Neuroblastoma Cell Lines Contain Pluripotent Tumor
Initiating Cells That Are Susceptible to a Targeted
Yonatan Y. Mahller1,2,4,5, Jon P. Williams2, William H. Baird1,2,4,5, Bryan Mitton1,4, Jonathan Grossheim3,
Yoshinaga Saeki6, Jose A. Cancelas2, Nancy Ratner2, Timothy P. Cripe1,2*
1Division of Hematology and Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America, 2Division of Experimental Hematology,
Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America, 3Division of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati,
Ohio, United States of America, 4Physician Scientist Training Program, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 5Graduate
Program of Molecular and Developmental Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 6Dardinger Laboratory for
Neuro-Oncology and Neurosciences, Department of Neurological Surgery and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States of
Background: Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common.
The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for
relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of
the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor
cells has been found in a variety of cancers.
Methodology/Principal Findings: Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell
media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three
lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a
verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere
formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next
generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may
represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex
virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in
athymic nude mice.
Conclusions/Significance: These results suggest that human neuroblastoma contains tumor initiating cells that may be
effectively targeted by an oncolytic virus.
Citation: Mahller YY, Williams JP, Baird WH, Mitton B, Grossheim J, et al. (2009) Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are
Susceptible to a Targeted Oncolytic Virus. PLoS ONE 4(1): e4235. doi:10.1371/journal.pone.0004235
Editor: Torbjorn Ramqvist, Karolinska Institutet, Sweden
Received August 28, 2008; Accepted December 10, 2008; Published January 21, 2009
Copyright: ? 2009 Mahller et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Cincinnati Children’s Hospital Medical Center Division of Hematology/Oncology, by TeeOffAgainstCancer.org, and NIH
grants R01-CA114004 and R21-CA133663 to TPC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org.
Neuroblastoma is the most common extracranial solid tumor
found in children, accounting for 8–10% of childhood cancers.
The median age of diagnosis is 17.3 months, with 40% of patients
diagnosed as infants and 98% by 10 years of age [1,2]. Most
children with low or intermediate risk neuroblastoma achieve
remission via a combination of surgery, radiation, and chemo-
therapy, and some children with high risk disease also benefit from
megadose chemotherapy with autologous hematopoietic stem cell
rescue . Unfortunately, due to therapy-resistant relapse, long
term survival in high risk cases is ,50%. Risk factors indicative of
poor prognosis include age .18 months, higher stage of the
disease, MYCN amplification, and unfavorable histology .
It has been hypothesized for many cancers that tumor cells
responsible for failures in long-term remission exhibit stem cell
properties. Considerable evidence supports the hypothesis that
leukemias arise from stem or progenitor cells, in an analogous
fashion to hematopoiesis . Although hematopoiesis is the most
thoroughly studied stem cell system, many other organs are
maintained by locally resident stem cells characterized by
asymmetric cell division and multi-lineage differentiation .
Reports have shown that cancer cells expressing stem markers
exhibit chemoresistance, thus supporting the notion that undiffer-
entiated cells are responsible for clinical relapse .
There is now mounting data supporting the cancer stem cell
theory for solid tumors . Historically researchers have injected
.106tumor cells to create xenograft models, but recent reports
PLoS ONE | www.plosone.org1 January 2009 | Volume 4 | Issue 1 | e4235
have identified sub-populations requiring as few as 10–200 cells to
form tumors [9,10,11,12,13]. Tumor formation may be analogous
to the development of normal tissues in that stem cells generate an
organ with cellular diversity in marker expression, differentiation,
therapeutic resistance and metastatic potential. Autonomous
growth in serum-free media, as non-adherent neurospheres, is
thought to be a surrogate marker of neural and malignant
progenitors [9,14,15,16,17,18]. Identification of normal and
tumorigenic stem cells has also been studied by side population
analysis in a technique that exploits the increased drug efflux
capacity of stem cells [19,20,21,22,23,24]. Highly tumorigenic
cells often express CD133, a five transmembrane domain
glycoprotein also expressed by untransformed hematopoietic and
neural progenitors [11,13,23,25]. CD133 expressing glioblastoma
cells were significantly resistant to chemotherapeutic agents
compared to cells not expressing this marker . Other cancer
stem cells have been described to express markers including CD20
and CD44+CD242/lineage low for melanoma and breast cancer
stem cells, respectively [10,12]. Cancer cell subpopulations based
only upon marker expression are likely to be inaccurate due to
antigenic plasticity and therefore studies based upon functional
assays are likely more reliable to identify tumor stem cells.
The prognostic significance of cellular heterogeneity of the
neural crest lineage cells in neuroblastoma has begun to be
described . A sub-population of so-called intermediate (I-type)
neuroblastoma cells showed increased tumorigenicity and patients
whose tumors contained a higher percentage of these cells had
increased relapse . Interestingly, these cells expressed CD133
and showed asymmetric cell division [28,29]. Other studies
revealed that neuroblastoma cells express neural precursor
markers including CD34, ABCG2, and nestin [7,16,18,30].
Further evidence that neuroblastoma is a stem cell disease is the
finding of side populations in 65% of primary neuroblastoma
samples . These data suggest that further characterization of
neuroblastoma stem cells is warranted and such studies may be
critical for improvement of anti-cancer therapeutics.
Tumor-targeted viral therapies have progressed rapidly to
clinical trials in recent years [31,32]. These biologic therapeutics
demonstrate widespread tumor tropism . We and others have
shown neural tumors are sensitive to oncolytic Herpes simplex
virus (oHSV) mutants and further hypothesized that these viruses
may effectively destroy drug-resistant neuroblastoma tumor
initiating cells [34,35,36,37,38,39]. Herein we identified and
isolated populations of human neuroblastoma cells that expressed
CD133 and grew as clonal spheres. Tumorsphere-derived cells
showed doxorubicin resistance and multi-lineage differentiation.
CD133 expressing cells demonstrated increased tumorsphere
forming ability and tumorigenicity in athymic nude mice. Finally,
a nestin-targeted oHSV effectively replicated within and killed
neuroblastoma tumor initiating cells as measured by prevention of
Materials and Methods
Cells and viruses
Vero and human neuroblastoma (LA-N-5, IMR-32, SKNBE(2),
CHP-134, SHSY5Y, SKNSH, CHLA-20, CHLA-79) cells were
gifts from Thomas Inge (Cincinnati Children’s Hospital Medical
Center, Cincinnati, OH) and Robert Seeger (Children’s Hospital
of Los Angeles, Los Angeles, CA); their origins and culture
conditions have been described [40,41]. MYCN status was
confirmed in each cell line by fluorescence in situ hybridization
(data not shown). rQLuc and rQNestin34.5 were created using a
BAC based approach and have been described [42,43]. Tumor-
sphere-media consisted of a 50:50 mix of F12 and DMEM
(Invitrogen, Carlsbad, CA), supplemented with 20 ng/ml EGF
(R&D systems, Minneapolis, MN), 40 ng/ml bFGF (R&D
systems), 1% B27 and N2 supplements (Invitrogen), 2 mg/ml
heparin (Sigma, St. Louis, MO), 0.1 mM b-mercaptoethanol
(Sigma) and 16 antibiotic/antimycotic (Mediatech, Herndon,
VA). Tumorspheres were dissociated using non-enzymatic disso-
ciation solution (Sigma) and strained over a 40 mM filter. For
tumorsphere formation assays erlotinib at 10 mM or L685,458
(Sigma) at 1 mM were utilized in DMSO at a final concentration
0.1%. Cell viability and HSV replication assays were performed as
previously described .
Flow cytometry and side population (SP)
100, 10 or 1 cell(s) were plated in 150 ml of neurosphere media
in 96-well dishes via FACS Vantage. Side population was
performed by staining cells at 16106/ml with 5 mg/ml Hoechst
33342 (Sigma) +/2verapamil at 75 ng/ml (Sigma) for 45–60 min
at 37uC. Cells were pelleted and stained with propidium iodide
(1 mg/ml) prior to analysis. Cell analysis and purification was
performed using a FACSVantage SE equipped with UV and 488
excitation lasers and DIVA software. Hoechst excitation and
emission were performed as described ; the UV laser was set to
50 mW and pressure was adjusted to 13 psi. Quantification of cell
markers were performed by FACSCaliber using anti-CD133
(Miltenyi Biotech, Gladbach, Germany), and ABCG2 (Stemcell
Technologies, Vancouver, Canada), anti-CD334 (BD Biosciences,
San Jose, CA), and anti-nestin (R&D Systems, Minneapolis, MN),
according to the manufacturers’ instructions, with cut-off gates
based on isotype controls. For the nestin studies, cells were first
permeabilized using the intracellular reagents protocol (R&D
Uninfected and rQNestin34.5-infected tumorspheres were fixed
in glutaraldehyde overnight. Scanning EM samples were sputter
coated with a Gold-Palladium alloy, sectioned and viewed using a
Hitachi Model S-3000N Scanning EM. Transmission EM samples
were heavy metal stained with Uranyl Acetate and Lead Citrate,
sectioned and viewed using a Hitachi Model H-7600 Transmission
EM. Sputter coater was a Denton Vacuum Desk IV.
Differentiation and staining
Clonal spheres were fixed (4% PFA overnight), cryoprotected
with 20% sucrose for 12 h, embedded in OCT and sectioned at a
thickness of 12 mM. Tumorsphere sections were incubated with
rabbit anti-nestin (Chemicon International, Temecula, CA) at
1:200 dilution followed by goat anti-rabbit FITC (Jackson
Immunoresearch, West Grove, PA). For differentiation experi-
ments, clonal spheres were dissociated and plated into chamber
slides coated with poly-lysine and fibronectin (R&D Systems). Cells
were grown in media as per Williams et al.  containing 10%
serum +/2the following factors: neurogenic, bFGF 20 ng/ml for
5–7 days then 10 ng/ml BDNF, NGF, and NT3 for 7 days;
gliogenic, forskolin 2 mM, b-heregulin 5 ng/ml, insulin 10 ng/ml;
or fibroblastic, bFGF20 ng/ml and TGF-b 10 ng/ml. Cells were
fixed and stained with rabbit anti-neurofilament-M (1:200,
Chemicon), mouse anti-GFAP (1:500, Chemicon), rabbit anti-
S100b (1:2000, Chemicon) and mouse anti-SMA (1:500, Sigma),
followed by host-appropriate FITC or TRITC secondary (Jackson
Immunoresearch) and co-stained with DAPI (Sigma). Images were
taken using a Zeiss inverted fluorescence microscope and Openlab
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In vivo models
Experiments were approved by the CCHMC Institutional
Animal Care and Use Committee. 5,000 CD133+or CD1332
neuroblastoma cells in 30% Matrigel (BD Biosciences) were
injected subcutaneously in 5–6 week old female athymic mice
(Harlan, Indianapolis, IN). To determine if oHSV infection could
prevent tumor formation, cells were infected ex vivo at 5 plaque
forming units per cell, incubated for 5 hrs, mixed with 30%
Matrigel (BD Biosciences) and injected subcutaneously in 5–6
week old female athymic mice (Harlan, Indianapolis, IN). Tumor
development was monitored 3 times per week and volume
calculated by V=(L6W2)6p/6.
Comparison between two means was performed with an
unpaired student’s t test and more than two means by ANOVA.
For small groups comparing tumor formation frequency, Fisher’s
Exact Test was used. All statistics were done using SPSS13.0.
Neuroblastoma cell lines show evidence of a cancer stem
We surveyed a panel of neuroblastoma cell lines for subpop-
ulations by assessment of stem cell marker (CD34, CD133, Nestin)
expression, the presence of a verapamil sensitive side population
and the ability to form clonal spheres. All cell lines expressed stem
cell markers and side populations were detected in most (Table 1).
Half of the lines readily formed tumorspheres when plated at
clonal density in serum-free neural stem cell conditions. Interest-
ingly, this result seemed to correlate with MYCN amplification
(Table 1). Tumorsphere-formation, cell surface marker expression
and side-population cells were further investigated using the LA-
N-5 cell line as it showed MYCN amplification, contained CD34/
CD133/nestin expressing cells and formed clonal tumorspheres.
Neuroblastoma cells form tumorspheres at clonal
When plated above clonal density (10 cells/ml) many spheres
formed by 1 week (Fig. 1A). Spheres ranging in size from 40 to
300 uM in diameter were observed and when dense often
coalesced. Compared with bulk cultured cells, sphere-derived cells
showed more cellular homogeneity in scatter patterns suggesting an
element of cell selection (Fig. 1B). Serial passage showed enrichment
for sphere-forming ability (p,0.0001 for passage 1 vs. 2 and 2 vs. 3),
followed by a plateau, likely due to super-clonal plating density and
sphere coalescence (confirmed by live video microscopy) (data not
shown), a feature of normal neurospheres . Also similar to bona
fide untransformed neurospheres [47,48], serial passage of LA-N-5
tumorspheres was dependent upon both gamma secretase activity
and epidermal growth factor receptor (EGFR) signaling (*p,0.003)
(Fig. 1C). To confirm clonality in tumorsphere formation,
neuroblastoma cells (LA-N-5, IMR-32, CHP-134) were plated at
100, 10 and a single cell per well, by FACS Vantage cell sorter. All
three cell lines showed clonal tumorsphere formation. LA-N-5
tumorsphere forming efficiency appeared steady regardless of
plating density, with 30–40% of cells capable of forming spheres
when plated at 100 or 10 cells per well, and 30% of wells receiving a
single cell showing clonally derived spheres (Fig. 1D). Therefore
human LA-N-5 neuroblastoma cells grown in serum-free media
exhibited c-secretase and EGFR-dependent clonal growth as
tumorspheres and were enriched via serial passage. These studies
highlight the heterogeneic nature of these tumorspheres as only
,30% of cells reformed secondary spheres.
Tumorsphere ultrastructure reveals dynamic three-
Electron microscopy (EM) on LA-N-5 neuroblastoma tumor-
spheres revealed similarities between tumorspheres and neuro-
spheres. Scanning and transmission EM of the tumorsphere
surface showed a smooth and uniform surface and revealed
microvilli-like structures and apopotic cells (Fig. 2), which have
also been reported in bona fide neurospheres [16,46].
Clonally-derived neuroblastoma tumorspheres show
Clonally-derived tumorspheres (LA-N-5, IMR-32, CHP-134)
were dissociated, plated on chamber-slides and grown in media
containing serum alone or supplemented with neurogenic,
gliogenic or smooth muscle fibroblastic growth factors. The
cultures were stained with antibodies against various cytoskeletal
and membrane proteins that have previously been validated as
markers of neural lineage differentiation pathways, including
neurofilament-M (NF-M) as a marker of neurogenic cell
differentiation, S100b as a marker of Schwannian cell differenti-
ation, glial fibulary acid protein (GFAP) as a marker of glial
Table 1. Stem cell-like characteristics of human neuroblastoma cell lines.
Cell Line MYCNamp
SK-N-BE(2)40.62.6 2.05.4 80.8 Yes
IMR-3280.1 21.321.0 n.d. 52.4Yes
21.31.6 1.3n.d.59.7 No
40.5 3.93.616.9 68.7No
5.1 2.40.7 11.0 49.3No
30.347.2 20.340.5 54.9No
MYCNamp, amplification of the MYCN gene.
DP, double positive for CD34 and CD133.
Side pop, presence of a verapamil-sensitive side population on flow cytometry with Hoescht stain.
n.d., not done.
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differentiation, and smooth muscle actin (SMA) as an indicator of
fibroblastic differentiation [6,45,49,50] (Fig. 3). All three cell lines
grown in both serum and serum supplemented with neurotrophic
factors expressed NF-M, though there was more evidence of
neuronal-like morphologic changes in the supplemented cultures
(Fig. 3, top row, best seen in IMR-32). There was a varied
response of the different lines under gliogenic conditions: in LA-N-
5 cells, GFAP but not S100b expression was clearly induced, in
CHP-134 cells both appeared albeit only faintly, and in IMR-32
cells both were present at baseline but only S100b appeared after
exposure to the factors (Fig. 3, middle row). Under fibroblastic
conditions, all three cell lines showed marked induction of SMA
(Fig. 3, bottom row). IMR-32 cells also showed a more elongated,
spindly morphology under these conditions. These data suggest
neuroblastoma cell lines harbor a subset of progenitor cells capable
of multi-lineage differentiation.
Tumorsphere cells are more resistant to doxorubicin and
are enriched for CD133 expression
We sought to determine if tumorsphere-derived cells were
resistant to a cytotoxic chemotherapeutic, as might be expected
for cancer stem cells . Growth of LA-N-5 cells as bulk or
tumorspheres in the presence of doxorubicin revealed that tumor-
sphere-derived cells exhibited relative resistance compared to bulk
cells (Fig. 4A). Investigationof cell surface marker expressionin bulk
and tumorsphere-derived LA-N-5 cells revealed that tumorspheres
are comprised of a more distinct subpopulation of cells expressing
the drug efflux channel ABCG2, but the percentage of positive cells
was only 2.9-fold increased, suggesting this is not likely to be a
predominantmechanismresponsibleforthe altered sensitivity.Cells
exposed to doxorubicin also showed a small increase in cells
expressing the CD133 (from 18.9% to 27.1%, Fig. 4B).
Side population studies in neuroblastoma cells reveal
asymmetric cell division
Expression of ATP-binding cassette transporters such as
ABCG2 have been associated on flow cytometric analysis of
Hoescht staining with the presence of a so-called ‘‘side population’’
(SP), thought to be enriched for stem cells in some tissues .
Thus, the presence of ABCG2 expression suggests that LA-N-5
cultures may contain an efflux channel-dependent SP. Indeed,
approximately 1% of bulk LA-N-5 cells appeared in a verapamil-
sensitive SP, which was nearly 2-fold enhanced following addition
of doxorubicin to culture media (10 ng/ml for 7–12 days) (Fig. 5A).
Most other tested neuroblastoma cell lines showed the presence of
a verapamil-sensitive SP, ranging from 2.5–40.5% (Table 1).
Characterization of cell surface expression of ABCG2 and CD133
in bulk and doxorubicin-treated LA-N-5 cultures revealed a 4.8-
fold increase of cells expressing ABCG2 and a 2.2-fold increase in
double-positive cells (Fig. 5B). Conversely, doxorubicin exposure
significantly reduced the percentage of CD133 expressing cells,
indicating that a large number of CD133 positive cells are indeed
doxorubicin-sensitive. Further, these studies imply that CD133
expressing cells will reside in both the SP as well as the non-SP.
We next evaluated sphere-forming ability of SP and non-SP cells
growninneurosphereconditionsfor2weeks.Sorted SPand non-SP
(NSP) cells from bulk cultured LA-N-5 showed no difference in
sphere-forming ability, while SP and non-SP cells from LA-N-5 cells
cultured with doxorubicin showed enrichment of sphere-forming
ability for SP cells and total loss of sphere formation in the non-SP
(Fig. 5C). Increased sphere-formation in the doxorubicin-cultured
SP may reflect the doxorubicin-induced increase in cells expressing
both CD133 and ABCG2, possibly cells with higher sphere-forming
ability. Further, decreased sphere-formation in the doxorubicin-
cultured non-SP cells may reflect the doxorubicin-mediated
decrease in total cells expressing CD133.
Figure 1. Neuroblastoma cells form tumorspheres in serum-free media. (A) LA-N-5 neuroblastoma cells plated in serum supplemented
media show adherent growth while those plated in serum-free media (with EGF and bFGF) formed non-adherent tumorspheres. (B) Dissociated bulk
and tumorsphere cells were subjected to FACS analysis. Tumorsphere cells showed increased uniformity in complexity (low side scatter) compared to
bulk cultured cells. (C) Tumorsphere formation over serial passage (plating at supra-clonal density) in neurosphere media alone or supplemented with
a c-secretase inhibitor (L685,458, 1 mM) or an EGFR inhibitor (erlotinib, 10 mM). The inhibitors were dissolved in DMSO, which was used as a negative
control. (D) Plating of dissociated LA-N-5 tumorsphere cells at 100 or 10 cells per well showed consistent sphere-forming efficiency regardless of
plating density; 30% of wells plated with a single cell contained a single sphere. Pictures show examples of clonally derived spheres.
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As a test to determine asymmetric cell division, we cultured LA-
N-5 SP and non-SP cells in serum containing media for 2 weeks and
then re-analyzed them by SP analysis. Cultures initially derived
from LA-N-5-SP cells regenerated both SP and NSP cells. In
contrast, LA-N-5-NSP derived cultures only regenerated NSP cells
(Fig. 5D). Similar findings of asymmetric cell division were observed
in doxorubicin-treated LA-N-5 cultures. These data reveal that
neuroblastoma cells are relatively resistant to doxorubicin, express
CD133 and ABCG2 and are capable of asymmetric cell division.
CD133 expressing cells show increased sphere and
To determine if CD133 expressing cells showed functional
differences from CD133 null cells, we sorted bulk LA-N-5 cells for
CD133 and analyzed their ability to form tumorspheres and
xenografts. At a plating density of 1.5 cells/ml, CD133 expressing
cells exhibited increased sphere formation (Fig. 6A). Animals
implanted with 5,000 CD133 expressing LA-N-5 cells demon-
strated earlier tumor formation and larger size compared to
CD133 null cells (p=0.03), though the frequency of tumors did
not reach statistical significance in this small experiment (p=0.13
by Fisher’s Exact test) (Fig. 6B, C).
A transcriptionally-targeted oncolytic HSV kills
neuroblastoma initiating cells
Based on nestin expression in primary neuroblastoma tumors
and tumorspheres, we hypothesized that a nestin-targeted oHSV
Figure 2. Ultrastructural characterization of neuroblastoma tumorspheres shows similarities to normal neurospheres. (A) Scanning
EM of neuroblastoma tumorsphere shows a smooth and uniform surface. (B) Microvilli-like structures shown on the tumorsphere surface by
transmission EM in cross-section. (C) Transmission EM shows tight packing of tumor cells in the tumorsphere core. (D) Apoptotic tumorsphere cell
shows nuclear blebbing, chromatin fragmentation and mitochondrial swelling.
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would be effective in killing both differentiated and tumor
initiating neuroblastoma cells. rQNestin34.5 was initially created
to target oHSV killing to nestin-positive brain tumor cells . In
this recombinant virus, expression of the HSV-1 neurovirulence
gene (encoding c134.5) is driven by the nestin enhancer , and
the virus remains attenuated in normal cells by a deletion in the
virally-encoded large subunit of ribonucleotide reductase (ICP6).
LA-N-5 neuroblastoma tumorspheres were positive for nestin by
immunostain and all other tested neuroblastoma cell lines showed
significant nestin expression (Fig. 7A and Table 1). Neuroblastoma
tumorspheres were readily transduced by rQNestin34.5 (Fig. 7B,
C). As predicted based on nestin expression, rQNestin34.5 caused
significant cell death of both bulk and tumorsphere-derived LA-N-
5 cells compared with the control virus, rQLuc (*p,0.05) (Fig. 8A).
Virus replication was similar in bulk cultured LA-N-5 cells. In
contrast, production of rQNestin34.5 was .10-fold increased
compared with rQLuc in tumorsphere-derived cells (*p,0.05)
(Fig. 8B). New virus production was confirmed to occur in
tumorsphere cells by transmission EM of a virus-infected tumor-
sphere, demonstrating that virus isn’t simply being produced
exclusively in non-sphere cells present in the cultures. These cells
showed viral nucleocapsids in the nucleus, particles in the
cytoplasm acquiring their envelope and fully enveloped HSV
particles (Fig. 7D, E).
Because the tumor initiating cells may represent only a small
subpopulation of the cultures, we sought to determine if oHSV
infection was truly affecting these cells. Cells were harvested,
infected with oHSV ex vivo and injected subcutaneously into mice
to detect tumorigenic cells. While rQLuc-treatment caused a
significant delay in tumor formation compared with saline (from a
mean of 25 to 35 days, p,0.001), treatment with rQNestin34.5
abolished tumor formation for .60 days (p,0.001, Fig. 8C),
suggesting that the virus was capable of destroying the neuroblas-
toma tumor initiating cells present in the culture.
Human neuroblastoma is known for its striking tumor cell
heterogeneity and ability to relapse. These features suggest that
neuroblastoma may be a stem cell disease. Herein we describe
studies using human neuroblastoma cell lines suggesting they
contain subpopulations of cells with characteristics shared with
normal neural stem cells. All cell lines tested contained cells that
Figure 3. Clonally-derived neuroblastoma tumorsphere cells show multi-lineage differentiation. Clonally-derived tumorspheres from
three different neuroblastoma cell lines were dissociated and cells were plated on poly-lysine and laminin coated chamber slides. Culture conditions
were media containing serum alone or serum with neurotrophic factors (top row), gliogenic factors (middle row) or fibroblastic factors (bottom row)
(except negative controls, which were serum alone without factors). Slides were stained with neurofilament-M (NF-M, green, top row), GFAP (red,
middle row) S100b (green, middle row), or smooth muscle actin (red, bottom row); each were also co-stained with DAPI (blue). Negative control
cultures were incubated without primary antibody and with secondary anti-mouse TRITC or anti-rabbit FITC. Arrows in the top and bottom rows
indicate spindle-like cell extensions consistent with either neuronal or fibroblastic differentiation, while arrows in the middle row indicate positively
stained cells. On close inspection of the GFAP/S100b stains, IMR-32 cells under serum conditions and CHP-134 cells supplemented with factors show
co-staining with a mixture of green/red signals. Scale bars=65 microns.
Figure 4. Neuroblastoma tumorsphere-derived cells are doxo-
rubicin resistant and express ABCG2 and CD133. (A) LA-N-5 bulk
and tumorsphere-derived cells were assessed for doxorubicin sensitivity
by MTT assay at day 7. (B) Analysis for ABCG2 and CD133 expression in
neuroblastoma cells grown as bulk culture or as tumorspheres.
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express markers previously described on neural stem cells such as
CD133, ABCG2, and nestin. Half of the cell lines were capable of
growing as ‘‘tumorspheres’’ in neural stem cell media, and three of
three tested were capable of multi-lineage differentiation. One line
tested further showed that the CD133 positive cells were enriched
for tumorigenicity and that the spheres were enriched for a
verapamil-sensitive side population, CD133 expression and
doxorubicin resistance. Infection with a nestin promoter-directed
oncolytc herpes virus prevented tumor formation, suggesting the
tumor initiating cells were targetable by virotherapy.
We observed clonal growth in serum-free neurosphere media in
four of eight neuroblastoma cells lines tested, which correlated with
their MYCN amplification status. It is likely that MYCN expression
plays a role in neuroblatoma stem cells given the recently described
role of myc-regulated gene networks in generating induced
pluripotent stem cells from somatic tissues  and the role of
myc in the maintenance of both normal hematopoietic stem cells
 and glioma cancer stem cells . Growth of LA-N-5
neuroblastoma cells as non-adherent, nestin positive, multi-potent
tumorspheres was dependent upon c-secretase and EGFR signal-
ing, similar to neural stem cells . Tumorsphere-derived
neuroblastoma cultures were enriched for stemness markers
CD133 and ABCG2 in comparison to bulk-grown cells. Tumor-
sphere-derived cells showed relative resistance to doxorubicin.
As predicted, addition of doxorubicin to culture media of
neuroblastoma cells increased the percentage of side population
cells and correspondingly enriched cultures for ABCG2 expressing
cells. Although CD133 positive and doxorubicin-sensitive cells
were dramatically reduced in the presence of doxorubicin, those
expressing ABCG2 and CD133 were enriched. These results
demonstrate that the CD133 positive population is heterogeneous
in expression of ABCG2 and that CD133 expression alone is not
sufficient to identify neuroblastoma tumor initiating cells. CD133
as been identified as a marker of cancer stem cells in some models
but not others, and its utility as a single marker of such cells is
controversial . Some studies have shown that CD133
expressing human cancer cells show chemoresistance [26,56,57].
In a tumorsphere formation assay, sorted SP and non-SP cells
Figure 5. Side population analysis reveals asymmetric cell division of neuroblastoma cells. (A) Side population analysis using Hoechst
33342, +/2verapamil, of bulk LA-N-5 cells (LAN5) and LA-N-5 cells grown in doxorubicin (LAN5-doxR). (B) Analysis of ABCG2 and CD133 expression in
LAN5 and LAN5-doxRcells. (C) Sphere-forming efficiency of sorted SP and non-SP (NSP) cells from LAN5 and LAN5-doxRcultures. (D) Sorted SP and
NSP cells, from cultures of LAN5 and LAN5-doxR, were plated in serum containing media for 2 weeks and re-evaluated by side population, +/
2verapamil. SP-derived cultures show regeneration of SP and NSP cells, while NSP-derived cultures showed only regeneration of NSP cells, not SP
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from bulk cultured LA-N-5 cells showed no difference, suggesting
that sphere-forming cells exist in both. In contrast, SP cells from
doxorubicin-treated LA-N-5 cultures showed enriched sphere
forming ability compared with bulk culture SP cells, likely due to
increased cells expressing ABCG2 and CD133. Further, non-SP
cells from doxorubicin-treated LA-N-5 cultures lost the ability to
form spheres, possibly due to doxorubicin-mediated destruction of
ABCG2 null, CD133 expressing cells. These results demonstrate
that doxorubicin treatment exerts multiple effects. First, it enriches
the culture for cells with drug efflux ability and those able to form
tumorspheres. Second, it diminishes the culture of cells lacking
drug efflux ability but capable of forming spheres. Thus, side
population or drug efflux status and sphere forming efficiency may
be related at least partially via CD133 expression.
Asymmetric cell division is a key property of both normal and
tumor stem cells. We observed that sorted SP-derived cultures
could regenerate their non-SP counterparts, while non-SP-derived
cultures could not. CD133 expressing cells showed increased
ability to form tumorspheres and xenograft tumors in immuno-
deficient mice, suggesting increased tumorigenicity. Overall, these
studies support the presence of cells with stem cell-like features in
neuroblastoma and suggest that multiple criteria be utilized in
parallel to further identify, isolate and characterize such cells.
Because cancer stem cells are believed responsible for tumor
metastasis, escape from anticancer therapies and ultimately disease
relapse, their therapeutic targeting is crucial [58,59]. Small
molecule inhibitors designed to target signaling pathways regulat-
ing stem cell renewal and maintenance may increase efficacy of
traditional therapies. Hypothetical targets for future therapies
include signaling via Notch, EGFR, Wnt, Hedgehog and bmi-1
. Alternatively, it is conceivable that tumor initiating cells may
be targeted via tumor cell-specific expression of stem cell surface
markers using toxin-coupled antibodies. Combination of current
chemotherapeutics with efflux blocking agents, such as calcium
channel blockers, may enhance antitumor efficacy.
Another strategy for targeting cancer stem cells could be to
utilize biologics. Oncolytic viruses are attractive anticancer
therapeutics due to their ability to replicate in vivo, thereby
amplifying the injected dose. These viruses have shown efficacy
and safety in clinical trials [61,62]. As drug-resistant tumor
initiating cells have been reported to exist in a perivascular niche,
intravenous administration of an oncolytic virus may be highly
effective to reach this site . In addition, because oncolytic
viruses circumvent traditional chemotherapy resistance mecha-
nisms, they have been thought to be ideal for targeting cancer stem
cells . As replication and toxicity of oncolytic viruses may be
targeted via tumor specific promoters, we rationalized that such a
virus could be targeted against neuroblastoma stem cells. In this
study we demonstrated that a nestin-targeted oHSV efficiently
infected, replicated and killed neuroblastoma tumorsphere cells.
Ex vivo infection of neuroblastoma cells with a nestin-targeted
oHSV resulted in death of tumor initiating cells as it prevented
tumor development in animals. Our study used cell lines derived
from neuroblastomas, so it will be important to verify our results
using primary human samples. Spheres derived from bone
marrow metastases of patients with neuroblastoma have been
shown to be highly enriched for tumor initiating cells ,
suggesting these cultures may be useful for such validation studies.
Like several other cancers, it is becoming increasingly evident
that neuroblastoma is a stem cell disease; thus, therapeutic
targeting of cells that cause relapse is vital to improve patient
outcome. Further study of neuroblastoma stem cells should reveal
their roles in tumor initiation, progression and metastasis. As
anticancer therapies incorporate anti-stem cell approaches, it will
be important to ensure that these treatments do not adversely
affect the function of normal tissue stem cells. Development of
such innovative strategies to target cancer stem cell populations in
human malignancies is likely to increase treatment success.
Figure 6. CD133 expressing neuroblastoma cells show in-
creased sphere and tumor formation. Cells sorted for CD133
expression were assayed for (A) tumorsphere formation and (B)
tumorigenicity following flank implantation of 5,000 cells in immuno-
deficient mice. Quantification of tumor volume and frequency were
performed at 21 days post-inoculation. Numerators indicate number of
flanks with tumors and denominators total number of flanks injected.
(C) Image of mouse injected with CD133 expressing cells on the left
flank and CD133 null cells on the right flank.
Figure 7. Neuroblastoma tumorspheres express nestin and are
efficiently infected by oHSV. (A) Immunohistochemistry on tumor-
sphere cryosection for nestin (green) and DAPI co-stain (blue). Scale
bar=10 microns. (B, C) A neuroblastoma tumorsphere infected with
rQNestin34.5 was imaged at 48 hours post-infection by (B) phase-
contrast and (C) fluorescent microscopy for GFP. (D, E) rQNestin34.5-
infected neuroblastoma tumorsphere at 48 hours post-infection
evaluated by transmission electron microscopy showing viral nucleo-
capsids in the nucleus (arrows), and mature HSV particles in the
cytoplasm in the process of acquiring their envelopes (arrows).
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PLoS ONE | www.plosone.org8 January 2009 | Volume 4 | Issue 1 | e4235
Special thanks to Dan Marmer, Sue Vergamini and Barb Lawall at the
Cincinnati Children’s Hospital Medical Center flow cytometry core, to Dr.
Jianqiang Wu for guidance in experimental design and to Mark Currier,
Jen Leddon, and Jill Fitzpatrick for laboratory assistance.
Conceived and designed the experiments: YYM JPW TPC. Performed the
experiments: YYM JPW WHB BM JG. Analyzed the data: YYM JPW
WHB BM JAC NR TPC. Contributed reagents/materials/analysis tools:
JPW YS JAC NR. Wrote the paper: YYM TPC.
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