Assessment of therapeutic efficacy and fate of engineered human mesenchymal stem cells for cancer therapy.
ABSTRACT The poor prognosis of patients with aggressive and invasive cancers combined with toxic effects and short half-life of currently available treatments necessitate development of more effective tumor selective therapies. Mesenchymal stem cells (MSCs) are emerging as novel cell-based delivery agents; however, a thorough investigation addressing their therapeutic potential and fate in different cancer models is lacking. In this study, we explored the engineering potential, fate, and therapeutic efficacy of human MSCs in a highly malignant and invasive model of glioblastoma. We show that engineered MSC retain their "stem-like" properties, survive longer in mice with gliomas than in the normal brain, and migrate extensively toward gliomas. We also show that MSCs are resistant to the cytokine tumor necrosis factor apoptosis ligand (TRAIL) and, when engineered to express secreted recombinant TRAIL, induce caspase-mediated apoptosis in established glioma cell lines as well as CD133-positive primary glioma cells in vitro. Using highly malignant and invasive human glioma models and employing real-time imaging with correlative neuropathology, we demonstrate that MSC-delivered recombinant TRAIL has profound anti-tumor effects in vivo. This study demonstrates the efficacy of diagnostic and therapeutic MSC in preclinical glioma models and forms the basis for developing stem cell-based therapies for different cancers.
- Molecular and cellular therapies. 08/2014;
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ABSTRACT: With the increasing recognition that stem cells play vital roles in the formation, maintenance, and potential targeted treatment of brain tumors, there has been an exponential increase in basic laboratory and translational research on these cell types. However, there are several different classes of stem cells germane to brain cancer, each with distinct capabilities and functions. In this perspective, we discuss the types of stem cells relevant to brain tumor pathogenesis, and suggest a nomenclature for future preclinical and clinical investigation.Oncoscience. 01/2014; 1(3):241-7.
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ABSTRACT: Cell-based therapy is a promising modality to address many unmet medical needs. In addition to genetic engineering, material-based, biochemical, and physical science-based approaches have emerged as novel approaches to modify cells. Non-genetic engineering of cells has been applied in delivering therapeutics to tissues, homing of cells to the bone marrow or inflammatory tissues, cancer imaging, immunotherapy, and remotely controlling cellular functions. This new strategy has unique advantages in disease therapy and is complementary to existing gene-based cell engineering approaches. A better understanding of cellular systems and different engineering methods will allow us to better exploit engineered cells in biomedicine. Here, we review non-genetic cell engineering techniques and applications of engineered cells, discuss the pros and cons of different methods, and provide our perspectives on future research directions.Advanced Drug Delivery Reviews 12/2014; · 12.71 Impact Factor
Assessment of therapeutic efficacy and fate of
engineered human mesenchymal stem cells for
Laura S. Sasportasa,b,1, Randa Kasmieha,b,1, Hiroaki Wakimotoc, Shawn Hingtgena,b, Jeroen A. J. M. van de Watera,b,
Gayatry Mohapatrae, Jose Luiz Figueiredob, Robert L. Martuzac, Ralph Weisslederb,f, and Khalid Shaha,b,d,2
aMolecular Neurotherapy and Imaging Laboratory,bCenter for Molecular Imaging Research (CMIR), Department of Radiology, Departments
ofcNeurosurgery,dNeurology, andePathology, andfCenter for Systems Biology, Department of Systems Biology, Massachusetts General
Hospital, Harvard Medical School Boston, MA 02114
Edited by Webster K. Cavenee, University of California at San Diego School of Medicine, La Jolla, CA, and approved January 16, 2009
(received for review July 11, 2008)
The poor prognosis of patients with aggressive and invasive cancers
combined with toxic effects and short half-life of currently available
treatments necessitate development of more effective tumor selec-
tive therapies. Mesenchymal stem cells (MSCs) are emerging as novel
cell-based delivery agents; however, a thorough investigation ad-
dressing their therapeutic potential and fate in different cancer
models is lacking. In this study, we explored the engineering poten-
tial, fate, and therapeutic efficacy of human MSCs in a highly malig-
nant and invasive model of glioblastoma. We show that engineered
MSC retain their ‘‘stem-like’’ properties, survive longer in mice with
gliomas than in the normal brain, and migrate extensively toward
gliomas. We also show that MSCs are resistant to the cytokine tumor
necrosis factor apoptosis ligand (TRAIL) and, when engineered to
express secreted recombinant TRAIL, induce caspase-mediated apo-
ptosis in established glioma cell lines as well as CD133-positive
primary glioma cells in vitro. Using highly malignant and invasive
human glioma models and employing real-time imaging with correl-
ative neuropathology, we demonstrate that MSC-delivered recombi-
nant TRAIL has profound anti-tumor effects in vivo. This study
demonstrates the efficacy of diagnostic and therapeutic MSC in
preclinical glioma models and forms the basis for developing stem
cell-based therapies for different cancers.
gliomas ? in vivo imaging ? TRAIL
of cell types (1, 2). A significant improvement in understanding
MSC biology in recent years has paved the way to their potential
clinical use. MSCs are attractive candidates for manipulation as
they can easily be isolated from patients, cultured in vitro, and
autologously transplanted into patients, thus overcoming the diffi-
culties related to immune rejection of transplanted cells (3, 4).
Modified MSCs have also been shown to have high metabolic
activity and strong expression of transgenes in vitro and in vivo (5).
Glioblastoma multiforme (GBM) is the most aggressive form of
glioma, with a median survival time of 10 to 12 months (6). Despite
considerable advances in glioma therapy, GBM remains one of the
most challenging diseases, particularly because of its invasiveness,
which precludes surgical removal. Glioblastomas consist of heter-
ogeneous population of cells, some of which have been shown to
extensively proliferate, self-renew, infiltrate, and be solely respon-
sible for the growth of main tumor mass (7, 8). Direct targeting of
invasive tumor cells by genetically modified stem cells could be a
promising therapeutic approach.
MSCs have been shown to migrate toward gliomas (9) and track
microscopic tumor deposits and infiltrating tumor cells in the brain
(10, 11). Furthermore, engineered MSCs have been shown to exert
potent inhibition of tumor growth in an in vivo glioma model and
also exhibit a protective effect for the normal brain (12). However,
most of these studies lack a thorough in vivo characterization of
uman mesenchymal stem cells (MSCs) are multipotent cells
engineered MSCs or their application in mouse models of cancer.
We have previously engineered lentiviral vectors bearing fluores-
cent and bioluminescent markers to stably label stem cells and
tumor cells to study their fate in real time in vitro and in vivo (13).
In this study, we have explored the possibility of modifying MSCs
with different combinations of fluorescent and bioluminescent
markers and engineered a tumor-specific secretable form of re-
combinant TRAIL, S-TRAIL (14). Furthermore, we have used
real-time optical imaging to follow the delivery, fate, and thera-
peutic efficacy of engineered MSCs and the pharmacodynamics of
therapeutic MSCs in real time in both invasive and malignant
mouse glioma models.
Human mesenchymal stem cells (Fig. 1A) were efficiently trans-
duced with our recently engineered lentiviral vector (LV) con-
structs encoding firefly luciferase (Fluc) and GFP fusion proteins
(13) as revealed by GFP fluorescence (Fig. 1B) and flow cytometry
(Fig. 1C). MSCs transduced with LV-GFP-Fluc retained fusion
protein expression through 3 weeks (Fig. 1D) and had a slightly
several passages in culture (transduced cells at day 2; 88% ? 5%;
Fig. S1A]. Genomic profiles generated by array comparative
genomic hybridization confirmed normal DNA copy number in
transduced (Fig. 1E) and non-transduced MSCs (data not shown).
Karyotype analysis performed on transduced (Fig. 1F) and non-
transduced MSC (data not shown) ruled out chromosomal rear-
rangements in lentiviral-transduced cells. These experiments dem-
and that LV modification does not result in altering DNA copy
number and arrangement of MSCs.
To determine MSC survival in vivo, we used MSCs expressing
GFP-Fluc. A direct correlation between cell number of the GFP-
Fluc-expressing MSCs and Fluc signal intensity in vitro (Fig. S1C)
and in vivo (Fig. S1D) within the ranges tested was observed. Next,
Gli36-EGFRvIII were implanted in the brain parenchyma and
survival was followed in real time by bioluminescence imaging. As
shown in the summary graph and representative images (Fig. 1G),
MSC survival was increased in the presence of glioma cells com-
Author contributions: K.S. designed research; L.S.S., R.K., H.W., S.H., J.A.J.M.v.d.W., G.M.,
J.L.F., and K.S. performed research; R.L.M. contributed new reagents/analytic tools; L.S.S.,
R.K., H.W., S.H., G.M., R.W., and K.S. analyzed data; and L.S. and K.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1L.S.S. and R.K. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
March 24, 2009 ?
vol. 106 ?
presence of GFP-expressing MSCs in day-6 brain sections from
mice implanted with MSCs only (Fig. 1H) and GFP-expressing
MSCs and Ki67-positive glioma cells in day-14 brain sections from
mice implanted with MSCs with gliomas (Fig. 1I) confirmed the
presence of MSCs in the brain. These results indicate that glioma
cells or host response modulates MSC survival in the brain after
Evidence suggests that MSCs may influence tumor progression
in several tumor types (15–17). To determine the effect of engi-
neered MSCs on glioma growth, we created a human glioma line
Gli36-EGFRvIII expressing Fluc-DsRed2 (Gli36-EGFRvIII-FD)
by transducing glioma cells with LV-Fluc-Dsred2. A direct corre-
lation between glioma cell number and Fluc signal intensity was
seen in vitro within the ranges tested (Fig. S1B). We implanted
Gli36-EGFRvIII-FD, or a mix of Gli36-EGFRvIII-FD and MSC-
GFP and followed glioma growth in real time. As shown in the
summary graph and representative images (Fig. 2A), there was no
significant effect of MSCs on glioma growth over time. Further-
brain sections from mice implanted with glioma cells only (Fig. 2B)
and with glioma cells and MSC-GFP (Fig. 2C), respectively, con-
firmed the presence of tumor cells and MSCs in mouse brains.
These results indicate that MSCs have no significant influence on
the progression of gliomas in the brain.
The formation of gap junctions between potential therapeutic
cells and tumor cells has been shown to be critical for bystander
effect, and this gap junction-mediated communication between
cells can be measured by flow cytometry using a membrane bound
marker, DI1, and a non-permeable marker, calcein-AM (18).
Human Gli36-EGFRvIII glioma cells were labeled with DI1 and
MSCs were labeled with calcein-AM. Two distinct populations of
red (DI1-labeled) and green (calcein-AM) cells were seen imme-
D). However, after 3 h of incubation, a double-positive red and
green fluorescent population of DI1-labeled glioma cells was seen
(Fig. 3 B, E, and F), whereas no green fluorescence was found in
MSCs as a result of the transfer of calcein-AM. To study homing
of MSCs in a mouse model of glioma, MSCs were transduced with
duced with LV-GFP-Fluc. MSCs expressing tdTomato (Fig. 3G)
were implanted at a 1-mm distance from established human
gliomas expressing GFP-Fluc (Fig. 3H). Fluorescence confocal
microscopy on brain sections from mice 2 and 10 days after
GFP-positive gliomas and not in the surrounding normal brain
culture and survive longer in mice bearing glio-
mas. (A) Light image of MSCs in culture. (B–D)
later, were FACS-sorted. Photomicrograph of
MSCs expressing GFP-Fluc (B). (C) Plot showing
the percentage of transduced MSCs expressing
Fluc in MSC-GFP-Fluc over time. (E) Genomic pro-
sities of MSC-GFP-Fluc implanted intraparenchy-
mally either alone or mixed with Gli36-EGFRvIII
mice with MSC-GFP-Fluc implanted with (?) or
without (-) glioma cells is shown. (H and I) Pho-
tomicrographs on brain sections from mice 16
days after implantation shows presence of GFP-
positive MSCs in normal brain (H) and the pres-
ence of Ki67-positive glioma cells (red) and GFP-
positive MSCs in glioma bearing brains (I).
(Original magnification: B, H, and I, ?20.)
Transduced human MSCs proliferate in
sities of intraparenchymally implanted mice
with Gli36-EGFRvIII-FD human glioma cells or a
mix of Gli36-EGFRvIII-FD and MSC-GFP. One
representative image of mice with Gli36-
EGFRvIII-FD implanted with (?) or without (-)
on brain sections from mice 16 days after im-
plantation shows expression of DsRed2 in gli-
oma cells (B) and the presence of GFP-positive
MSCs in mice bearing gliomas (C). (Original
magnification: B and C, ?20.)
Sasportas et al.PNAS ?
March 24, 2009 ?
vol. 106 ?
no. 12 ?
tissue (Fig. 3 I and J). Intravital microscopy confirmed the robust
migration of MSCs toward and into gliomas within 10 days of MSC
implantation (Fig. 3K). Immunohistochemical analysis on brain
sections from mice bearing gliomas and implanted with MSCs
expressing tdTomato 2 weeks after implantation showed no ex-
or the neuronal marker MAP-2 (Fig. 3O) in MSCs. In contrast,
robust Ki67 expression was seen in glioma cells (Fig. 3L), and
GFAP (Fig. 3N) and MAP-2 (Fig. 3O) expression was seen in
normal brain. These results show that MSCs implanted in the mice
with established glioma home to tumors, do not proliferate, and
remain in un-differentiated state.
To verify whether MSCs can serve as cellular vehicles to deliver
a secretable form of TRAIL, we first evaluated the expression of
death domain-containing TRAIL binding receptor, TRAIL-R1
(DR4), on both MSCs and glioma cells. Immunoreactive proteins
of expected size were present in the glioma cells and absent in the
S-TRAIL 24 h after incubation (Fig. 2B). To convert MSCs into
therapeutic vehicles, we transduced MSCs with LV-S-TRAIL. A
high number of transduced cells was revealed by GFP fluorescence
(Fig. 2C) and FACS sorting (Fig. S2D). Quantification of TRAIL
in the cell culture medium confirmed secretion of 250 ng/106
cells (Fig. S2E). Furthermore, Gli36-EGFRvIII cells exposed to
conditioned medium from MSC-S-TRAIL and MSC-GFP showed
activation of caspases (Fig. S2 F–J) and PARP (Fig. S2H) and
in MSC-S-TRAIL and not MSC-GFP-treated cells (Fig. S2J).
These results show that MSCs are resistant to TRAIL and engi-
neered MSCs secrete S-TRAIL that induces caspase-mediated
apoptosis in glioma cells in culture.
used a recently engineered N-terminal Gaussia luciferase (Gluc)
protein that is well suited for extra-cellular detection as it does not
require ATP for its activity (20). Gluc bioluminescence imaging on
MSCs transduced with LV-Gluc-S-TRAIL showed that MSCs
secreted Gluc-S-TRAIL in the culture medium (Fig. 4A). To
simultaneously track MSCs survival and S-TRAIL secretion in
vivo, MSCs were co-transduced with LV-Gluc-S-TRAIL and LV-
GFP-Fluc. A linear correlation between the Gluc activity depicting
S-TRAIL secreted in the culture medium and the Fluc activity
depicting the number of MSCs secreting S-TRAIL was shown by
dual in vitro bioluminescence imaging (Fig. 4A). When a mix of
human glioma cells, Gli36-EGFRvIII, and GFP-Fluc MSCs secret-
ing Gluc-S-TRAIL was implanted s.c. in mice, dual biolumines-
cence imaging revealed that both MSCs and the release of S-
S-TRAIL expression is continuous and stable over time for at least
2 weeks (Fig. 4B). These experiments demonstrate that kinetics of
both the apoptotic agent and the delivery vehicle can be followed
in real time in vivo. Furthermore, these experiments also demon-
strate that tumor cells have the prolonged access to the S-TRAIL
delivered by MSCs in vivo.
To assess the effect of S-TRAIL delivered via engineered MSCs
on the formation of gliomas, we used the human glioma line
implanted with a mix of Gli36-EGFRvIII-FD and S-TRAIL or
control GFP expressing MSCs (3:1 ratio) revealed a significant
reduction in glioma burden in animals bearing MSCs expressing
S-TRAIL compared with controls (P ? 0.001; Fig. 4 C–I). His-
topathological analysis on day-6 brain sections revealed the pres-
cells in MSC-S-TRAIL-treated tumors and not in MSC-GFP-
treated tumors (Fig. 4 J–L). Also, a significant decrease in the
number of proliferating tumor cells was seen in MSC-S-TRAIL-
treated tumors compared to the controls (Fig. 4 M–O). These
MSCs. (A–F) Human Gli36-EGFRvIII glioma cells
were labeled with DI1 and MSCs were labeled
with calcein-AM, mixed, and FACS-sorted after
5 min and after 3 h. FACS-sorted plots and
graphs reveal different populations of labeled
cells after 5 min (A, C, and D) and 3 h (B, E, and
F). (G and H) Photomicrographs of MSCs ex-
pressing tdTomato (G) and human Gli36-
EGFRvIII glioma cells expressing GFP-Fluc (H).
MSCs expressing tdTomato were implanted in-
tracranially at a 1-mm distance from estab-
lished human gliomas expressing GFP-Fluc. (I
and J) Photomicrographs showing MSC-tdTo-
day 10 (J) in brain sections and by intravital
microscopy on day 10 after MSC implantation
(K). (L–O) Immunohistochemistry on day-14
mato. Representative images of brain sections
immunostained for Ki67 (L), nestin (M), GFAP
(N), and MAP-2 (O). (Green, GFP expression;
blue, nestin, Ki67, GFAP, or MAP-2 expression;
Bystander effect and migration of
www.pnas.org?cgi?doi?10.1073?pnas.0806647106Sasportas et al.
results show that MSC-delivered S-TRAIL strongly inhibits tumor
growth in vivo by activation of caspase-mediated apoptosis.
To assess the effect of S-TRAIL on established gliomas, we
characterized a CD133-positive human primary brain tumor cell
line (GBM8) for its DR4, Akt, and PTEN status (Fig. 5A). GBM8
cells expressed a higher level of DR4 and pAkt than Gli36 human
glioma cells. GBM8 cells exposed to conditioned medium from
To follow GBM8 cells in vivo, we transduced GBM8 cells with
LV-GFP-Fluc. Flow cytometry analysis on GBM8-GFP-Fluc cells
stained with phycoerythrin-conjugated anti-CD133/2 antibody re-
in transduced GBM8 (Fig. S3A). A direct correlation between
GBM8-GFP-Fluc cell number and Fluc signal intensity was seen in
vitro within the ranges tested (Fig. S3B). Transduced GBM8
stained positive for nestin (Fig. 5E) and GFAP (Fig. 5F), thus
retaining the characteristics of GBM8. Histopathological analysis
on the sections from the brain with GBM8 implantation also
confirmed their capability of forming invasive tumors in vivo (Fig.
5 G and H). Next, mice bearing established GBM8-GFP-Fluc
gliomas were implanted with MSC-S-TRAIL or control MSC-
DsRed2 and serial Fluc bioluminescence imaging was performed
every 2 weeks for a period of 5 weeks. As a result of the diffuse and
invasive nature of GBM8 cells, no Fluc signal could be seen until
week 3 of glioma cell implantation in both control and MSC-S-
TRAIL-treated tumors (data not shown). A significant Fluc signal
intensity revealing the growth of GBM8 in the brain was seen in
MSC-DsRed2 control-treated mice at 3 and 5 weeks, whereas mice
implanted with MSC-S-TRAIL showed no Fluc signal (P ? 0.001;
Fig. 5I). Kaplan-Meier survival analysis revealed statistically sig-
nificant prolongation of survival in the group receiving MSC-S-
Fig. 5J). Histopathological analysis on brain sections revealed the
presence of MSCs (Fig. 5K) and a significantly higher number of
proliferating glioma cells in MSC-DsRed2-treated mice than in
MSC-S-TRAIL-treated mice (Fig. 5 L–N). Furthermore, a signif-
icantly higher number of activated caspase-3-positive cells was seen
in MSC-S-TRAIL-treated gliomas compared with MSC-DsRed2-
treated gliomas (Fig. 5 O and P). These results show that MSC-
thus leading to a significant increase in survival times in mice.
MSCs with diagnostic and therapeutic proteins to study their fate
show that engineered human MSCs retain their characteristics,
home to glioma tumors, and have significant anti-tumor effect on
both malignant and invasive primary gliomas in vivo.
Stem and progenitor cell-mediated gene delivery is emerging as
a strategy to improve the efficacy and minimize the toxicity of
current gene therapy approaches. Multiple potential sources for
clinically useful stem and progenitor cells have been identified,
including autologous and allogeneic embryonic cells and fetal and
adult somatic cells from neural, adipose, and mesenchymal tissues.
MSCs harvested from bone marrow are easy to obtain and highly
for immuno-suppression, and, in contrast to embryo-derived stem
cells, MSCs pose few ethical problems. Because of their high
amphotrophic receptor levels, MSCs are readily transducible with
integrating vectors and lead to stable transgene expression in vitro
and in vivo (20). Recent studies have shown that lentiviral trans-
duction is more efficient than onco-retroviral transduction and
improves engraftment of MSCs (21) without affecting their stem
cell properties (22). In this study, we have shown that human MSCs
can be efficiently transduced with bi-modal lentiviral vectors and
engineered MSCs maintain transgene expression and can be cul-
tured long-term in vitro, without losing their ‘‘stem-ness.’’ Further-
more, our studies reveal that engineered MSCs retain transgene
expression in vivo; however, their survival is affected by the
secretion of a number of bioactive growth factors and cytokines,
such as VEGF, transforming growth factor-b, or IL-10 (23, 24),
secreted by the tumor microenvironment, which have been shown
to exert a profound immunosuppressive activity on antigen-
presenting cells and T-effector cells. Tumors are also known to be
efficacy of MSC-S-TRAIL. (A) MSCs were co-
transduced with LV-Gluc-S-TRAIL and LV-GFP-
were imaged for Fluc and Gluc activity, respec-
tively. Plots show correlation between the dif-
ferent concentrations of cells-Fluc activity and
(B) MSCs expressing Gluc-S-TRAIL and GFP-Fluc
were mixed with Gli36-EGFRvIII glioma cells
and implanted s.c. in nude mice. Mice were
imaged for Fluc and Gluc activity every week
for a period of 2 weeks. (C–H) Serial in vivo
lowing intracranial implantation of Gli36-
EGFRvIII-FD glioma cells mixed with MSCs ex-
pressing S-TRAIL (MSC-S-TRAIL; D, F, and H) or
mouse image from each group is shown. (I)
Relative mean bioluminescent signal intensi-
Photomicrographs show presence of cleaved
mice (K and N) 6 days after implantation. Plot
Ki67 (O) cells in MSC-S-TRAIL and MSC-GFP-
purple, Ki67 or cleaved caspase-3 expression;
original magnification: J, K, M, and N, ?20.)
Pharmacodynamics and therapeutic
Sasportas et al.PNAS ?
March 24, 2009 ?
vol. 106 ?
no. 12 ?
infiltrated by regulatory T cells and myeloid suppressor cells, which
actively inhibit T cell responses at the tumor site through direct
cell-cell contact (25), secretion of nitric oxide, or reactive oxygen
species (24). All these factors favor conditions that allow tumors—
and might also allow tumor-associated MSCs in our model—to
escape immune recognition and foster their proliferation and
survival. A number of studies have shown enhancement of tumor
growth and development, potentially through immunomodulatory
no apparent effect of MSCs or have demonstrated inhibition of
tumor growth and extended survival (15–17). Our results indicate
that MSCs have no significant influence on the progression of
gliomas in the brain.
In this work, we demonstrate that MSCs are able to migrate to
tumors in a mouse model of human glioblastoma, remain in an
un-differentiated state, and do not proliferate. This is in line with
studies by Miletic et al. on exogenously administered MSCs in a
mouse glioma model (10). Although MSCs have been shown to
migrate to glioma tumors in mouse models, the molecular mech-
yet been completely elucidated. Based on previous studies on stem
cell homing to lesions and tumors, a number of cytokine/receptor
pairs including SDF-1/CXCR4, SCF/c-Kit, HGF/c-Met, VEGF/
VEGF receptor MCP-1/CCR2, and HMGB1/RAGE (26-32) and
adhesion molecules, ?1- and ?2-integrins, and L-selectin (28) have
known to functionally express various chemokine receptors such as
CCR1, CCR4, CCR7, CCR9, CCR10, CXCR4, CXCR5, CXCR6,
CX3CR1, and c-met, which might be responsible for the homing of
MSCs in different organs following tissue damage (28, 33). Recent
studies by Birnbaum et al. (34) show that IL-8, TGF-ß1, and NT-3
(besides VEGFA) mediate MSC recruitment by glioma.
A number of studies have shown the therapeutic effect of MSCs
in mouse models of cancer. MSCs have been used for delivery of
therapeutic cytokines like IFN-? (9), IL-12 (11, 35), cytosine
deaminase (36), and oncolytic adenovirus (37). The ability of
TRAIL to selectively target tumor cells while remaining harmless
apoptotic therapy in highly malignant brain tumor treatment.
TRAIL signals via two pro-apoptotic death receptors (DR4 and
DR5), inducing a caspase-8-dependent apoptotic cascade in tumor
cells (38) However, because of its short biological half-life and
limited delivery across the blood-brain barrier, most promising
studies using TRAIL lack applicability (41, 42). In an established
glioma model, most of the tumor cells lose the ability to proliferate
CD133-positive primary brain tumor cells are essential in main-
cells, a CD133-positive primary brain tumor cell line in vitro, and,
when implanted into established GBM8 tumors, resulted in a
significant increase in survival of mice bearing gliomas. These
results reveal that MSC-S-TRAIL cytotoxic therapy is highly
efficient in inducing apoptosis in the ‘‘proliferating and maintain-
ing’’ fraction in established non-xenographic gliomas. As opposed
to systemic delivery of therapeutic agents, MSCs have the advan-
tages of offering a continuous and concentrated local delivery of
secretable therapeutic molecules like TRAIL, thus reducing the
non-selective targeting, and allowing higher treatment efficiency
and potency for a longer time period.
of CD133-positive human primary glioma cell line (GBM8) and human Gli36 glioma line (B and C) GBM8 cells were incubated with the conditioned medium from
viability of GBM cells incubated with different concentrations of S-TRAIL. (E and F) Photomicrographs of GFP-Fluc-expressing GBM8 cells stained with anti-nestin (E)
(J) Survival curves of GBM8-GFP-Fluc-bearing mice treated with MSC-DsRed2 and MSC-S-TRAIL. (K–M) Photomicrographs showing presence of DsRed2 MSCs in brain
sections from control mice (K) and Ki67 (L and M) and cleaved caspase-3 (O and P) cells in brain sections from control and MSC-S-TRAIL mice 2 weeks after the second
MSC implantation. (N and Q) Plot shows the number of Ki67 (N) and cleaved caspase-3 (Q) cells in MSC-S-TRAIL- and MSC-DsRed2-treated tumors. (Original
magnification: B, G, and H, ?10; E, F, K–M, O, and P, ?20.)
www.pnas.org?cgi?doi?10.1073?pnas.0806647106Sasportas et al.
A number of previous studies by others and ourselves (4, 13, 14,
44, 45) have shown that in vivo bioluminescence imaging of tumor
during, and after treatment of mice with highly malignant intra-
cranial tumors. Despite the advantages and convenience of biolu-
minescence imaging for monitoring tumor growth, there are some
therefore, the number of luciferase-expressing cells and their lo-
calization within the body is critical to obtain a detectable signal to
follow the fate of cells in vivo—the deeper the tumors within the
body (or if they are intracranial tumors), the greater the signal
attenuation. In our studies on invasive glioma cells, we show that
luciferase signal is undetectable in control tumors to 2 weeks and
is not detectable at all in MSC-S-TRAIL-treated tumors. This
could be a result of the dispersed nature of the GBM cells that are
of engineered MSCs in a mouse model of glioma using engineered
lentiviral vectors and novel imaging methods. Using this study as a
template, advances can be made in the way stem cells can be
engineered and used in clinics in patients with brain tumors. We
envision neurosurgical removal of the main tumor mass at the time
of surgery and implantation of the patient’s own therapeutically
would result in killing of both residual and invasive tumor cells.
Materials and Methods
communication), LV-S-TRAIL (4), and LV-Gluc-STRAIL (van Ekelen, personal com-
munication). Both LV-S-TRAIL and LV-Gluc-S-TRAIL has an IRES-GFP element in
293T/17 cells using a helper virus-free packaging system as described (46). The
transduction of MSC and glioma cells is described in detail in SI Text.
(MSC) (kindly provided by David Prockop, Tulane University, New Orleans) were
grown in Alpha-MEM (Invitrogen/GIBCO) with 16.5% FBS 2–4 mM L-glutamine
active variant of EGFR (Gli36-EGFRvIII), and Gli36EGFRvIII engineered to express
Fluc-DsRed2 (Gli36-EGFRvIII-FD) and 293T/17 cells were grown as described (45).
GBM8 glioma cells were derived from surgical specimens of glioblastoma col-
lected at Massachusetts General Hospital (GBM series) with approval by the
Institutional Review Board. Mechanically minced tissues were digested as de-
scribed in ref. 47 and were grown as ‘‘neurospheres’’ in Neurobasal medium
(Invitrogen/GIBCO) supplemented with 3 mM L-glutamine (Mediatech), 1 ? B27
(Invitrogen/GIBCO), 2 ?g/mL heparin (Sigma), 20 ng/ml human EGF (R and D
Systems), and 20 ng/ml human FGF-2 (Peprotech).
Statistical Analysis. Data were analyzed by Student’s t test when comparing 2
than 2 groups. Data were expressed as mean ? SEM, and differences were
considered significant at P ? 0.05. Survival times of the mouse groups treated
with MSC-S-TRAL and MSC-DsRed2 were compared using the log-rank test.
Other methods are described in detail in SI Text.
ACKNOWLEDGMENTS. We thank Rainer Koehler for his help with intravital
microscopy and Fil Swirski for his help with FACS analysis. This work was sup-
ported by grants from the American Brain Tumor Association (K.S.), Goldhirsh
Society (K.S.), and by National Institutes of Health Grant P50 CA86355 (to R.W.).
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no. 12 ?