Neuro-Oncology Advance Access published June 9, 2016

Neuro-Oncology
Neuro-Oncology 2016; 0, 1 – 13, doi:10.1093/neuonc/now096

Stem cell-based therapies for tumors in the brain: are we there yet?
Khalid Shah

Stem Cell Therapeutics and Imaging Program, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts (K.S.);
Molecular Neurotherapy and Imaging Laboratory, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
(K.S.); Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts (K.S.); Department
of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts (K.S.); Harvard Stem Cell Institute,
Harvard University, Cambridge, Massachusetts (K.S.)

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Corresponding Author: Khalid Shah, MS, PhD 5403, Bldg 149, 13th street, Charlestown, MA 01810 (kshah@mgh.harvard.edu).

Advances in understanding adult stem cell biology have facilitated the development of novel cell-based therapies for cancer. Re-
cent developments in conventional therapies (eg, tumor resection techniques, chemotherapy strategies, and radiation therapy)
for treating both metastatic and primary tumors in the brain, particularly glioblastoma have not resulted in a marked increase in
patient survival. Preclinical studies have shown that multiple stem cell types exhibit inherent tropism and migrate to the sites of
malignancy. Recent studies have validated the feasibility potential of using engineered stem cells as therapeutic agents to target
and eliminate malignant tumor cells in the brain. This review will discuss the recent progress in the therapeutic potential of stem
cells for tumors in the brain and also provide perspectives for future preclinical studies and clinical translation.

Keywords: brain tumors, glioblastoma (GBM), receptors, stem cells, therapeutics.

Tumors in the brain are categorized as either primary brain
tumors, defined by their origin from cells autochthonous to
the brain, and secondary brain tumors, which originate from
metastatic cells from peripheral tumor sites.1 Although the
exact incidence and ratio are controversial, metastatic brain
tumors are known to be far more common than primary
brain tumors and have been estimated to outnumber primary
brain tumors by more than 4 to 1.2 Primary brain tumors are
commonly further subcategorized according to their cell of
origin, which can be within the brain tissue itself (eg, glioma),
the membranes around the brain (eg. meningioma), the
nerves (eg, nerve sheath tumors), glands (eg, pituitary tu-
mors), and vessels (eg, choroid plexus tumors) within the cra-
nium.3 The most frequent type of primary malignant brain
tumor is glioma, of which . 50% may be categorized as
WHO grade IV glioblastoma (GBM). 4 GBM may either arise
de novo (referred to as primary GBM) or much less often
(about 10%) may progress from lower-grade or anaplastic as-
trocytomas (secondary GBM).1 GBM is characterized as malig-
nant, mitotically active, and predisposed to necrosis, and its
very low median survival (14.6 months) is attributed to
unique treatment limitations such as a high average age of
onset, tumor location, and poor current understanding of
the tumor’s pathophysiology.5 With a low median survival,
both primary and secondary GBMs are both associated with
an extremely poor prognosis, with virtually all patients
suffering from recurrence and ,5% of patients surviving
5 years after diagnosis.6,7
The current standard therapy for GBM is a combination of
cytoreductive surgery followed by concurrent and adjuvant
chemotherapy with temozolomide (TMZ) and radiation thera-
py.8 Current limitations in the treatment of GBM involve hin-
drances in achieving complete tumor resection, tumor
resistance, challenges in penetrating the blood brain barrier
(BBB), and insufficient accumulation of therapeutic agents at
the tumor site (www.ncbi.nlm.nih.gov). Thus, patients with
GBM are in dire need of alternative treatments that can circum-
vent the limitations posed by the current standard of care.
Many adult stem cells (SCs) display intrinsic tumor-tropic prop-
erties, making them attractive candidates for targeted delivery
of anticancer biologics by modifying them to stably express/re-
lease a variety of anticancer agents. This review aims to high-
light the most recent advances in SC-based treatments for
treatment of tumors in the brain, particularly GBM, and discuss
the most recent SC-based therapeutic clinical trials for these
tumor types.
Stem Cell Sources
Stem cells are the natural source of embriogenetic tissue gen-
eration and continuous regeneration throughout adult life. Over
the last few decades, 3 major stem cell types (ie, embryonic,

Received 22 January 2016; accepted 8 April 2016

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Shah: Stem cell-based therapies for brain tumors
adult [mesenchymal and neural], and induced pluripotent)
have been identified and extensively characterized. Embryonic
stem cells (ESCs) are derived from the inner cell mass of
blastocyst-stage embryos, and closely related embryonic
germ cells were among the first cell types identified as possible
sources for stem cell therapy.9 However, owing to numerous
concerns related to the use of ESCs, adult stem cell types
(namely mesenchymal stem cells [MSCs], neural stem cells
[NSCs], and induced pluripotent stem cells [ipSCs]) have been
explored extensively for therapeutic purposes.10 MSCs are
spindle-shaped, fibroblast-like multipotent stem cells that are
responsible for regeneration and cellular homeostasis in almost
all tissues and have the potential of converting to tissue types
of other lineages both within and across germ lines.11 MSCs
have been isolated from a number of organs including bone
marrow, adipose tissue, fetal tissues, dental pulp, umbilical
cord, Wharton’s jelly, and other tissue types.12 Most of the pre-
clinical studies to date have been performed with bone
marrow-derived MSCs; however, adipose tissue and umbilical
cord blood are other MSC sources that have received consider-
able attention in recent years. NSCs, also referred to as neural
precursor cells (NPCs), are a heterogeneous population of self-
renewing and multipotent cells of both the developing and the
adult CNS13 and are especially enriched in the subventricular
zone (SVZ) and the subgranular zone in the hippocampal den-
tate gyrus DG. NSCs have been proven to be an invaluable
source for developing cell-based therapies, especially for neuro-
degenerative disorders and also for other conditions, particular-
ly certain cancer types.10,14 Induced pluripotent stem cells
(iPSCs) generated from somatic cells have emerged as popular
and effective stem cell types for therapy.15 The iPSCs are gen-
erated by allowing a differentiated somatic cell to revert to em-
bryonic stage via induced expression of transcription factors
OCT4, SOX2, KLF4, and C-MYC associated with pluripotency.16
iPSCs can be derived from a wide variety of starting cells,
even though easy accessibility to fibroblasts makes them the
most common source for iPSC generation.17 Recent studies
have shown the ability of ectopic expression of cell type-specific
transcription factors to directly switch cell fates between
somatic cells, thus circumventing the pluripotent state and
eliminating any risk of malignant transformation.18 Similarly,
MSCs derived from induced pluripotent stem cells (iPSC-MSCs)
have also evolved as a promising alternative cell source for
MSCs and regenerative medicine. Several studies have revealed
successful derivation of functional iPSC-MSCs19,20 that were
shown to have similar characteristics as bone marrow-derived
MSCs,21 including expression of typical mesenchymal markers
and the capacity to efficiently differentiate into osteogenic
and chondrogenic lineages.
Stem Cell Homing and Migration to Tumors
The intrinsic homing property of a variety of stem cell types to
brain pathologies such as ischemic, neoplastic, and demyelinating
lesions has been unraveled during the past decade.22 – 25 A
number of studies have shown tumor tropism of both NSCs
and MSCs when injected via different routes in mouse tumor
models of brain tumors (reviewed in10). The G-protein coupled
receptor CXCR4 and its only known ligand, stromal cell-derived
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factor 1 (SDF1; also known as CXCL12) is one of the best-
studied mediators of stem cell tropism. SDF1 and CXCR4 are
expressed at high levels, particularly in the DG and SVZ
where they are involved in regulating the tropism of endoge-
nous NSCs.26 The migration of exogenously modified thera-
peutic stem cells has been shown to proceed in a similar
fashion to that of endogenous NSCs toward tumors in the
brain.27,27–30 The chemokine stem cell factor (SCF) has been
shown to be upregulated by cells that reside in and around le-
sioned areas and induce the migration of exogenous NSCs to-
ward pathology within the brain through interaction with the
tyrosine receptor kinase c-Kit, as demonstrated in a GBM
model.31 NSCs also express CCR2 and migrate in the direction
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of a MCP-1 gradient toward neoplastic lesions within the
brain.32 Hypoxia is known to promote NSC tropism in vitro33
and in vivo,34 mainly due to the upregulation of vascular en-
dothelial growth factor (VEGF) by hypoxic cells, which has
been observed to result in increased expression of chemotac-
tic factors Ang2 and GROa.35 These 2 proteins promote the
migration of NSCs toward regions of hypoxia within the
brain. VEGF can induce NSC migration in a reactive oxygen spe-
cies (ROS)- and focal adhesion kinase (FAK)-dependent man-
ner. 36 Other influential signaling pathways involved in SC
homing have been elucidated and include hepatocyte growth
factor (HGF)/c-MET receptor, 37 urokinase-type plasminogen
activator (uPA)/uPA receptor (uPAR),38 platelet-derived growth
factor (PDGF)/uPAR/b1 integrin, 39 and transforming growth
factor b (TGFb)/TGFb receptor (TGFbRII).40 Macrophage mi-
gration inhibitory factor (MIF)/CXCR4 has been recently identi-
fied as the dominant chemotactic axis for recruiting human
MSCs to tumors. 41 Although the tumor-homing ability of
MSCs and NSCs has been extensively studied and established,
understanding the ability of iPSC-derived SCs to home to tu-
mors is still in its infancy. Recently, iPSC-derived NSCs have
been shown to have potent glioma tropism.42 The degree of
SC homing to tumors in vivo is influenced by diverse factors
including the nature of the SC and the tumor microenviron-
ment.10 In addition to homing to the primary tumor mass,
both NSCs and MSCs have been shown to efficiently track ma-
lignant microinvasive deposits in the brain.24,27 Therefore ,the
potential for migration of SCs to the main tumor mass and the
microinvasive deposits offers a strong rationale for the use of
SCs in treating brain tumors.
Therapeutic Applications for Brain Tumors
The major limitations of current GBM therapies are (1) margin-
al chance of curative resection due to the highly invasive na-
ture of GBM, which allows tumor cells to infiltrate deeply into
normal tissue; (2) the frequent relapses after seemingly cura-
tive resection due to the seeding of microsatellite deposits in
the surrounding neuropil; (3) the pharmacokinetic problems
associated with the blood-brain-barrier that impede the accu-
mulation of therapeutic drug concentrations in the tumoral
interstitium; and (4) the caution that has to be incorporated
with high-dose drug treatment and radiation therapy due to
the vulnerability of normal brain tissue and the serious conse-
quences of brain damage to the patient’s quality of life. Trans-
planted SCs, engineered to release therapeutic agents, can be
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used as an engraftable, highly mobile, “organic” delivery vehi-
cle for tumor treatment. This is reliant on the biological and
physiological differences between normal and malignant
cells and also on the GBM tumor-homing properties of engi-
neered SCs. The latter is a crucial factor in clinical effective-
ness as engineered SCs can “home in” on microsatellite
deposits of escaping GBM cells that are known to give rise to
recurrent tumors in the brain. Both unmodified SCs, which
possess intrinsic antitumor effects attributed to their released
factors and physical interactions with tumor cells,43 and mod-
ified SCs have been utilized to treat tumors in the brain. Some
of the most promising are outlined in Fig. 1 and are discussed
below.
Modifying Stem Cells Genetically to Release Direct and
Indirect Effector Proteins
SCs are typically modified by viral transduction, although non-
viral methods to express transgenes encoding secretable effec-
tor proteins have been reported.25,44 Depending on whether the
therapeutic proteins act directly on malignant tumor cells in the
brain or upon tumor-supporting cells in the tumor microenvi-
ronment, SC secretion of such proteins can be divided into 2
Fig. 1. Modifying stem cells for brain tumor therapy. Stem cells can be expanded and manipulated in vitro to express a variety of
tumor-antagonizing transgenes to establish antitumor effect following stem cell implantation in vivo.
Neuro-Oncology
Shah: Stem cell-based therapies for brain tumors
broad categories: direct and indirect effectors. The direct effec-
tors include proapoptotic and antiproliferative proteins.
Proapoptotic Proteins
TRAIL (tumor necrosis factor-related apoptosis inducing ligand)
binds to death receptors (DR)4/5 specifically expressed on
tumor cells and induces caspase-mediated apoptosis. 24,45
Previous studies on intratumoral implantation of murine pri-
mary NSCs engineered with full length TRAIL in established
GBM revealed a significant reduction of tumor burden.46 How-
ever, TRAIL is a type 2 receptor membrane protein, and its re-
lease into the SC environment requires accidental cleavage
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from its membrane docking site. We have engineered recom-
binant TRAIL that consists of a fusion between the extracellu-
lar domain of TRAIL and the extracellular domain of the hFlt3
ligand, which is a ligand for Flt3 tyrosine kinase receptor and
mediates both secretion through the endoplasmic retic-
ulum. 47 Both MSCs and NSCs transduced with a lentiviral
expression cassette for secretable (S)-TRAIL have shown anti-
tumor effects in mouse tumor models of both nodular and in-
vasive GBM.24,48 Recent studies have confirmed the cytotoxic
effects of TRAIL-engineered SCs on GBMs, 49 indicating the
therapeutic potential of this strategy. Recently, we have
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Shah: Stem cell-based therapies for brain tumors
developed in vivo imageable breast-to brain metastasis
tumor models and shown that intra-arterial injection of
SC-S-TRAIL suppressed metastatic tumor growth and
prolonged the survival of mice bearing metastatic tumors in
the brain.50
Antiproliferative Proteins
Among the number of antiproliferative molecules, a limited
number of molecules can be expressed and released in the
extracellular milieu. The antitumor effects of SCs expressing
cytokines such as interferon (INF)-b in brain tumors was
shown in an early study revealing that human SCs expressing
IFN-b were able to track murine GBM tumors when adminis-
tered systemically.51 IFN-b, which can also act as an alterna-
tive immunogenic candidate, 52,53 and (IFN)-a 54 have both
been shown to negatively regulate tumor growth in a variety
of preclinical cancer models. Recent studies have further
shown that the release of biologics from SCs can outcompete
or sterically block the binding of endogenous ligands to their
cognate receptors such as epidermal growth factor receptor
(EGFR) or its tumor-specific antigen EGFRvIII variant, 55,56
resulting in the inhibition of proliferation pathways in
tumor cells.
Bone morphogenetic proteins (BMPs) activate their cognate
receptors (BMPRs), and in vivo delivery of BMP4 has been shown
to effectively block the tumor growth post intracerebral grafting
of human GBM cells.57 In a recent study, human adipose-
derived MSCs engineered to express BMP4 were shown to prolong
survival in mice bearing GBM.58 Other studies on the regulated
expression of proteins that induce cell cycle arrest, such as
growth-arrest specific-1 (Gas1) in SCs, have shown antitumor ef-
fects in mouse tumor models of GBM.59
Indirect effectors of tumor cells include antiangiogenic
thrombospondin-1 (aaTSP-1),60 PEX (a fragment of metallopro-
teinase-2),61 and immunostimulatory molecules such as inter-
leukins (ILs).
Antiangiogenic Agents
Tumor angiogenesis represents the ability of a tumor to stimu-
late new blood vessel formation for self-sustained growth.62
Previous clinical studies have shown that intravascular delivery
of antiangiogenic drugs transiently normalizes the abnormal
vasculature, thus reducing tumor-associated vasogenic brain
edema and providing a clinical benefit for GBM patients.63
The decrease in mean vessel diameter and permeability64
and increased pericyte coating of small vasculature65 have
been shown to be associated with vessel normalization. Previ-
ous studies have shown that MSCs localize to tumor vascula-
ture upon intratumoral implantation,66 and recent findings
demonstrated that MSCs display pericyte markers and intratu-
morally grafted MSCs could possibly function as tumor peri-
cytes.67 Human NSCs expressing PEX, a naturally occurring
fragment of human metalloproteinase-2 when injected directly
into murine GBM, have shown efficacy post-treatment.61 In our
previous studies, we have shown that NSCs engineered to ex-
press antiangiogenic repeats of anti-angiogeneic three type 1
repeats (3TSR) of thrombospondin (TSP)-1 markedly reduced
tumor vessel-density, resulting in the inhibition of tumor
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progression in mice bearing highly malignant human GBM.60
These studies suggest that SCs engineered to express antian-
giogenic agents such as PEX and aaTSP-1 could be exploited
for enhanced therapeutic benefit.
Immunostimulatory Molecules
The rationale for utilizing immunostimulatory molecules is
based on shifting the immunosuppressive tumor milieu to-
wards directing an immune response against the cancer.68 In-
terleukins are known to regulate inflammatory and immune
responses and have antitumor effects.69 Both murine and
human primary NSCs engineered to express interleukin-12
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have been shown to have treatment efficacy on GBM in mice
and rats, respectively.70,71 Human MSCs secreting IL-12 or
IL-18 have also shown efficacy in mice-bearing GBM.72–74
The sustained presence of SC-delivered ILs resulted in the
activation of natural killer cells and the recruitment of
tumor-specific T cells.
Immunotherapy employing peripherally implanted cyto-
kine-secreting inactivated tumor cells in syngeneic tumor
models has shown promising results by potentially eradicating
intracranial GBM through the induction of a local proinflam-
matory response in the tumor microenvironment.75,76 Several
reports show that MSCs can potentially synergize with this ap-
proach when exposed to cytokines such as interferon-gamma
(IFNg). 77,78 As such, peripheral immunotherapy using
cytokine-secreting tumor cells has been shown to eradicate
experimental GBM by inducing a proinflammatory tumor mi-
croenvironment. 76,79 Recent studies have shown that the
intratumoral implantation of MSCs into rats bearing GBM, in
addition to peripheral immunotherapy with interferon-
gamma (IFNg)-secreting inactivated tumor cells, significantly
improved animal survival.80 Although the complete mecha-
nism of action in this context remains unclear, in vitro results
suggest that MSCs in response to IFNg act as conditional
antigen-presenting cells, upregulate MHC classes I and II mol-
ecules, and secrete low amounts of immunosuppressive mol-
ecules, ultimately inducing an intratumoral antigen-specific
cytotoxic CD8+ T cell response.
Modifying Stem Cells Genetically to Release
Tumor-targeted Toxins
Pseudomonas exotoxin (PE) potently blocks protein synthesis by
catalyzing the inactivation of elongation factor-2 (EF-2). Target-
ed PE-cytotoxins have been used as antitumor agents; howev-
er, their off-target delivery, systemic toxicity, and short
chemotherapeutic half-life have confounded their effective
clinical translation in GBM patients. 81 In a recent study, we
have created toxin-resistant SCs by modifying endogenous
EF-2 and engineered them to secrete PE-cytotoxins that specfi-
cally target expressed interleukin (IL)-13 receptor subunit
alpha-2 or overexpressed EGFR in GBM. The release of IL13-PE
from SCs in a clinically relevant GBM resection model resulted
in tumor cell-specific killing and led to increased long-term
survival of mice compared with IL13-PE protein infusion.82
A recent study has shown that intratumoral injection of
MSCs expressing a bispecific immunotoxin, VEGF165-ephrin
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A1-PE38KDEL, was effective at inhibiting tumor growth in a ma-
lignant GBM tumor model.83
Modifying Stem Cells Genetically to Induce Suicide
Therapy
SC-mediated suicide therapy is a strategy whereby SCs are en-
gineered to express an enzyme that converts a nontoxic pro-
drug into a cytotoxic drug, resulting in efficiently killing
surrounding cells by the bystander effect. Three major prodrug
systems are currently being used: cytosine deaminase (CD)/
5-fluorocytosine (5-FC); herpes simplex virus thymidine kinase
(HSV-TK)/ganciclovir (GCV); and carboxylesterase (CE)/
camptothecin-11 (CPT-11). Both MSCs and NSCs have been
modified with the CD/5-FC system, tested in mouse models
of GBM84,85 and medulloblastoma,86 and shown to result in
tumor regression and prolonged survival. The HSV-TK system
has been used in modified MSCs and relies on the formation
of gap junctions between the SCs and surrounding target
cells for an efficient bystander effect. MSCs expressing HSV-TK
have shown efficacy in animal models of GBM.87 – 89 Human
NSCs bearing the CE/CPT-11 system have proven effective in
preclinical models of medulloblastoma.90 The feasibility of
treating GBM with SCs expressing double prodrug enzymes
has been explored. Human NSCs expressing CD and TK were
shown to have efficient antitumor efficacy and eradication of
the SCs.91 In an effort to develop efficient SC-based therapeutic
strategy that simultaneously allows killing of GBM tumor cells
and eradication of SCs post tumor treatment, we have engi-
neered MSCs to co-express HSV-TK and S-TRAIL and shown
that they induce caspase-mediated GBM cell death and can
be selectively eradicated post tumor treatment in mouse GBM
models.88 Similar studies utilizing SCs expressing CD/IFNb57,92
and CE/TRAIL93 have also shown efficacy in malignant GBM
mouse models.
Stem Cells Carrying Nanoparticles
Nanoparticles are emerging as novel therapeutic and diagnos-
tic tools with great potential in cancer biology. The surface of
nanoparticles can be modified, thus making them ideally suited
to contain high concentrations of often insoluble therapeutic
reagents.94 Although the use of nanoparticles offers consid-
erable delivery benefits, their efficient clearance, inefficient
dissemination in solid tumors, and inability to target microme-
tastatic lesions94 have presented a number of challenges. SCs
have the potential to overcome these barriers and can be load-
ed with nanoparticles and administered in vivo, where they can
deposit the loaded nanoparticles in close proximity to the
tumor.95 Previous studies have shown the potential for loading
cell membranes of MSCs with porous silica “nanorattles” con-
taining doxorubicin (DOX)96 and their efficacy in intracranial tu-
mors.96 Recent studies have shown that NSCs can improve the
tumor-selective distribution and retention of nanoparticles
within invasive brain tumors.97 The release of nanoparticles
from SCs is either due to membrane rupture or by facilitating
death via photoinduction98,99 or inducing hyperthermia.100
The combination of nanoparticle-based therapeutics with SC
delivery has exciting prospects for the treatment of tumors in
the brain.
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Shah: Stem cell-based therapies for brain tumors
Stem Cells Loaded With Oncolytic Virus
Oncolytic viruses (OVs) are natural or genetically modified vi-
ruses that, upon infection, selectively replicate in and kill neo-
plastic cells while sparing healthy cells.101 However, clearance
of the virus by host defense mechanisms post systemic ad-
ministration and insufficient viral spread post intratumoral
administration confound their therapeutic efficacy. In an ef-
fort to improve delivery of OV-based therapeutics and circum-
vent antiviral immunity, a number of studies have explored
the possibility of using SCs as delivery vehicles.11,102 Different
SC types have been utilized for local release of intact replicat-
ing oncolytic adenoviruses administered systemically or intra-
tumorally into mouse models of GBM.103 – 105 Previous studies
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have shown effective homing of NSCs loaded with the onco-
lytic adenovirus, CRAd-Survivin-pk7, to intracranial GBM, re-
sulting in increased animal survival. 106 Recently, we have
shown that hyaluronan or hyaluronic acid (HA) is highly ex-
pressed in a majority of tumor xenografts established from
patient-derived GBM lines that present both invasive and nod-
ular phenotypes. 107 Intratumoral injection of MSCs loaded
with a conditionally replicating adenovirus expressing soluble
hyaluronidase (ICOVIR17) into GBM enhanced viral spread
and resulted in a significant antitumor effect and mice sur-
vival.107 Oncolytic herpes simplex virus (oHSV) is inherently
neurotropic and has been modified for tumor-selective repli-
cation, making it a promising candidate for brain tumor ther-
apy. 108 Previous clinical studies have revealed suboptimal
response rates in patients after delivery of oHSV in GBM post
tumor resection,109 which could be attributed to “wash out”
of the oHSV in the tumor resection cavity. We have utilized
MSCs for local release of oHSV and its proapoptotic variant
oHSV-TRAIL into mouse models of GBM and shown prolonged
median survival in mice as compared with direct injection of
purified oHSV.110
Stem Cell-released Extracellular Vesicles
Extracellular vesicles (EVs) (exosomes and shedding micro-
vesicles) are released by various cell types and contain
mRNA, miRNA, proteins, and lipids. 111 Accumulating evi-
dence supports the notion that MSCs act in a paracrine man-
ner112 by transferring the biological material to adjacent or
distant cells to facilitate communication between different
cell types.113 Previous studies have shown that MSCs secrete
and transfer microvesicles containing specific miRNAs that
can serve in intercellular communication. 114 The ability of
adult MSCs to transfer miRNA (miR-124 and miR-145) mim-
ics to GBM cells, which express very low levels of these miR-
NAs, has been explored and shown to have antitumor effects
in a mouse model of GBM.115 Similarly, intratumoral injection
of exosomes derived from miR-146-expressing MSCs signifi-
cantly reduced tumor growth in a rat model of primary
GBM. 116 Recent studies have reported that MSCs are able
to package and deliver active drugs such as paclitaxel
(PTX) through their exosomes and increase survival of mice
bearing GBM. 117,118 Additional, recent studies have shown
that exosomes derived from MSCs engineered to express dif-
ferent miRs have antitumor effects in mouse tumor
models.119
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Shah: Stem cell-based therapies for brain tumors
Synergistic Approaches Enhancing Therapeutic Stem Cell
Efficacy in Combination With Other Antitumor Agents
Brain tumors comprise a heterogeneous population of cells
that are genetically and epigenetically unstable.120 Given the
GBM heterogeneity, it is unlikely that any one effective strategy
will provide a satisfactory treatment regimen for such tumors.
Therefore, using a combination approach would be more effec-
tive for treating GBM. Because brain tumor cells and the cells in
the tumor microenvironment either specifically express or over-
express cell surface receptors, one strategy is to create SCs that
simultaneously express/secrete different therapeutic agents
targeting these receptor types. This will allow targeting multiple
pathways in tumor cells and associated cells in the microenvi-
ronment and thus increase the efficacy of targeted therapies.
As such, there are a growing number of studies using bimodal
SCs or SCs that secrete bifunctional molecules. We have shown
that 3TSR of TSP-1 upregulates death receptor (DR) 4/5 expres-
sion in a CD36-dependent manner and primes resistant GBMs
to TRAIL-induced apoptosis. A single administration of MSCs ex-
pressing 3TSR and S-TRAIL targets both tumor cells and vascu-
lar component of GBMs and extends survival of mice bearing
intracranial TRAIL-resistant and highly vascularized GBM.121 Re-
cent studies have shown that SCs coexpressing immunostimu-
latory cytokines IL-18 and (IFN)-b significantly prolonged the
survival and inhibited tumor growth in a rat intracranial GBM
Fig. 2. Enhancing stem-cell (SC) efficacy by engineering bifunctional molecules. The efficacy of SC-delivered therapeutic payload can be enhanced
by engineering bispecific molecules that target multiple cell surface receptor mediated signaling pathways in glioblastoma cells.
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model by promoting cell apoptosis, antitumor cytokine produc-
tion, and CD4+ and CD8+ T-cell infiltration.122
An emerging branch of cancer therapies is the use of smaller
antibody fragments such as single-chain variable fragments
(scFv) and nanobodies (consisting of just the VHH domain)
that bind to epitopes overexpressed on tumor cells123 and cre-
ate bifunctional proteins that are able to perturb multiple sig-
naling pathways specifically in tumor cells (Fig. 2). In one
study, we fused S-TRAIL to an EGFR-specific nanobody to
make a proapoptotic immunoconjugate (ENb-TRAIL) that con-
currently targets cell proliferation and death pathways.56 SCs
engineered to express ENb-TRAIL were shown to efficiently in-
hibit EGFR signaling and induce caspase-mediated cell death in
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a panel of GBM expressing varying levels of EGFR and DR4/5.56
In vivo, when mice bearing intracranial GBM were treated with
stem cell-delivered ENb-TRAIL, tumor burden was significantly
decreased and mice survival was prolonged.56 Taken together,
bifunctional antibody fragments possess many attributes,
making this a novel and potentially effective means for treating
malignancies.
A number of different studies have combined an additional
agent that synergizes with SC therapy or sensitizes an other-
wise resistant population to SCs delivering a single therapy,
thus increasing the effectiveness of locally delivered biological
agent to the tumor site. As such, different drugs that can be
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 Shah: Stem cell-based therapies for brain tumors

delivered systemically have been shown to synergize with

SC-delivered TRAIL to potentiate its p53-independent proapop-
totic mode of action in brain tumors. These include proteasome
inhibitors such as bortezomib, HDAC inhibitors, genotoxic drugs
such as cisplatin, the PI3-kinase/mTOR inhibitor PI-103, shRNA,
and micro-RNA inhibitors.48,124 We have also designed addi-
tional supplementary treatments such as microRNA-21
(miR-21) inhibitors52 and novel PI3-kinase/mTOR inhibitor
PI-103125 to augment the antitumor effect of different
SC-mediated S-TRAIL therapy in mouse models of GBM in
vivo. The combination of valproic acid (VPA) with MSCs express-
ing HSV-TK was shown to enhance the bystander effect, allow-
ing enhanced cellular transmission of GCV-monophosphate in

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culture and increased survival in preclinical models of GBM.89
GBM patients are often treated by maximal surgical resec-
tion followed by ionizing radiation and TMZ.6 Therefore, it is im-
perative to initially test SC therapies with preclinical models and
study designs that incorporate current standard of care in an
effort to be translated into the clinic. Previous studies have
shown that tumor irradiation enhances the tumor tropism of
human umbilical cord blood-derived MSCs mediated by in-
creased IL-8 expression on glioma cells.126 Similarly, MSCs ex-
pressing TRAIL127 or interferon-b128 were shown to be more
effective at killing malignant GBM in the presence of TMZ. In an-
other study, NSCs carrying OV used in combination with radia-
tion and TMZ129 were shown to prolong the survival of mice
bearing patient-derived GBM xenografts.
Promising Preclinical Studies and Clinical Trials
The CNS lesion site(s) (focal vs multifocal) very much define(s)
the route of SC transplantation.14 Direct local transplantation is
suggested as a preferred route of NSC transplantation for focal
CNS disorders, whereas the multifocality of certain CNS dis-
orders represents a major limitation for intralesional cell-
transplantation approaches. In mouse models of GBM, our
earlier studies have shown that the intraventricular route of
delivery is much more efficacious than the intravenous or intra-
peritoneal route.33 Recent studies suggest that intranasal deliv-
ery of SCs is an emerging noninvasive means for the treatment
of brain tumors.130,131 Given that about 75% of GBM patients
undergo tumor debulking,31,132 another innovative strategy is
the encapsulation and implantation of SCs in the resected
GBM tumors in intracranial mouse GBM models. Biodegradable
hydrogels and synthetic extracellular matrix (sECM) materials
composed of hyaluronic acid, alginate, agarose, and other poly-
mers133 have been utilized to encapsulate SCs in a variety of
rodent cancer models133,134 and thus offer an important pros-
pect for harnessing on-site delivery of tumor-specific agents.
We have investigated a new approach to GBM treatment by en-
capsulating therapeutic SCs in GBM resection xenograft mouse
models that recapitulate the clinical scenario of surgical
debulking of GBM31 (Fig. 3). Human MSCs and NSCs encapsulat-
ed in sECM were retained in the resection cavity and permitted
tumor-selective migration. sECM-encapsulated SCs engineered
to express proapoptotic TRAIL protein or loaded with oHSV-
TRAIL were placed into the mouse GBM tumor resection cavity
and shown to increase survival significantly.31,110 Recent stud-
ies have further shown significant therapeutic efficacy of MSCs
loaded with conditionally replicating adenovirus expressing
Fig. 3. Encapsulating engineered therapeutic stem cells in clinically
relevant mouse tumor models of GBM. Stem cells expressing
bifunctional molecules or loaded with oncolytic viruses can be
encapsulated in biodegradable synthetic extracellular matrix and
placed in the tumor resection cavity created after GBM tumor
debulking in mouse tumor models of GBM.
soluble hyaluronidase (ICOVIR17) compared with direct injec-
tion in a clinically relevant mouse model of GBM resection.107
Similarly, we have shown that a low dose of cisplatin in combi-
nation with sECM-encapsulated SC-TRAIL transplanted in the
GBM resection cavity significantly decreases tumor regrowth
and increases survival in mice bearing GBM.135 Together,
these studies provide a platform for clinical translation of engi-
neered SCs to target residual brain tumor cells post surgery.
Currently, a few trials are testing if modified SCs have active
antitumor activity in brain tumor patients (Table 1). Genexine,
Inc is sponsoring NCT02079324, which is intended to establish
the safety and efficacy of MSCs expressing IL-12 (GX-051) ad-
ministered intratumorally in patients with advanced head and
neck cancer. In another trial, allogeneic NSCs modified to ex-
press CD are being tested in recurrent GBM (NCT01172964).
Two more complementary trials have been approved that
combine NSC-CD therapy with either leucovorin calcium
(NCT02015819) or irinotecan hydrochloride (NCT02055196)
to further sensitize GBM cells.
Prospects and Caveats on the way to the Clinics
The adult SC represents a potentially powerful tool and offers
great potential for use in the treatment of tumors in the
brain. Transplantation experiments have revealed that both
NSCs and MSCs migrate extensively towards both primary and
metastatic tumors in the brain. These intrinsic abilities have
been harnessed to deliver apoptosis-inducing proteins,

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Shah: Stem cell-based therapies for brain tumors
Table 1. Stem cell therapies for cancer in clinical trials
Stem-cell Type Therapeutics Loaded Phase (study start)
NSC Cytosine deaminase I (12/2013)
NSC Carboxylesterase I (01/2016)
MSC IL-12 I (03.2014)
MSC MV-NIS I/II (03/2014)
MSC IFNb I (03/2016)
MSC ICOVIR5 I/II (01/2013)
HSPC rHIV7-shl-TAR-CCR5RZ N/L (08/2015)
HSPC LVsh5/C46 (CAL-1) I (11/2015)
HSPC LV-C46/CCR5/P140K I (03/2016)
Abbreviations: HSPC, hematopoietic stem/progenitor cell; ICOVIR5, oncolytic adenovirus; IFNb, Interferon-b; L-12, interleukin-12; LV-C46/CCR5/
P140K, lentiviral vector encoding multiple inhibitors for HIV replication; LVsh5/C46 (CAL-1), dual anti-HIV lentiviral vector; MSC, mesenchymal
stem cell; MV-NIS, oncolytic measles virus encoding thyroidal sodium iodide symporter; N/L, not listed in clinical trial database; NSC, neuronal
stem cell; rHIV7-shl-TAR-CCR5RZ, lentiviral vector encoding multiple anti-HIV RNAs.
immunostimulatory signals, antiangiogenic factors, cell cycle
modulators, inducers of differentiation, and OVs. However,
the integration of nascent therapeutic technologies such as ge-
netically modified SCs would require considerable understand-
ing of fundamental mechanisms before they can be efficiently
translated into clinical settings. The use of a particular SC type
in clinical trials will depend on the ability SCs to preferentially
home to tumors in the brain, their easy availability, and their
relative ease of manipulation in vitro without requiring immor-
talization. A major advantage of using autologous stem cells is
their immunological compatibility, which has a profound effect
on cell survival post transplantation. MSCs with multilineage
potential show a broad migratory capacity for different brain
tumor types including glioblastoma, medulloblastoma, epen-
dymoma, and astrocytoma.136,137 Hence, they have been stud-
ied as a better alternative to NSCs that, despite their strong
tumor-tropic properties, have limited availability and ethical is-
sues.138 Due to their abundant availability, easy isolation and
expansions, fewer ethical concerns and, most importantly,
eligibility for autologous transplantation, human adipose
tissue-derived MSCs are one of the most attractive vehicles
for the delivery of therapeutic agents.138 Although iPS-derived
NSCs have been considered a feasible, effective, and autolo-
gous source for clinical applications, their therapeutic ability
has not yet been fully addressed.19 The selection of the SC
type, its purity, thorough understanding of its biology, and
the effect of any modifications on its function should be estab-
lished in preclinical studies before its consideration for clinical
translation.
In recent years, patient-derived primary GBM lines have
been created from isolated human brain tumor tissue and uti-
lized for preclinical studies. A number of studies have shown
that xenografts of these primary patient-derived cell lines
often recapitulate clinical GBM more faithfully, thus providing
additional insights and more stringent testing of promising
new anti-GBM therapies.139 A thorough biological understand-
ing of such GBM tumor types will improve the chances of
therapeutic success by yielding new therapeutic targets
and enabling mechanism-based SC therapies to be developed,
tested, and refined in response to a specific cancer profile.140
8 of 13
National Clinical Trial Number Targeted Cancer Types
NCT02015819 Recurrent high-grade glioma
NCT02192359 Recurrent high-grade glioma
NCT02079324 Head and neck cancer
NCT02068794 Recurrent ovarian cancer
NCT02530047 Ovarian cancer
NCT01844661 Metastatic and refractory solid tumor
NCT02337985 AIDS-related non-Hodgkin’s lymphoma
NCT02378922 Relapsed or refractory acute myeloid leukemia
NCT02343666 Lymphoma with HIV infection
Downloaded from http://neuro-oncology.oxfordjournals.org/ at University of Cambridge on June 11, 2016
The use of genetically engineered mouse brain tumor models
in which tumors arise in situ or incorporation of surgical resec-
tion into GBM tumor studies might hold more validity and need
exploration.
In clinical settings, the safety of the grafted SC is a major
concern as there are still scientifically contentious issues re-
garding basic SC biology. Of particular concern is whether SCs
promote the growth of certain tumors141 – 143 or indeed form
tumors themselves.144,145 Importantly, non-immortalized
adult stem cells provide fewer safety concerns than immortal-
ized adult stem cells and may be used without posing a risk to
the patient. The incorporation of suicide genes such as HSV-TK
into therapeutic SCs would enable their controlled eradication
post treatment and alleviate this concern. Because patient
safety is vital, these issues should be resolved before starting
the next generation of clinical trials.
Funding
This work was supported by R01CA138922 and R01CA173077.
Acknowledgments
We thank Dr. Sung Hugh Choi and Dr. Clemens Reinshagen with their
help with literature search for the manuscript and Anusha
Venogopalan for their help with the references for this review. We
also thank Dr. Daniel Stuckey for his help with literature search and
Fig. 2 of the review.
Conflict of interest statement. There is no conflict of interest.
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