TISSUE-SPECIFIC STEM CELLS

Human Bone Marrow-Derived Mesenchymal Stem Cells Suppress

Human Glioma Growth Through Inhibition of Angiogenesis
IVY A.W. HO,a HAN C TOH,b WAI H. NG,c YUAN L. TEO,a,d CHANG M. GUO,e KAM M. HUI,f,h PAULA Y.P. LAMa,g,h
a
Laboratory of Cancer Gene Therapy; bLaboratory of Cell Therapy and Cancer Vaccine, and fBek Chai Heah
Laboratory of Cancer Genomics, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore;
c
Department of Neurosurgery, National Neuroscience Institute, Singapore; dNational Institute of Education,
Singapore; eDepartment of Orthopedic, Singapore General Hospital, Singapore; gDepartment of Physiology,
National University of Singapore, Singapore; hCancer and Stem Cells Biology Program, Duke-NUS Graduate
Medical School, Singapore

Key Words. Angiogenesis • Mesenchymal stem cells • Glioma • Platelet derived growth factor • Interleukin-1b

ABSTRACT
Tumor tropism of human bone marrow-derived mesenchy-
mal stem cells (MSC) has been exploited for the delivery of
therapeutic genes for anticancer therapy. However, the
exact contribution of these cells in the tumor microenviron-
ment remains unknown. In this study, we examined the bio-
logical effect of MSC on tumor cells. The results showed
that MSC inhibited the growth of human glioma cell lines
and patient-derived primary glioma cells in vitro. Coadmi-
nistration of MSC and glioma cells resulted in significant
reduction in tumor volume and vascular density, which was
not observed when glioma was injected with immortalized
normal human astrocytes. Using endothelial progenitor
cells (EPC) from healthy donors and HUVEC endothelial
cells, the extent of EPC recruitment and capacity to form
endothelial tubes was significantly impaired in conditioned
media derived from MSC/glioma coculture, suggesting that
Disclosure of potential conflicts of interest is found at the end of this article.
MSC suppressed tumor angiogenesis through the release of
antiangiogenic factors. Further studies using antibody
array showed reduced expression of platelet-derived growth
factor (PDGF)-BB and interleukin (IL)-1b in MSC/glioma
coculture when compared with controls. In MSC/glioma
coculture, PDGF-BB mRNA and the corresponding pro-
teins (soluble and membrane bound forms) as well as the
receptors were found to be significantly downregulated
when compared with that of glioma cocultured with normal
human astrocytes or glioma monoculture. Furthermore,
IL-1b, phosphorylated Akt, and cathepsin B proteins were
also reduced in MSC/glioma. Taken together, these data
indicated that the antitumor effect of MSC may be medi-
ated through downregulation of PDGF/PDGFR axis, which
is known to play a key role in glioma angiogenesis. STEM
CELLS 2013;31:146–155

INTRODUCTION
Human mesenchymal stem cells (MSC) are a population of
nonhematopoeitic stem cells in the bone marrow microenvir-
onment that have the ability to self-renew and differentiate
into multiple lineages. MSC are increasingly being developed
for potential clinical usage due to the ease of isolation from
adult donors, thus obviating the ethical issues that plague
embryonic stem cell research [1]. Because of its strong che-
motactic response to injury, MSC have emerged as an attrac-
tive homing vehicle for the delivery of therapeutic genes to
tumors and diseased tissues. MSC secrete a variety of cyto-
kines that have both paracrine and autocrine functions in the
tumor milieu. These include suppression of local immune
response, inhibition of fibrosis and apoptosis, modulation of
angiogenesis, and stimulation of mitosis and differentiation of
cells [2]. MSC could also cause a direct effect through inter-
cellular signaling via physical contacts with neighboring cells,
which may consist of tumor cells, stromal fibroblast, infiltrat-
ing immune cells, blood, and lymphatic networks that ulti-
mately determine the fate of the tumor growth kinetics.
Angiogenesis, the formation of new blood vessels from
pre-existing vessels, is essential for normal development and
maintenance of homeostasis. Tumor angiogenesis is also cru-
cial for tumor growth; a tumor cannot grow beyond the size
of 2–3 mm3 without blood supply [3]. Tumor angiogenesis
could arise from the recruitment of existing endothelial cells
(EC) or endothelial progenitor cells (EPC) in response to
proangiogenic factors. In the latter example, vascular endothe-
lial growth factor (VEGF) [4], fibroblast growth factor (FGF)
[4], granulocyte macrophage-colony stimulating factor [5],
platelet derived growth factor (PDGF) [6], hypoxia-induced
macrophage migration inhibitory factor [7] have been shown

Author contributions: I.A.W.H.: conception and design, data analysis and interpretation, and manuscript writing; H.C.T., C.M.G., and
W.H.N: provision of study material; Y.L.T.: collection and assembly of data; K.M.H.: administrative support; P.Y.P.L.: conception and
design, data analysis and interpretation, manuscript writing, financial support, and final approval of manuscript.
Correspondence: Paula Y.P. Lam, Ph.D., Laboratory of Cancer Gene Therapy, Division of Cellular and Molecular Research, Humphrey
Oei Institute of Cancer Research, National Cancer Centre, 11, Hospital Drive, Singapore 169610, Singapore. Telephone: 65-64368357;
Fax: 65-62265694; e-mail: cmrlyp@nccs.com.sg Received February 2, 2012; accepted for publication August 1, 2012; first published
online in STEM CELLS EXPRESS October 3, 2012. V
C AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.1247

STEM CELLS 2013;31:146–155 www.StemCells.com

Ho, Toh, Ng et al.
to mobilize EPC. Alternatively, cytokine-stimulated MSC [8]
and MSC that have undergone transdifferentiation into EC or
smooth muscles cells [9–11] have been shown to participate
in tumor angiogenesis and growth.
Having said this, MSC are known for their unique biologi-
cal property of expressing low levels of major histocompatibil-
ity complex antigens, which may contribute to their low immu-
nogenicity and anti-inflammation effect when introduced
exogenously [2]. Using chemically burned corneas in rat as a
model, Oh et al. [12] showed that MSC mediated an anti-
inflammatory and antiangiogenic effects on the injured corneal
surface. Secchiero et al. [13] demonstrated that intraperitoneal
injection of MSC improved the survival of lymphoma-bearing
mice through induction of EC apoptosis. Murine MSC was
observed to intercalate into the capillary networks of EC and
caused capillary degeneration in a MSC/EC coculture system in
vitro and abrogated melanoma growth in vivo [14]. More
recently, the antitumor effect of cord blood-derived MSC was
also demonstrated by Dasari et al. [15] in a glioma tumor
model. Given the numerous conflicting reports on the ability of
MSC to support tumor growth, it is important to further explore
the role of MSC in the tumor microenvironment.
In this study, we demonstrated that MSC elicited an antitu-
mor effect in patient-derived primary glioma cells as well as in
human glioma xenograft model. The antitumor effect is medi-
ated through the secretion of soluble factors that inhibit EPC
recruitment and impaired tumor angiogenesis. Using antibody
array, we have identified and subsequently confirmed that the
antiangiogenic effect of MSC might be mediated through the
downregulation of PDGF/PDGF Receptor (PDGFR) axis,
which is known to play a key role in glioma angiogenesis
MATERIALS AND METHODS
Cell Culture
Human MSC were isolated, cultured, and characterized as previ-
ously described [16]. The culturing of human glioma cells
DGli36 was described previously [16]. Normal human astrocytes
(NHA) was purchased from Lonza (Basel, Switzerland, http://
www.lonza.com) and cultured in astrocyte basal medium (ABM)
supplemented with recombinant human EGF, insulin, ascorbic
acid, gentamycin sulfate, amphotericin, L-glutamine, and fetal bo-
vine serum (FBS) as recommended by the supplier. Immortalized
NHAs that overexpress E6, E7, and human telomerase reverse
transcriptase (hTERT) were kindly provided by R.O. Pieper (Uni-
versity of California, San Francisco, CA). Human ECs, Human
umbilical vein endothelial cells (HUVEC), was purchased from
Lonza and cultured in endothelial growth medium supplemented
with recombinant epidermal growth factor (EGF), hydrocortisone,
VEGF, basic FGF, insulin growth factor (IGF), heparin, ascorbic
acid, gentamycin sulfate, amphotericin-B, and 2% FBS.
Isolation of Primary Human Glioma Cells
This study has been approved by the SingHealth Centralized
Institutional Review Board, Singapore. Primary human glioma
cells were isolated, after informed consent, from the brain tumor
tissues of patients undergoing brain tumor surgery at the National
Neuroscience Institute, Singapore. The harvested tissue was sepa-
rated into small pieces in the presence of complete medium
(ABM) supplemented with 10% FBS, penicillin/streptomycin,
normocin, and L-glucose (Cambrex Bio Science Walkersville,
Inc., Walkersville, MD, http://www.cambrex.com). The tissue
suspensions were first passed through a 5 ml serological pipette,
followed by a 1 ml pipette and finally a flame-polished pasteur
pipette until no clumps were visible. Following trypsin digestion,
the homogenate was filtered through a 70-lm cell strainer (BD
www.StemCells.com
147
Biosciences, San Jose, CA, http://www.bdbiosciences.com) and
then subjected to centrifugation. The collected cells were cultured
in complete ABM.
Isolation and Characterization of EPC
EPC was isolated according to Brunt et al. [17]. Briefly, periph-
eral blood mononuclear cells were isolated by centrifugation
(400g for 20 minutes) in the presence of Histopaque-1077 (1.077
g/ml, Sigma, http://www.sigma-aldrich.com). After centrifugation,
the buffy coat was isolated, rinsed twice with phosphate buffered
saline (PBS), and resuspended in endothelial growth medium
(EGM-2MV) containing 5% serum, VEGF, FGF-2, EGF, IGF,
hydrocortisone, and ascorbic acid. Cells were cultured at 37
C
with 5% CO2 in a humidified incubator for 3 days. Nonadherent
cells were removed after 3 days, and adherent cells were cultured
for a further 10–20 days until colonies appeared.
EPC was characterized by the expression of CD133, CD34,
Tie2, vascular endothelial (VE)-cadherin (CD144), VEGF recep-
tor (VEGFR)-2 and for the uptake of acetylated-low density lipo-
protein (Ac-LDL) [17, 18]. Briefly, DiI-labeled acetylated-LDL
(DiI-Ac-LDL; 10 lg/ml; Invitrogen, Carlsbad, CA, http://www.
invitrogen.com) was added to live cells and incubated at 37
C for
1 hour. Cells were rinsed twice with PBS to remove excess dye.
The number of DiI-Ac-LDL positive cells was counted under a
wide-field fluorescence microscope equipped with a CCD camera
(Nikon Eclipse 90i; Nikon, Tokyo, Japan, http://www.nikon.com).
Flow Cytometry
For quantification of the glioma cells in the coculture experi-
ments, carboxyfluorescein diacetate N-succinimidyl ester (CFDA-
SE)-labeled-DGli36 glioma cells were coincubated with carboxy-
methyl-DiI-labeled MSC for 48 hours. Labeling of the cells was
described previously [16]. Cells were detached with 0.25% tryp-
sin/EDTA and washed twice with PBS. Cell pellet was then
resuspended in 1 ml of PBS. Fluorescence-activated cell sorting
analysis was performed using Cell Quest.
In Vivo Tumor Model
To establish the subcutaneous tumor model for monitoring the effect
of MSC on glioma growth, immunodeficient BALB/c-nu/nu mice
(Animal Resource Center, Canningvale, Western Australia, http://
www.arc.wa.gov.au) were inoculated with equal ratio of DGli36 and
carboxymethyl-DiI-labeled-MSC in the presence of Matrigel (BD
Biosciences). Tumor volume was calculated using the formula: tu-
mor volume (mm3) ¼ 0.52  (width [mm2]  (length [mm]) [19].
All animal experiments were performed according to the guidelines
and protocols approved by the Institutional Animal Care and Use
Committee at the Singapore General Hospital, Singapore.
Immunofluorescence and Immunohistochemistry
Staining
Slides were visualized using wide-field microscopy using an
upright microscope (Eclipse 90i; Nikon), and images were
acquired on a CCD color digital camera (DS-U2; Nikon) using
NIS Element AR v2.3 software (Nikon). To assess the cell local-
ization with higher resolution, images were obtained with a con-
focal system (LSM 510 Meta; Carl Zeiss, G€ottingen, Germany,
http://www.zeiss.com). Images were obtained using either a 40/
0.75 numerical aperture (N.A.) Plan-Neofluar or a 63/1.25 N.A.
Plan-Neofluar oil immersion objective (Carl Zeiss). The following
antibodies were used for the various staining: CD31 (1 lg/ml;
BD Biosciences), cathepsin B (5 lg/ml; R&D Systems, Minneap-
olis, MN, http://www.rndsystems.com), single-stranded DNA
(ssDNA; 1 lg/ml; clone F7-26, Millipore, Temecula, CA, http://
www.millipore.com), a-smooth muscle actin (SMA; 1:100 dilu-
tion; Sigma Aldrich, St. Louis, MO, http://www.sigmaaldrich.
com), CD34 (1 lg/ml), CD133 (1:100 dilution), Tie2 (1 lg/ml),
VE-cadherin (1 lg/ml) and VEGFR-2 (1 lg/ml) (Santa Cruz Bio-
technology, Santa Cruz, CA, http://www.scbt.com), and CD45
(1:100 dilution; BD Biosciences). Mouse IgG1 (Dako Denmark
148 MSC Restrict Glioma Angiogenesis

Figure 1. Coculture of MSC with glioma cell lines or patient-derived glioma cells induced tumor cell death in vitro. (A): Equal ratio of
CFDA-SE-labeled-DGli36 glioma cells were cocultured with either carboxymethyl-DiI-labeled-MSC or carboxymethyl-DiI-labeled-NHA. Flow
cytometry FL1-H and FL2-H analysis of the DGli36/MSC coculture demonstrating the separation of the two populations by color. Total number
of CFDA-SE-labeled-DGli36 human glioma cells was counted using fluorescence-activated cell sorting analysis after 48 hours. (B): Equal ratio
of patient-derived glioma cells NNI32 were cocultured with either carboxymethyl-DiI-labeled-MSC (MSC-1 and MSC-8) or carboxymethyl-DiI-
labeled-iNHA . Images were captured from 10 random fields under 100 original magnification using Nikon TE300 wide-field microscope
equipped with a CCD camera after 48 hours. Total number of NNI32 glioma cells was counted using NIS Elements version 3.0. Data shown are
averages of triplicates 6 SEM. Abbreviations: CFDA-SE, carboxyfluorescein diacetate N-succinimidyl ester; iNHA, immortalized NHA; MSC,
mesenchymal stem cell; NHA, normal human astrocytes. **, p < 0.01; ***, p < 0.0001.

A/S, Glostrup, Denmark, http://www.dakocytomation.com) and
rabbit serum (Dako Denmark A/S) were used as isotype control.
Mice were sacrificed 21 days post-tumor implantation and
immediately perfused through the heart with ice-cold saline.
Tumors were harvested and kept in 4% paraformaldehyde (PFA)
for 4 hours at 4
C, transferred to 30% sucrose in PBS overnight
at 4
C, and embedded in Jung Tissue Freezing Medium (Leica
Microsystems Nussloch, Heidelberg, Germany, http://www.leica-
microsystems.com), snap-frozen in isopentane immersed in liquid
nitrogen, and stored at 80
C.
For immunofluorescence staining, frozen sections were fixed
with ice-cold acetone or 4% PFA, blocked for 30 minutes with
10% goat serum, and incubated for 1 hour at room temperature
with primary antibodies. To assess the pericyte coverage of
microvessels, tumor sections were double-stained for a-SMA
expression to detect pericytes and CD31 (1 lg/ml) to detect ECs.
To determine the microvessels density (MVD), the number of
CD31þ cells was counted. To express MVD counts microscope-
independent, counts were transformed and expressed as the num-
ber of microvessels per mm2.
For immunohistochemistry staining, following incubation
with primary antibodies, sections were stained with goat anti-
mouse or anti-rabbit polymer, followed by detection using 3,30
diaminobenzidine substrate solution (Dako Denmark A/S). Anti-
body detection in sections stained with mouse monoclonal anti-
bodies was performed using the Zenon mouse IgG labeling kit
(Invitrogen). Sections were then washed and coverslipped in
mounting medium. Parallel samples were incubated with no pri-
mary antibody or an isotype as a specificity control.
Immunoblot Analysis
Tumor lysates (DGli36, DGli36/iNHA, and DGli36/MSC) (50 lg)
were harvested for immunoblot analysis. The following antibodies
were used: total and activated PDGFR-b (1:1,000 dilution; Cell Sig-
naling Technology, Beverly, MA, http://www.cellsignal.com), total
and activated Akt (1:1,000 dilution; Cell Signaling Technology), b-
tubulin (1:5,000 dilution; Sigma Aldrich), and interleukin (IL)-1R1
(1:100 dilution; Epitomics, Burlingame, CA, http://www.epitomics.
com). Chemiluminescence detection was performed using Western
Lightning chemiluminescence kit (Perkin Elmer, Waltham, MA,
http://www.perkinelmer.com). The band intensity of specific proteins
was quantified after normalization with the density of b-tubulin with
MetaVue imaging software (version 6.1; Sunnyvale, CA).
Cytokine Array Analysis
RayBio Human Cytokine Array C2000 (RayBiotech, Inc., Nor-
cross, GA, http://www.raybiotech.com) that contains antibodies
targeting to 174 proteins were used to detect proteins that are dif-
ferentially expressed in DGli36/MSC-CM in comparison to that
of DGli36-CM and MSC-CM. One hundred microgram of condi-
tioned media (CM) was used according to manufacturer’s instruc-
tions. Signals were detected by chemiluminescence reaction and
quantified using Quantity-One software (Bio-Rad Laboratories,
Hercules, CA, http://www.bio-rad.com).
ECs Tube Formation Assay
Tube formation assay was performed according to manufacturer’s
instructions. Briefly, on the day of the assay, 10 ll of EC matrix
(Millipore) was added to the ibidi l-Slide angiogenesis dish (ibidi,
Munich, Germany, http://www.ibidi.de) and incubated for 1 hour
at 37
C with 5% CO2. During the incubation time, HUVEC was
harvested. The cells were counted and 50 ll of cells suspension
containing 5,000 cells was added to the wells containing the EC
matrix. The slides were then incubated at 37
C for 6 hours. The
tubes formed are visualized and captured using the Zeiss 200M
phase contrast microscope at 50 original magnification (Carl
Zeiss). Tube formation was quantified after 6 hours by measuring
the total tube length using ImageJ software (National Institutes of
Health, Bethesda, MD, http://rsb.info.nih.gov/ij/). For checking the
effect of the CM on HUVEC tube formation abilities, cells were
resuspended in the respective medium, namely, DGli36-CM, MSC-
CM, NHA-CM, DGli36/NHA-CM, and DGli36/MSC-CM.
Ho, Toh, Ng et al. 149

Figure 2. Antitumor effect of MSC in glioma/MSC coculture in vivo. (A): DGli36 cells (5  105), MSC (5  105), iNHA (5  105), DGli36/
iNHA (5  105:5  105), DGli36/MSC-1 (5  105:5  105), and DGli36/MSC-7 (5  105:5  105) were implanted into the flank of nude mice
in the presence of Matrigel. Tumor volume was monitored at various time points. Data shown are averages of seven mice 6 SEM. *, p < .05;
**, p < .01 (B): Tumor weight comparison between DGli36 and DGli36/MSC-1 tumors. (C): Photomicrograph of DGli36 and DGli36/MSC
tumors. H&E staining was performed on the tumor sections. Blue arrow indicated the presence of pockets of DGli36 tumor cells. Scale bar ¼
100 lm. (D): Photomicrograph of DGli36 tumor coinjected with carboxymethyl-DiI-labeled-MSC. Sections were counterstained with DAPI nu-
clear stain; red color indicates the presence of carboxymethyl-DiI-labeled-MSC. Photomicrograph showed composite image from three separate
images (original magnification 40, scale bar ¼ 250 lm.). I, inset, showed the localization of carboxymethyl-DiI-labeled-MSC (original magnifi-
cation 200; scale bar ¼ 100 lm). Images were captured using Nikon TE300 wide-field microscope equipped with a CCD camera. (E): Photomi-
crograph of tumor sections stained for the presence of apoptotic cells using anti-single-stranded DNA in DGli36, DGli36/MSC, and DGli36/iNHA
tumor. Sections were counterstained with DAPI nuclear stain (blue); green color indicates anti-single-stranded-DNA-positive cells. Sections were
shown at original magnification 200. Scale bar ¼ 50 lm. Abbreviations: H&E, hematoxylin and eosin; iNHA, immortalized NHA; MSC, mes-
enchymal stem cell; N, necrotic region; NHA, normal human astrocytes; T, tumor; DAPI, 40 ,6-diamidino-2-phynylindole.

Transwell EPC Recruitment Assay and IL-1b was quantified using QuantiTech SYBR Green PCR
4 kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). All
For determining the recruitment potential of EPC, EPC (1  10 )
qPCR reactions were performed in duplicate. Standard curves for
were cultured in EBM-2MV media with 0.5% FBS in a 24-well
each gene were generated independently. The relative copy num-
tissue culture insert with a 8-lm pore size membrane (BD Bio-
ber of each sample was calculated according to the corresponding
sciences). CM from DGli36 human glioma cells (DGli36-CM),
standard curve using RotorGene software version 6.0. The rela-
MSC (MSC-CM), or DGli36/MSC (DGli36/MSC-CM) coculture
tive expression levels were calculated by arbitrarily designating
were added to the bottom well. After 6 hours, the filter membrane
the lowest normalized value to 1. Quantification of PDGF-BB,
was fixed with 4% PFA, and cells were mounted in mounting
IGF-1, FGF-2, and IL-1b protein expression was performed using
medium containing propidium iodide (PI, 100 lg/ml; Sigma-
Quantikine human ELISA kit (R&D Systems) according to manu-
Aldrich) containing RNase A. Migration of EPC was determined
facturer’s suggestions.
by counting the number of PI-stained nuclei on the underside of
the membrane under 200 magnification.
Statistical Analysis
Quantitative Real Time Polymerase Chain Reaction Statistical analysis was performed using Prism 3.0 (Graphpad
and ELISA Software Inc., San Diego, CA, http://www.graphpad.com). One-
Quantitative real time polymerase chain reaction (qPCR) was per- way ANOVA followed by Bonferroni multiple comparisons test
formed as described previously [20]. Primers used are listed in were used for comparing statistical significance for more than
supporting information Table S1. Primers for the amplification of two groups. Two-way ANOVA was used for comparing statistical
PDGF-BB and IL-1b were purchased from Qiagen (QuantiTech significance for more than two factors. p value <.05 was consid-
Primer Assay). The expression level of PDGF-BB, IGF-1, FGF-2, ered statistically significant.
www.StemCells.com
150 MSC Restrict Glioma Angiogenesis

Figure 3. Reduction of angiogenesis in DGli36 cocultured with MSC. (A): Angiogenic vessels were identified by immunostaining using anti-
body against CD31 in DGli36, DGli36/MSC, and DGli36/iNHA tumor. Microvessel density of the tumor sections were determined by expressing
the cumulative CD31þ vessels to the area of the tumor sections. Data shown are averages of five randomly selected fields 6 SEM. *, p < .05;
**, p < .01. Scale bar ¼ 100 lm. (B): DGli36 and DGli36/MSC tumor sections were double immunostained with CD31 (green) and a-SMA
(blue). White arrow indicates colocalization of CD31 and a-SMA staining. Scale bar ¼ 20 lm. (C): Confocal images of DGli36/MSC tumor sec-
tions stained for the colocalization of CD31 (green), a-SMA (blue), and carboxymethyl-DiI-labeled MSC (red). Scale bar ¼ 20 lm. Abbrevia-
tions: iNHA, immortalized NHA; NHA, normal human astrocytes; MSC, mesenchymal stem cells; SMA, a-smooth muscle actin.

Fig. 2A). By contrast, there was no statistically significant dif-

RESULTS
MSC Induced Cell Death in Glioma Cells in an In
Vitro and In Vivo System of Coculture
To evaluate the influence of MSC on the growth of human
glioma cells, CFDA-SE-labeled MSC (green fluorescence)
was cocultured with human glioma DGli36 cells prelabeled
with carboxymethyl-DiI (red fluorescence) at equal ratio for
48 hours. Using flow cytometry analysis, we detected a
69.4% reduction in the number of glioma cells in DGli36/
MSC coculture in comparison to that of DGli36/NHA cocul-
ture (DGli36/NHA; Fig. 1A). Similar findings were observed
in primary glioma cells derived from human tumor biopsy
materials (denoted as NNI32 henceforth). In comparison to
NNI32/immortalized NHA (iNHA) coculture, the percentage
of NNI32 was greatly reduced by 52.3% and 56.6% when the
cells were cocultured with two independent MSC isolates,
MSC-1 and MSC-8, respectively (Fig. 1B), suggesting that
MSC exert an inhibitory effect on glioma cells.
Because in vitro coculture conditions are not representa-
tive of the tumor microenvironment, we performed in vivo
coculture experiment to monitor the effect of MSC on the
tumorigenicity of DGli36 glioma cells. For ease of monitoring
changes in the tumor volume, both cell types were implanted
in equal ratio subcutaneously. The tumor volumes and tumor
weights in mice injected with equal ratio of MSC and DGli36
were significantly smaller than control animals injected with
either DGli36 cells or DGli36/iNHA coculture (Fig. 2A, 2B,
respectively). This phenomenon was consistently observed in
two isolates of MSC, thus indicated that the antitumor effect
was not pertaining to a particular MSC isolates. Two-way
ANOVA analysis indicated that the differences among the six
groups (DGli36, iNHA, MSC, DGli36/iNHA, DGli36/MSC-1,
and DGli36/MSC-7) were statistically significant (p < .0001;
ference in the tumor volumes between DGli36/MSC-1 and
DGli36/MSC-7 groups. It is important to note that none of the
MSC isolates became tumorigenic; tumor nodule was not
detectable in mice injected with the same MSC cell number
in the presence of Matrigel up to 1 month. Gross anatomy of
the tumors clearly showed that DGli36/MSC tumors were
visibly less vascularized than those of the DGli36 control
tumors (Fig. 2C). Hematoxylin and eosin (H&E) staining per-
formed on the cocultured tumor sections revealed a peripheral
rim of tumor cells cuffing a zone of necrosis (N) (Fig. 2C).
Furthermore, analysis using antibodies against ssDNA indi-
cated that the regions of the apoptotic cells in the cocultured
tumor (Fig. 2E) coincided with the localization of the car-
boxymethyl-DiI-labeled-MSC (Fig. 2D, inset (I)), thus sug-
gesting that the presence of MSC causes cell death in the gli-
oma. On the contrary, apoptosis was not detected in DGli36
tumor and DGli36/iNHA tumor (Fig. 2E; supporting informa-
tion Fig. S1). Taken together, the results demonstrated that
specific interactions between MSC and DGli36 human glioma
cells induced apoptosis of the glioma cells.
MSC Restrict Vascular Growth in Glioma Cells In
Vivo and Reduced Angiogenesis In Vitro
Tumors obtained from coinjection of MSC and DGli36 human
glioma cells were further evaluated for vessel morphology
and microvessel density. The microvessel density as revealed
by CD31 staining was significantly higher in DGli36 and
DGli36/iNHA tumor when compared with that of DGli36/
MSC (p < .01; Fig. 3A). To assess the functional status of
the tumor neovasculature, tumor sections were simultaneously
stained for CD31 expression and pericytes coverage (a-SMA).
The results showed the presence of CD31þ cells in regional
areas of the representative tumor sections of DGli36 and
DGli36/MSC. Expression of a-SMAþ cells could only be
Ho, Toh, Ng et al. 151

Figure 4. Conditioned medium from DGli36/MSC coculture prevented recruitment of EPC and endothelial tube formation. (A): EPCs were
immunostained with antibodies against CD133, CD34, VE-cadherin, VEGFR-2, Tie2, and CD45. Mouse IgG1 and rabbit serum were used as neg-
ative control for the first five antibodies and CD45, respectively. Scale bar ¼ 100 lm. (B): EPC and MSC were assessed for their ability to
uptake DiI-Ac-LDL (red fluorescence). Cells were counterstained with DAPI (blue fluorescence). Representative images were presented. Scale
bar ¼ 50 lm. (C): EPC recruitment assay was performed using DGli36-CM, MSC-CM, and DGli36/MSC-CM. The number of recruited cells to
the bottom chamber were counterstained with propidium iodide and counted. Data shown are averages of triplicates 6 SEM. *, p < .05. (D):
Angiogenesis assay was performed to determine whether DGli36-CM, MSC-CM, NHA-CM, DGli36/NHA-CM, or DGli36/MSC-CM could induce
tube formation of HUVEC. 50 ng/ml of recombinant human VEGF was used as positive control. Tubes formed were visualized and captured
using the Zeiss 200M wide-field microscope at 50 original magnification. Scale bar ¼ 200 lm. (E): Tube formation index was scored using
ImageJ software (National Institutes of Health, Bethesda, MD). Data were obtained from four replicates of two independent experiments and pre-
sented as ratio of coculture to monoculture. Abbreviations: CM, conditioned medium; EPC, endothelial progenitor cells; LDL, low density lipo-
protein; NHA, normal human astrocytes; MSC, mesenchymal stem cell; VE, vascular endothelial; VEGFR, vascular endothelial growth factor
receptor; DAPI, 40 ,6-diamidino-2-phenylindole; HUVEC, Human umbilical vein endothelial cells.

found in DGli36/MSC but not in DGli36 (Fig. 3B), indicating
the presence of a reactive microvasculature in the DGli36
tumors. In DGli36/MSC tumors, a-SMAþ pericytes were
present within the tumor vasculature. These cells did not
overlap with the staining against CD31 (Fig. 3B). As shown
in Figure 3C, MSC (red cells) could be detected close to
CD31þ regions, suggesting a possible role in angiogenesis.
To further investigate whether MSC may be involved in
EPC recruitment during angiogenesis, we isolated EPC from
peripheral blood obtained from healthy donor using density
gradient centrifugation. EPC expressed high level of CD133,
which is indicative of early population of EPC [17]. In addi-
tion, the isolated cells were immune-positive for CD34, Tie2,
VE-cadherin (CD144), VEGFR2 (CD309) but expressed very
low level of CD45 (Fig. 4A; supporting information Fig. S2).
Moreover, cellular uptake of DiI-Ac-LDL, which is indicative
of functional EPC, was also observed in these cells and was
absent in MSC (Fig. 4B). Collectively, these markers con-
firmed the endothelial lineage of the isolated cells [17, 18].
As shown in Figure 4C, the recruitment of EPC was severely
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impaired by DGli36/MSC-CM in comparison to DGli36-CM.
Similar findings were obtained with another isolate of MSC
(data not shown), indicating that the suppressive effect of
MSC on EPC recruitment is not specific to one MSC isolate.
Interestingly, the extent of EPC recruitment in MSC-CM was
similar to those in DGli36/MSC-CM, thus suggesting that the
observed reduced tumor vasculature in the presence of MSC
is likely due to an inhibition on the recruitment of EPC and
its associated angiogenic inducing factors.
Next, we investigated whether the antitumor effect was
mediated by antiangiogenic factors in DGli36/MSC coculture
using in vitro angiogenesis assay. Our results showed that
incubation of HUVECs in DGli36-CM, MSC-CM, NHA-CM,
and DGli36/NHA-CM stimulated tube formation (Fig. 4D),
which is comparable with the known angiogenic molecule,
recombinant human VEGF (data not shown). As shown in
Figure 4E, increased tube formation index was observed in
DGli36/NHA coculture when compared with that of NHA
monoculture (ratio of DGli36/NHA vs. NHA is 1.5), whereas
tube formation index is similar for DGli36/NHA and DGli36
152 MSC Restrict Glioma Angiogenesis

ture (ratio of DGli36/MSC vs. DGli36 or MSC is 0.5), thus

Table 1. Cytokines that are differentially expressed in DGli36/ indicating that MSC impaired tumor angiogenesis through the
mesenchymal stem cells coculture as determined by antibody release of antiangiogenic factors.
array analysis
Fold change MSC Downregulate the Expression of Angiogenic
(normalized Molecules in Glioma Cells
Protein to DGli36-CM) Biological process
To determine which factors are expressed by DGli36/MSC co-
IGF-1 0.431 Angiogenesis, culture, a proteome array was performed using CM harvested
differentiation from DGli36, MSC, and DGli36/MSC. Cytokines that are dif-
PDGF-BB 3.576 Angiogenesis, ferentially expressed in DGli36/MSC coculture were shown in
cell migration, Table 1. These candidates included activated leukocyte cell
and proliferation
adhesion molecule (also known as CD166 antigen), PDGF-
IL-1b 0.06 Angiogenesis,
inflammatory response BB, FGF-2, macrophage inflammatory protein-1-a, Eotaxin-3
MIP-1a (CCL3) 1.26 Chemotaxis, (also known as CCL26), regulated on activation normal T-cell
inflammatory response expressed and secreted (also known as CCL5), tissue inhibi-
Eotaxin-3 (CCL-26) 0.251 Chemotaxis, tors of metalloproteinases-2, interleukin-1b (IL-1b), and IGF-
inflammatory response 1 (Table 1). Because angiogenic index was reduced in the in
RANTES (CCL-5) 0.293 Chemotaxis, vivo coculture, we narrowed down the candidate proteins to
inflammatory response those that were downregulated in angiogenesis. The mRNA
TIMP2 1.473 Cell growth and expression of these genes was further validated by qPCR in
proliferation, ECM
the coculture cells. Significant downregulation of PDGF-BB
disassembly
ALCAM 88.51 Cell adhesion, and IL-1b mRNA expression was observed in DGli36/MSC
axon guidance coculture in comparison to that of DGli36/NHA and DGli36
FGF-2 0.389 Angiogenesis, (Fig. 5A). In line with the qPCR results, lower level of
differentiation PDGF-BB proteins were also detected in DGli36/MSC when
compared with DGli36/NHA and DGli36, thus confirmed our
Fold change shown was normalized to DGli36-CM.
antibody array results (Fig. 5B). However, IGF-1, IL-1b, and
The contribution from MSC-CM was removed by subtracting the
values obtained from that of DGli36/MSC-CM. FGF-2 protein expression failed to show similar trend as
For purpose of completeness, the expression of these cytokines observed in qPCR. IGF-1 and FGF-2 protein expressions were
was also normalized to basal levels in MSC-CM. The results are lower in DGli36/MSC when compared with the controls,
IGF-1 (3.74); PDGF-BB (1.53); IL-1b (4.86); MIP-1a while the expression of IL-1b protein was similar for the
(1.1); Eotaxin-3 (3.42); RANTES (8.53); TIMP2 (0.48); three culture conditions (Fig. 5B).
ALCAM (18.41); FGF-2 (4.08)
Abbreviations: ALCAM, activated leukocyte cell adhesion Impaired IL-1b and PDGF Signaling Cascades in
molecule; CM, conditioned medium; ECM, Extracellular Matrix; Glioma Cocultured with MSC
FGF-2, fibroblast growth factor-2; IL-1b, interleukin 1b; IGF-1,
insulin growth factor-1; PDGF, platelet-derived growth factor; IL-1b, a proinflammatory cytokine, binds to its cell-surface
MIP, macrophage inflammatory protein; RANTES, regulated on receptor and initiates signaling cascade that involves the acti-
activation normal T-cell expressed and secreted; TIMP, tissue vation of nuclear factor kappa B, which in turn activates the
inhibitors of metalloproteinases. cysteine protease, cathepsin B. Cathepsin B has been shown
to be overexpressed in malignant gliomas, which are charac-
terized by aberrant neovascularization [21]. Thus, we first
(ratio of DGli36/NHA vs. DGli36 is 1.0). However, the pres- examined whether MSC may downregulate angiogenesis
ence of MSC in the DGli36 coculture inhibited tube formation through IL-1b signaling. IL-1b protein expression was 40%
by 50% when compared with either DGli36 or MSC monocul- and 80% lower in DGli36/MSC tumor in comparison to that

Figure 5. Crosstalk between MSC and DGli36 human glioma cells downregulate the expression of angiogenic molecules. (A): QPCR was per-
formed on total RNA isolated from DGli36, DGli36/MSC, and DGli36/NHA coculture using primers against PDGF-BB, IGF-1, IL-1b, and FGF-
2. (B): ELISA was performed on CM harvested from DGli36, DGli36/MSC, and DGli36/NHA coculture. For both qPCR and ELISA, the results
shown are normalized to DGli36. Data shown are averages of triplicate 6 SEM. *, p < .05. Abbreviations: CM, conditioned medium; FGF-2,
fibroblast growth factor-2; IL-1b, interleukin-1b; IGF, insulin growth factor; NHA, normal human astrocytes; PDGF-BB, platelet-derived growth
factor.
Ho, Toh, Ng et al. 153

Figure 6. Glioma/MSC coculture resulted in downregulation of PDGF and IL-1b signaling. (A): IL-1b and IL1-R1 expression were determined
using ELISA and Western blot, respectively, on tumor lysate harvested from DGli36, DGli36/MSC, and DGli36/iNHA tumors. Amount of protein
loaded was normalized against b-tubulin. (B): Photomicrograph of tumor sections stained for the presence of cathepsin B in DGli36, DGli36/
MSC, and DGli36/iNHA tumor. Sections were counterstained with hematoxylin and shown at original magnification 200. Scale bar ¼ 50 lm.
(C): PDGF-BB expression was determined using ELISA on DGli36, DGli36/MSC, and DGli36/iNHA tumor lysates. Data shown are averages of
triplicate 6 SEM. (D): Immunoblot was performed on tumor lysates harvested from DGli36, DGli36/MSC, and DGli36/iNHA tumors using
antibodies against Akt, phospho-Akt, PDGFR-b, and phospho-PDGFR-b. Amount of protein loaded was normalized against b-tubulin. *, p < .05.
Immunoblots were performed using tumor lysates harvested from two representative animals per group. Abbreviations: IL-1b, interleukin-1b;
iNHA, immortalized NHA; MSC, mesenchymal stem cells; PDGF-BB, platelet-derived growth factor.

of DGli36/iNHA and DGli36, respectively (Fig. 6A). How-
ever, using a representative of two animals per group, we
detected significantly lower level of IL1-R1 in one of the
DGli36/MSC sample (Fig. 6A). Immunohistochemistry stain-
ing confirmed that cathepsin B expression was lost in the
DGli36/MSC tumor, whereas abundant cathepsin B expression
was detected in DGli36 and DGli36/iNHA tumor alone (Fig.
6B), indicating that MSC exert an inhibitory effect on tumor
vasculature, in part, through the downregulation of IL-1b sig-
naling cascade.
In a recent report, the downregulation of cathepsin B
was shown to decrease the expression of PDGFR-b expres-
sion and correspondingly increase the apoptotic index of gli-
oma cells [22]. Since PDGF-BB is also known to play an
important role in glioma angiogenesis [23], we further exam-
ined whether the reduction in microvessel density may be
modulated through PDGF-BB/PDGFR-b signaling. As shown
in Figure 6C, PDGF-BB protein expression was significantly
reduced in the DGli36/MSC tumor section when compared
with that of DGli36 and DGli36/iNHA tumors (Fig. 6C). Im-
munoblot analysis further showed that the levels of activated
PDGFR-b were correspondingly reduced in DGli36/MSC tu-
mor in comparison to control tumors (using a representative
of two animals per group as shown in Fig. 6D). This reduced
PDGFR-b activation correlated with downregulation of
activated Akt (Fig. 6D) in DGli36/MSC tumors, suggesting
www.StemCells.com
the involvement of PDGF/PDGFR signaling in MSC-induced
tumor suppression.
DISCUSSION
In the field of cancer therapy, the most attractive feature of
MSC is its inherent ability to track tumor cells and its immu-
nosuppressive nature, suggesting that MSC modified with the
appropriate therapeutic gene of interest could home to micro-
scopic tumors and deliver long-term transgene expression
prior to eradication by the immune system. However, the mul-
tifaceted role of MSC in the tumor microenvironment is still
not fully understood. Results of this study suggested that the
inhibitory effect of MSC on glioma tumor angiogenesis might
be mediated through paracrine pathways that resulted in
impaired EPC recruitment and downregulation of proangio-
genic factors such as PDGF-BB, IGF-1, FGF-2, and IL-1b.
MSC has been shown to have the capacity to transdifferentiate
into EC-like [10] and pericytes-like cells [24] to stimulate angio-
genesis and promote tumor growth [25]. Direct coculture of adi-
pose-derived MSC (AMSC) with prostate cancer was shown to
enhance tumor growth through increase in tumor vascularity
mediated by FGF-2 [26] and differentiation of AMSC into endo-
thelial-like cells [27]. By contrast, Otsu et al. [14] showed that
154
coculture containing 1:1 ratio of EC to MSC resulted in EC apo-
ptosis and degeneration of EC capillaries due to the generation of
reactive oxygen species. In our system, neither differentiation of
MSC into EC (Fig. 3) nor pericytes-like cells (Fig. 3) was
observed in glioma/MSC cocultured tumors. Rather, our results
showed decreased microvessel density in vivo that correlated with
reduced tube formation index and recruitment of EPC in vitro,
suggestive of an antiangiogenic response elicited by the MSC.
The presence of the a-SMAþ cells, which is indicative of peri-
cytes, and corresponding reduction of CD31þ microvessel density
in glioma/MSC when compared to glioma alone suggested that
the presence of MSC in the EC environment reduces vascular per-
meability, a finding that is consistent with that of Pati et al. [28].
Pericytes are known to contribute to the integrity of mature
blood vessels. Absence of pericytes results in a more reactive ves-
sel that is irregular, leaky and supports metastasis [29, 30]. It is
interesting to note that although a-SMAþ cells are present in gli-
oma/MSC coculture, the level of PDGF-BB, which has been
shown to recruit pericytes to glioma [23], is downregulated.
PDGF-BB is expressed in the endothelial tip cells or cell that
leads the new sprout at the forefront during angiogenesis, which
stimulates both recruiting pericytes with PDGFR-b expressed on
their surface and the subsequent creation of the wall of the newly
formed capillary [31, 32]. However, PDGF-BB is not the only
factor that mediates pericyte recruitment because mice that are
deficient in either PDGF or PDGFR-b have been shown to have
normal pericyte coverage in the perisinusoidal capillaries [33],
suggesting the involvement of other factors, including secreted
protein acidic and rich in cysteine [34] and endothelial nitric ox-
ide [35]. On the other hand, the reduced PDGF expression in the
presence of MSC may be a consequence of the downregulation of
angiogenesis pathways. Gliomas are highly invasive and malig-
nant tumors and displayed aberrant PDGF signaling [36]. PDGFs
bind and signal through the receptor tyrosine kinases, PDGFR-a
and b. Binding of PDGFs to its receptors trigger receptor dime-
rization that leads to receptor autophosphorylation and subse-
quent signal propagation. Inhibition of PDGF signaling with the
receptor tyrosine kinase inhibitor SU6668 [37] or SU5416 [38]
has been shown to inhibit tumor growth through vessel regression
with or without pericyte involvement.
Inflammation and angiogenesis are pivotal processes in tu-
mor progression. Inflammatory molecules such as IL-1b are fre-
quently upregulated in cancer cells and macrophages found in
the tumor microenvironment. In the coculture of DGli36/MSC,
the IL-1b mRNA levels were significantly reduced when com-
pared with that of DGli36 or DGli36/NHA (Fig. 5A). Although
we observed a slight decrease in IL-1b proteins in the CM
derived from DGli36/MSC when compared with the controls,
the difference was not significant possibly due to the relatively
low levels of proteins (8.74–9.93 pg/ml). However, the lower
IL-1b proteins in DGli36/MSC versus DGli36 or DGli36/NHA
were statistically significant in vivo (Fig. 6A). IL-1b has been
shown to interact with PDGF-BB to induce sustained phospho-
rylation of PDGFR-b and its association with IL1-R1 [39],
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CONCLUSION
Our results have clearly demonstrated that MSC exhibited an
antiglioma effect. Analysis indicated that PDGF signaling,
which is known to play a key role in glioma angiogenesis, is
one of the pathways that is likely involved in the observed
reduced glioma angiogenesis.
ACKNOWLEDGMENTS
This research is supported by grants from the SingHealth Foun-
dation and National Medical Research Council, Singapore.
DISCLOSURE OF POTENTIAL
CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.
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