Supplementary Data
Supplementary methods and materials
Differentiation Assays
The adherent cells were trypsinized and replated in six-well plates for differentiation assays in
vitro according to the following culture conditions.
Osteogenesis. MSCs were seeded in six-well plates at 3,000 cells per square centimeter. After 2
days, the normal growth medium was replaced with osteogenic medium containing 100 nM
dexamethasone, 50μM ascorbate-2-phosphate, and 10 mM β-glycerol phosphate (Sigma-Aldrich).
The osteogenic medium was replaced twice weekly. After 4 weeks, the medium was aspirated and
the cells were examined by Alirazin Red S stain for calcium deposition in the differentiated cells34.
Adipogenesis. MSCs were plated in six-well plates at 10,000 cells/ml. Adipogenic medium
contained 0.5 mM isobutyl-methylxanthine (IBMX), 1 μM dexamethasone, 10 μM insulin, and
200 μM indomethacin (Sigma-Aldrich). After 2 weeks, adipogenic characterized neutral lipid
deposits cells and the cells were stained with 0.5% Oil Red O for microscopic evaluation34.
Flow cytometry
hGC-MSCs (1.0x106 cells) were trypsinized, washed twice with PBS and immunostained for 30
min on ice with monoclonal antibodies against CD14, CD19, CD29,CD34, CD44, CD45， CD90,
CD105,
and
CD133
(FITC-conjugated)
(Becton
Dickinson,
San
Jose,
CA,
www.bdbiosciences.com). The labeled cells were analyzed using a flow cytometer (FACS Calibur,
Becton Dickinson). Background signals were established using control cells incubated with
isotype-specific IgGs.
Electron Microscopy.
For electron microscopy, the cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate
buffer, fixed in 0.5% osmium tetroxide, and dried using graded ethanol. The samples were cut into
500 nm sections that were embedded in epoxyresin and then analyzed (JEM-2000EX, JEOL,
Tokyo, Japan). For scanning electron microscopy (SEM) analyses the samples were coated with
gold and viewed on an S-3400N scanning electron microscope （EOL, Tokyo, Japan）.
Western blot analysis
The cells were washed twice with PBS and lysed directly in radio-immunoprecipitation assay
buffer (50 mM Tris–HCl (pH 7.4), 1% (v/v) Triton X-100, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 10 mM NaF, and 1 mM Na3VO4). The lysates were centrifuged at
12,000 rpm for 10 min at 4°C and the supernatants were collected.
Thirty micrograms of protein were loaded onto an 8% SDS-PAGE gel and subjected to
electrophoresis at 20 mA for 80 min under denaturing conditions and then transferred to
nitrocellulose. The membrane was blocked using 5% non-fat dry milk in PBS for 2 hours, and
probed
using
the
following
(1:1000,Sigma-Aldrich);
Twist
primary
antibodies:
(H-81);
Snail
vimentin
(E-18);
(A2547);α-SMA (V5255)
E-cadherin
(G-10);
N-cadherin
(D-4);β-Catenin (H-102) (1:100, SantaCruz, CA, UAS. www.scbt.com); MMP16 (ZA-0266, 1:100,
BA1280，Boster, Wuhang, China. www.boster.com.cn). After repeated washing, the membranes
were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary
antibodies (Sigma-Aldrich) that were diluted 1:5000. The proteins were detected using the
enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Arlington Heights,
IL, USA. www.gelifesciences.com).
Immunofluorescence Staining
Immunofluorescence analysis. The cells were plated on sterilized coverslips in 24-well plates. The
cells were fixed in 4% paraformaldehyde (room temperature, 10 min), washed with PBS, treated
with 0.25% Triton for 15 minutes, blocked with 10% goat serum for 30 minutes, and then
incubated with the appropriate primary antibody overnight and thawed for 1 hour at RT. Cells
were immunostained for E-Cadherin (1:50), β-catenin (1:100),α-SMA (1:250), or vimentin (1:200).
Secondary antibodies (1:400) included donkey anti-mouse IgG-Cy3 and anti-mouse IgM-FITC
(Molecular Probes, Oregon, USA. www.invitrogen.com). After several more washes, the cells
were counterstained with DAPI nuclear dye (Sigma) and examined using a fluorescence
microscope. The mean intensity fluorescence evaluated by Image-pro Plus (SUM (IOD) /
SUM(area) ). IOD=integrated optical density
Real-time PCR
Total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions
(Invitrogen). Reverse transcription (RT) reactions were performed using 2 ug of total cellular RNA
using the PrimeScript RT reagent kit and SYBR premix Ex Taq (Takara, Dalian, China.
www.takara.com.cn) was used to detect the target genes. RT-PCR amplification was performed as
follows: an initial denaturation for 2 min at 95°C, followed by 45 cycles of PCR (95°C for 15 s,
56°C for 20 s, and 72°C 15 s). The housekeeping gene, GAPDH, was used as the control gene.
Primers:
Gene name
S100A4
vimentin
β-catenin
MMP-16
GAPDH
CXCR4
SDF-1
5'-3'
upstream
downstream
upstream
downstream
upstream
downstream
upstream
downstream
upstream
downstream
upstream
downstream
upstream
downstream
GGGTGACAAGTTCAAGCTCAACAA
ATCATGGCGATGCAGGACAG
TGAGTACCGGAGACAGGTGCAG
TAGCAGCTTCAACGGCAAAGTTC
CACGTGCAATCCCTGAACTGA
CGCATGATAGCGTGTCTGGAA
TGATGGACCAACAGACAGA
GCAGGGAATGACAATAGC
GCACCGTCAAGGCTGAGAAC
TGGTGAAGACGCCAGTGGA
AATGGGCTCAGGGGACTA
GATGGTGGGCAGGAAGAT
TGGCTGTTGTGCTTACTTG
CTGTGCCCTTCAGATTGT
PKH26 staining
According to the PKH26 guideline, a single cell suspension of 1x107 MKN28 cells or MSCs was
mixed with 2x10-6 molar dye and incubated at 25 °C for 2 to 5 minutes. The staining process was
stopped by adding an equal volume of serum and washed two times. Finally, the stained cells were
resuspended in PBS ande injected into nude mice.
Gene Expression Profiling
Cells were harvested after exposure to tumor cell CM, bone marrow or control medium, and RNA
was isolated using the Invitrogen RNA isolation kit. Total RNA was processed for microarray
analysis after verifying the RNA quality at the DNA microarray core facility at the Cancer
Institute of New Jersey/RWJMS. Briefly, the RNA was reverse-transcribed and hybridized to
Affymetrix Human Genome U133 Plus 2.0 Arrays comprised of more than 47,000 transcripts
(Affymetrix, Santa Clara, CA, USA. www.affymetrix.com). Comparative analyses of the
expressed genes were performed using KEGG (Kyoto Encyclopedia of Genes and Genomes) and
MAS (Bioassay Laboratory of CapitalBio Corporation, China).
Immunohistochemical Analysis
The microarray assay was explored using a gastric cancer-specific tissue microarray consisting of
192 cases (Fanpu-bio, Guilin, China. http://www.fanpu.com) (Approved by the ethical committee
of Xijing Hospital No: 2010-8-H102). Full clinical history (age, sex, tumor size, grade, TNM
stage, and 57 cases of survival data) was provided. For immunohistochemical detection, antibody
binding was visualized using 3,3’-diaminobenzidine (DAB) (ZhongShan Gold-bridge, Beijing,
China, www.zsbio.com). Endogenous peroxidase activity was blocked using 3% H2O2 (10 min at
RT). After washing once with water and twice with PBS (pH 7.4), the primary antibodies were
incubated overnight at 4℃. The normal gastric and intestinal mucosa were used as positive
controls, and PBS was used as negative control. After washing three times with PBS, the sections
were incubated with PV-6000-G (ZhongShan Gold-bridge; China) for 30 min at RT. Two
independent pathologists judged the histology and immunohistochemistry.
Supplement figures and table
Supplementary Figure 1.
Identification of MSCs using a surface marker assay and a differentiation assay. (A) FACS
analysis of MSCs surface makers and typical hematopoietic and endothelial markers. (B)
Histochemical staining of adipocytes (Oil Red O, B), osteocytes (Alizarin Red, C) and myogenic
cells with long myotube-like structure (Arrowhead, D) using phase-contrast microscopy. Scale,
200μm.
Supplementary Figure 2.
Representative images of the co-culture system. Different cancer cells were co-cultured with
MSCs (ratio 1:10) to assay the dispersion and phenotype changes. There were no changes
observed in the NIH3T3 cells grown with MKN28 (A, Asterisk). MCF-7 cells were dispersed on
the surface of MSCs (G, arrow). Scale, 200μm.
Supplementary Figure 3.
MCF-7 cells mixed with MSCs exhibited higher expression levels of vimentin and β-catenin were
translocated into the nucleus (Asterisk, MSCs). Scale, 200μm.
Supplementary Figure 4.
MKN28 mixed with MSCs induced β-catenin nuclear translocation. Scale, 200μm.
Supplementary Figure 5.
MKN45 displayed nuclear-translocated β-catenin and the loss of the E-cadherin membrane marker
in MSCs and MKN28 co-culture conditioned medium. (Magnification, ×200)