primary human MSCs (BM-MSCs) were isolated using
RosetteSep Human Mesenchymal Stem Cell Enrichment Cocktail (STEMCELL, Vancouver,
Canada) from normal human BM isolates. Human MDA-MB231 and MCF7 breast cancer cell
lines, purchased from American Type Culture Collection (ATCC, Manassas, VA), were
maintained in DMEM with 10% FBS. Lentiviral transduction (GenTarget Inc, San Diego, CA)
was used to generate dual-labeled RFP-Luc-MB231 and RFP-Luc-MCF7 cells for use in animal
studies. Further manipulation using lentiviral transduction generated MCF7 cells that
constitutively express OPN (RFP-Luc-MCF7-lvOPN). Human skin fibroblasts (CCD-1140SK),
human lung fibroblasts (MRC-5), and murine embryonic fibroblasts (CF-1) were purchased from
ATCC. Human mammary fibroblasts were a gift from Dr. Kuperwasser, Tufts University.
Co-culture: For all co-culture experiments, a 1:1 ratio of MDA-MB-231 or MCF-7 cells to
MSCs were plated in Boyden Chamber (Corning Inc, Corning, NY) wells with 0.4m pores that
allows cytokine and growth factor passage but prevents cell movement.
Adhesion and invasion assays: Adhesion assays were performed on 96-well microtiter plates
coated with 10 µg/ml Matrigel. Cells were trypsinized and re-suspended in DMEM with 1%
BSA, 1 mM MgCl2, 0.5 mM CaCl2 at a concentration of 1x 106 cells/ml. 1x105 cells (100 µl)
were added into each well and placed for 30 min at 37°C in 5% CO2 humidified air incubation.
Non-adherent cells were removed by gently washing the wells three times with phosphatebuffered saline (PBS) with 1 mM MgCl2 and 0.5 mM CaCl2. Adherent cells were fixed with
3.7% paraformaldehyde for 10 min at room temperature, followed by rinsing with PBS, and
stained with 0.4% crystal violet for 10 min. After extensive rinsing, the dye was released from
the cells by addition of 30% acetic acid, and the microtiter plates were read in a microplate
reader (Molecular Devices, Berkeley, California) at 590 nm. The invasion assay was carried out
in a Boyden Chamber system (Corning, NY). Cells were seeded at a density of 105 cells per well
in triplicate in the upper chamber of 12 well transwells (8 µm pore). The cells were fixed in 3.7%
paraformaldehyde in phosphate-buffered saline for 10 min. The cells on the top surface of the
filters were wiped off with cotton swabs. Following three wash with PBS, the filters were stained
with 0.4% crystal violet for 10 min, and the dye was detected as described for the in vitro
adhesion assay.
Quantitative Real-time PCR: Quantitative Real-time polymerase chain reaction (qRT-PCR) was
performed with the two-step reaction protocol using iQ SYBR Green detection kit (Bio-Rad
Laboratories, Hercules, CA). First-strand cDNA were synthesized from 1.0 μg of total RNA
using the iScript Select cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). The
following reaction protocol was used: 25◦C for 5 minutes, 42◦C for 30 minutes, and 85◦C for 5
minutes. The qRT-PCR parameters used were: 95◦C for 3 minutes; 95◦C for 30 seconds, 55◦C
for 35 seconds for 40 cycles; 95◦C for 1 minute, and 55◦C for 10 minutes, and the primer sets are
listed below. PCR was performed with iQ SYBR Green super mix, using the iCycler iQ Realtime PCR Detection System (Bio-Rad Laboratories, Hercules, CA). -actin was used as the
endogenous control. The ΔΔCT values were calculated after -actin normalization.
Quantitative Real-time PCR Primer Sequences:
SMA
PCR product length: 166bp
5’-TAGCACCCAGCACCATGAAGAT-3’
5’-GAAGCATTTGCGGTGGACAATG-3’
Vimentin
PCR product length: 200bp
5’-AGAACGTGCAGGAGGCAGAAGAAT-3’
5’-TTCCATTTCACGCATCTGGCGTTC-3’
Tenascin-C
PCR product length: 119bp
5’-AGCATCACCCTGGAATGGAGGA-3’
5’-TGTGGCTTGTTGGCTCTTTGGA-3’
FSP-1
PCR product length: 188bp
5’-TGTCCTGCATCGCCATGATGTGTA-3’
5’-TGAACTTGCTCAGCATCAAGCACG-3’
TGF-1
PCR product length: 405bp
5’-TGGCGATACCTCAGCAACC-3’
5’-CTCGTGGATCCACTTCCAG-3’
MZF1
PCR product length: 137bp
5’-AGTGTAAGCCCTCACCTCC-3’
5’-GGGTCCTGTTCACTCCTCAG-3’
-actin
PCR product length: 135bp
5’-AGAGGGAAATCGTGCGTGAC-3’
5’-CAATAGTGATGACCTGGCCGT-3’
Enzyme-linked immunosorbent assay: All TGF- enzyme-linked immunosorbent assay kits were
used according to manufacturer guidelines (R&D Systems, Minneapolis, MN). Cells were grown
in serum free media as fetal bovine serum contains TGF-. Cell culture supernatants were
collected and analyzed for either active TGF-1, 2, and 3 or total TGF-1, 2, and 3. The
measurement of the active form required no manipulation of samples, whereas the measurement
of the total protein required acidification using 1 N HCl followed by neutralization with 1.2 N
NaOH/0.5 M HEPES prior to assay.
Construction of promoter plasmids: Human genomic DNA was isolated from a normal human
lung fibroblast cell line (ATCC CCL-210) and the human TGF-1 promoter region was cloned
using polymerase chain reaction (PCR). The promoter region was defined using the NCBI
Reference Sequence NM_000660.4. The full-length promoter (-3360/+124) and five deletion
constructs (-1196/+124; -691/+124; -624/+124; -551/+124; -220/+124) were generated, setting
the ATG start site as 0. All promoter fragments were then cloned into the pGL4.10(luc2) vector
(Promega, Madison, WI) to generate luciferase reporter constructs.
Construction of promoter mutations: Point mutations of the putative binding site of MZF1 on the
TGF-1 promoter were made using the QuikChange II Site Directed Mutagenesis Kit (Agilent
Technologies, Santa Clara, CA) according to manufacturer guidelines. These mutated promoter
fragments were also cloned into pGL4.10(luc2) vectors and their sequence was verified with
direct sequencing analysis.
Dual luciferase reporter assay: To measure promoter activity, 4 ug of each of the luciferase
reporter constructs or 4 ug of empty pGL4.10(luc2) vector were co-transfected with 0.4 ug of
Renilla into MSCs. Six hours after transfection, cells were exposed to OPN for 48 hours. Using a
Dual-Luciferase assay system (Promega, Madison, WI), the cells were passively lysed and the
Firefly luciferase and Renilla luciferase activity was measured in each cell lysate using the
Modulus Luminometer (Turner Biosystems Inc, Sunnyvale, CA). The reporter activity of each
construct is shown as a ratio of Firely:Renilla luciferase activity.
ChIP RT-PCR: For both ChIP assays, cells were treated with recombinant human OPN and
cultured for 48 hours prior to DNA cross-linking and lysis. Chromatin was fixed and
immunoprecipitated using the EZ-ChIP assay kit (EMD Millipore) as recommended by the
manufacturer. For the RNA Polymerase II ChIP assay, purified chromatin was
immunoprecipitated using 1 ug of RNA Polymerase Ab or 1 ug normal mouse IgG (negative
control). For the MZF1 ChIP assay, purified chromatin was immunoprecipitated using 1 ug of
MZF1 Ab, 1 ug of RNA Polymerase Ab (positive control), or 1 ug normal rabbit IgG (negative
control). The DNA was sonicated using previously determined settings to 500bp fragments.
After DNA purification, for the RNA Polymerase II ChIP assay, qRT-PCR was used to
determine the presence of RNA polymerase II bound to the TGF-1 TATA box and the final
TGF-1 exon. For the MZF1 ChIP assay, qRT-PCR was used to determine the presence of
MZF1 bound to the putative binding site for MZF1 on the TGF-1 promoter. All CT values were
normalized to -actin to calculate a CT value. The products from qRT-PCR were then run on
agarose gel to visualize bands. Primers used for qRT-PCR for ChIP assays are as follows:
TATA box:
F 5’-CCCAGCCTGACTCTCCTTCCGTTC-3’
R 5’-GGTGATCCAGATGCGCTGTGGCTT-3’
Final exon:
F 5’-GGTGGAGCAGCTGTCCAACATGAT-3’
R 5’-GCACGGGTGTCCTTAAATACAGCC-3’
MZF1:
F 5’-GAGCTCGCCCCAGAGTCTGAG-3’
R 5’-CGCGCCTCCGGCTCGCAGCGG-3’
Zymography: Gelatin zymography was carried out as described by Novex Zymogram Gel
manual (Life Technologies). Briefly, the concentrated serum-free MEM collected from MSC
cultures was mixed with Novex Tris-Glycine SDS sample buffer and directly loaded on 10%
Gelatin Novex Zymogram Gel. After running for 1.5 h, the gel was incubated in Zymogram
renaturing buffer for 30 min at room temperature with gentle agitation. After incubating the gel
with fresh Zymogram developing buffer at 37°C overnight and staining the gel with Colloidal
Blue Staining kit (Invitrogen), images were acquired by AlphaImager 3400 (Protein Simple,
Santa Clara, CA).
Mouse xenograft model: Animal handling and procedures were approved by the Loyola
University Animal Care and Use Committee. Six-week-old female NOD-scid mice were
purchased from the Jackson Laboratory (Bar Harbor, Maine). The 106 MB231, or MCF7 RFPluciferase-expressing cells and/or 106 GFP-labeled MSCs were suspended in PBS and implanted
into the R4 positions of the mice mammary fat pad (four per group). APT (10 mg/kg), or
MuAPT (10 mg/kg) were injected, respectively, into the mouse tail vein every 2 days following
tumor cell implantation. The mice were anesthetized with intraperitoneal ketamine (75 mg/kg)
and xylazine (10 mg/kg). For bioluminescence imaging, animals were placed in a light-tight
chamber in which grayscale reference images were obtained under dim conditions. A
pseudocolor image acquired in the dark was superimposed on the grayscale image to represent
photons emitted from tumors. Bioluminescence is reported as the sum of detected photons per
second from a constant region of interest (photons/second/region of interest). Ten minutes after
administration of luciferase substrate (D-luciferin, 150 mg/kg), anesthetized mice were imaged
with the IVIS 100 Imaging System (PerkinElmer, Waltham, MA). Initial in vivo images at day 2
were obtained to establish baseline tumor volume as measured by photon emission. For ex vivo
imaging, D-luciferin (150 mg/kg) was injected into the mice before necropsy. Primary tumor,
lung lobes and liver were excised and placed into tissue culture plates with D-luciferin (300
μg/ml) in PBS. For fluorescence optical imaging, the small animal imaging System 200 (Caliper
Lifesciences, Hopkinton, MA) containing an MT-20 light source and GFP filter was used. Highresolution GFP images were captured. Images were analyzed with the use of software
AxioVision LE (Carl Zeiss MicroImaging, Thornwood, NY) After imaging, tissues were either
kept in −80°C freezer for RNA and protein analysis, in 1% formalin solution for
immunohistochemical analysis or immediately prepared for flow cytometry cell sorting.
Fluorescence activated cell sorting: Fresh primary tumor, lung or liver tissue was obtained.
Single cell suspension was prepared as reported by Keng et al. The tissues were finely minced
with surgical scissors and transferred to 10 ml collagenase–PBS solution (1 × PBS, PH7.4;
0.025% collagenase, 0.05% pronase and 0.04% DNAse). After 1 h incubation at 37°C, the tissue
pellets were centrifuged at 1000 rpm for 10 min at 4°C and washed three times with 5 ml PBS.
The tissue homogenate was gently passed through 70 um pore nylon mesh filter at 4°C. Cells
were sorted using BD FACSAriaIII (BD Biosciences, Franklin Lakes, NJ). For GFP, cells were
excited using a 488 nm laser, with emission collected through a 530/30 bandpass filter. For RFP,
cells were excited using a 561 nm laser, with emission collected through a 610/20 bandpass
filter.
GFP-positive cells and RFP-positive cells were collected in PBS and stored at −80°C.
Immunohistochemistry: Human breast cancer tissue samples (IRB approved) formalin
fixed, paraffin embedded were sectioned to 5m thickness and mounted on Starfrost/Plus
slides. Slides were hydrated through a Xylene and alcohol gradient. Slides were rinsed in
distilled water followed by antigen unmasking utilizing Leica ER2 (Leica Biosystems,
Richmond, IL) concentrated EDTA retrieval solution at pH 8.9, and then rinsed in Leica
wash buffer. Tissues were stained on Leica Bond with OPN, TGF-1, MZF1 and SMA
antibodies using modified Protocol F for 60 minutes at room temperature. Staining was
detected using Leica refine DAB kit. Slides were rinsed in distilled water, counterstained
and dehydrated through an alcohol gradient and mounted with micromount.
Statistical Analysis: All experiments were done in triplicate. Data are presented as mean  SD.
Student’s t-tests and ANOVA were used for analysis. Values of p<0.05 were considered
significant.