Notch Pathway Inhibition Depletes Stem-like Cells and
Blocks Engraftment in Embryonal Brain Tumors
3and Charles G. Eberhart
Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York
2Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland and
The Notch signaling pathway is required in both nonneo-
plastic neural stem cells and embryonal brain tumors, such
as medulloblastoma, which are derived from such cells. We
investigated the effects of Notch pathway inhibition on
medulloblastoma growth using pharmacologic inhibitors of
;-secretase. Notch blockade suppressed expression of the
pathway target Hes1 and caused cell cycle exit, apoptosis, and
differentiation in medulloblastoma cell lines. Interestingly,
viable populations of better-differentiated cells continued to
grow when Notch activation was inhibited but were unable to
efficiently form soft-agar colonies or tumor xenografts,
suggesting that a cell fraction required for tumor propagation
had been depleted. It has recently been hypothesized that a
small population of stem-like cells within brain tumors is
required for the long-term propagation of neoplastic growth
and that CD133 expression and Hoechst dye exclusion (side
population) can be used to prospectively identify such tumor-
forming cells. We found that Notch blockade reduced the
CD133-positive cell fraction almost 5-fold and totally abol-
ished the side population, suggesting that the loss of tumor-
forming capacity could be due to the depletion of stem-like
cells. Notch signaling levels were higher in the stem-like cell
fraction, providing a potential mechanism for their increased
sensitivity to inhibition of this pathway. We also observed that
apoptotic rates following Notch blockade were almost 10-fold
higher in primitive nestin-positive cells as compared with
nestin-negative ones. Stem-like cells in brain tumors thus
seem to be selectively vulnerable to agents inhibiting the
Notch pathway. (Cancer Res 2006; 66(15): 7445-52)
Medulloblastoma and other embryonal brain tumors are thought
to arise primarily from neural stem/precursor cells of the
ventricular zone and cerebellar external germinal layer (1–3).
Pathways such as Wingless, Hedgehog, and Notch, which control
the specification, proliferation, and survival of nonneoplastic
neural precursors, are also all aberrantly activated in such tumors,
suggesting a molecular link between neural stem cells and
medulloblastoma (4–9). These developmentally significant signal-
ing pathways are attractive therapeutic targets. For example,
several groups have shown that pharmacologic inhibitors of Hh
signaling block the proliferation and survival of medulloblastoma
in vitro and in vivo (10–12). Such therapeutic advances are clearly
needed, as current embryonal brain tumor treatments are
associated with significant side effects and often do not result in
In addition to arising from stem or precursor cells, medullo-
blastoma may also contain functionally important subsets of
cells with stem-like properties. It has recently been hypothesized
that in many types of cancer, including medulloblastoma, such
stem-like cells are uniquely capable of propagating tumor growth
(16–18). This ‘‘cancer stem cell’’ hypothesis is based, in part, on
paradigms explaining the development and repair of normal
organs, in which only stem cells are thought to have a long-term
capacity for self-renewal and are therefore necessary for the
generation and regeneration of tissues. Experimental support for
the existence of a discrete cancer cell subpopulation required
for tumor propagation initially came from clonogenic assays,
which showed that only a small portion of tumor cells could form
colonies in vitro or engraft in vivo (19–21). Only recently the
prospective isolation of cells uniquely capable of propagating
neoplasms has been achieved, providing firmer support for
the presence of cancer stem cells (22, 23). This has been made
possible by the characterization of markers that identify stem-like
cells in tumor specimens. Two such markers, CD133 and side
population, have been used to prospectively isolate a small
percentage of cells in brain tumors uniquely able to generate
tumor neurospheres and xenografts (24–27). Interestingly,
both markers were initially identified in nonneoplastic stem cells
(28–31), highlighting the similarities between normal and
malignant stem cells.
To date, no agent has been shown to target the cancer stem cell
subpopulation in solid tumors. However, similarities in the growth
characteristics and gene expression patterns of benign neural
stem cells and brain tumor stem cells suggest that the same
signaling pathways may be required for survival and growth in
both (18, 32–35). Notch is known to promote the survival and
proliferation of nonneoplastic neural stem cells and to inhibit
their differentiation (36, 37). Signaling is initiated by ligand
binding, followed by intramembranous proteolytic cleavage of the
Notch receptor by the g-secretase complex. Inhibitors of this
complex slow the growth of Notch-dependent tumors such as
medulloblastoma and T-cell leukemia (8, 9, 38). We therefore used
a potent small-molecule g-secretase inhibitor to further investi-
gate the role for Notch signaling in medulloblastoma, comparing
the effects on overall tumor cell mass to those on cells expressing
stem-like markers. Our data indicate that stem-like brain tumor
cells may be especially vulnerable to attacks on molecular
pathways, such as Notch, which are required in their nonneo-
plastic cognate cells.
Requests for reprints: Charles G. Eberhart, Department of Pathology, Johns
Hopkins University School of Medicine, Ross Building 558, 720 Rutland Avenue,
Baltimore, MD 21205. Phone: 410-502-5185; Fax: 410-955-9777; E-mail: ceberha@
I2006 American Association for Cancer Research.
Cancer Res 2006; 66: (15). August 1, 2006
Materials and Methods
Cell culture. The DAOY, PFSK, D283Med, and D425Med cell lines were
obtained from the American Type Culture Collection and maintained in
MEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum
(FBS) unless otherwise noted. Cell pools and stable subclones transfected
with Notch2 intracellular domain (NICD2) were generated as previously
described, and, unless otherwise noted, the DAOY:NICD2 subclone used is
the same as the one previously reported (8). The low-passage medulloblas-
toma line MB2 was derived from a tumor resected at Johns Hopkins
Hospital and analyzed at passage 8 to 9. It was finely minced, triturated, and
maintained in MEM with 10% FBS. For treatment studies, cells were plated
and allowed to grow overnight in medium containing 10% FBS; then
medium was replaced the next morning with low-serum (0.5% FBS) MEM
containing g-secretase inhibitor dissolved in DMSO at the concentrations
indicated. RNA and protein extractions and all cell-based assays were done
48 hours after drug application unless otherwise noted. All experiments
with error bars were done in triplicate and shown as mean values with SE
unless otherwise noted. Cell mass was measured using CellTiter assays
according to the instructions of the manufacturer (Promega, Madison, WI).
Cell number and viability were assessed using the Guava PCA and Viacount
reagent according to instructions (Guava Technologies, Heyward, CA). Soft-
agar assays were done as previously described, with colonies counted using
an automated reader (8).
;-Secretase inhibitor synthesis. The potent g-secretase inhibitor
thiophene-2-sulfonamide was listed as compound 18 in the recent report by
Lewis et al. (39) describing its synthesis and testing, and we refer to it as
GSI-18. It was synthesized as previously described (39) and its identity and
quality were confirmed by nuclear magnetic resonance and mass spectral
analysis. The biological activity of this inhibitor against g-secretase was
confirmed using in vitro and cell-based assays as previously described (40).
Immunocytochemistry. The cell lines DAOY and MB2 were seeded in
12-well tissue culture plates and allowed to adhere overnight. After treating
with 2 Amol/L GSI-18 for 48 hours, cells were washed with PBS and fixed
using 4% paraformaldehyde solution in PBS at room temperature for
30 minutes. Cells were then permeabilized with 0.4% Triton X-100 in PBS for
5 minutes at room temperature, washed with PBS, and incubated in
5% bovine serum albumin (BSA)-PBS for 1 hour and then in 1:1,000 anti-
nestin antibody (Chemicon, Temecula, CA) or 1:500 anti–cleaved caspase-3
(Cell Signaling Technology, Beverly, MA) in 1% BSA-PBS for 2 hours. After
washing with PBS, a final 60-minute incubation with a 1:300 dilution of
Cy3-conjugated goat anti-mouse and Cy2-conjugated goat anti-rabbit
secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove,
PA) diluted in PBS containing 1% BSA was done. After washing with PBS,
cells were then counterstained with 4¶,6-diamidino-2-phenylindole (Vector
Laboratories, Burlingame, CA), mounted, and visualized with fluorescence
microscope (Zeiss, Jena, Germany). In the single-immunolabeling study
(Fig. 6A and C), six high-power fields of DAOY cultures containing 147 to
333 (mean, 193) cells were photographed and the percentages of cells
positive for nestin were scored in a blinded fashion. In the double-
immunolabeling study (Fig. 6D-F), 10 high-power fields were photographed
Flow cytometric analyses. Flow cytometric analysis of S-phase fraction
and cell cycle kinetics was done following fixation and staining with
propidium iodide using a FACSCalibur (Becton Dickinson, San Jose, CA)
with CELL Quest version 3.3 software (8). CD133 studies were done using
the same instrument, with antibodies from Miltenyi Biotec (Auburn, CA)
according to the instructions of the manufacturer. In brief, cells were
blocked in Fc receptor blocking reagent and incubated with CD133/1
(AC133)-phycoerythrin antibody (Miltenyi Biotec) for 10 minutes in the dark
at 4jC. Then cells were washed and resuspended in 500 AL buffer (PBS
containing 0.5% BSA and 2 mmol/L EDTA). Cells expressing levels of CD133
higher than those seen in immunoglobulin G (IgG) controls were considered
positive. Experiments measuring CD133 were repeated two to three times
for each line and error bars represent the mean and SD. For analyses of side
population, cells (106/mL) were incubated with Hoechst 33342 (3 Amol/L;
Molecular Probes, Carlsbad, CA) for 90 minutes at 37jC in DMEM
containing 2% FBS. Before analysis, cells were washed and resuspended in
2 Ag/mL propidium iodide (Sigma, St. Louis, MO). Cells were analyzed on a
LSR flow cytometer equipped with 424/44-nm band pass and 670-nm long
pass optical filters (Omega Optical, Brattleboro, VT). To ensure proper
identification of side population cells, cells were incubated as above with
the addition of 50 Amol/L verapamil (Sigma).
Real-time reverse transcription-PCR. Quantitative reverse transcrip-
tion-PCR (RT-PCR) for Notch1, Notch2, and Hes1 was done as previously
described (31), with all reactions normalized to actin (Applied Biosystems,
Foster City, CA). Commercially available Assay on Demand TaqMan primers
and probes were used to measure mRNA for Tuj1 and the a6subunit of the
g-aminobutyric acid type A receptor (GABRA6). Each quantitative RT-PCR
reaction was done in triplicate and error bars represent SE.
Protein analysis. Western blots contained 20 Ag of protein per lane on
a 10% Tris-glycine SDS-PAGE gel (Invitrogen) and electrophoresing for
several hours in 1? TG-SDS buffer (Amresco, Solon, OH). Proteins were
transferred to 0.45-Am Optitran nitrocellulose (Schleicher & Schuell,
Keene, NH) in 1? Tris-glycine buffer (Amresco). Blots were blocked in
PBS containing 5% nonfat dry milk powder and incubated overnight at
4jC with antibodies directed against Hes1 (kind gift of Dr. Tetsuo Sudo,
Toray Industries, Tebiro, Kamakura, Japan; 1:2,000) or glyceraldehyde-3-
phosphate dehydrogenase (Research Diagnostics, Flanders, NJ; 1:20,000).
Blots were then washed several times with PBS containing 0.1% Tween 20
and incubated in peroxidase-conjugated IgG diluted 1:2,000 in blocking
solution. After washing several times in PBS with 0.1% Tween 20, blots were
developed with enhanced chemiluminescence reagent (Pierce, Rockford, IL)
and exposed to film.
Statistical analysis. Statistical analyses were done using GraphPad
Prism 4 (GraphPad Software, San Diego, CA). Data graphed with error bars
represent mean and SE from experiments done in triplicate unless
otherwise noted. A two-sided Student’s t test was used to determine the
significance of any differences.
Growth of medulloblastoma cultures is slowed but not
arrested by Notch blockade. Experiments were done using GSI-
18, a potent g-secretase inhibitor with a sulfonamide core (39). We
first sought to determine what concentration of GSI-18 effectively
inhibited Notch activity in tumor cells by measuring expression
of the pathway target Hes1. GSI-18 at 2 Amol/L reduced both
mRNA and protein levels of Hes1 in DAOY cells by >70% (Fig. 1A
and B), suggesting that this concentration should be sufficient to
cause antitumor effects mediated by Notch pathway inhibition.
GSI-18 levels <0.4 Amol/L did not affect Hes1 mRNA expression
(data not shown).
Notch pathway inhibition using GSI-18 slowed the growth of
DAOY medulloblastoma cultures. The increase in viable cell mass
over 2 days was reduced in a dose-dependent fashion by GSI-18
(Fig. 1C) but many tumor cells survived Notch pathway inhibition
and continued to proliferate over this period. JC2, a g-secretase
inhibitor with a benzodiazepine core, also slowed but did not arrest
growth in medulloblastoma cell mass when used at concentrations
that effectively inhibited the Notch pathway, indicating the findings
were not specific to one structural class of compound (data not
shown). DAOY cells stably transfected with a NICD2 were used to
further control for the specificity of the pharmacologic effects.
This truncated Notch receptor does not require ligand binding or
g-secretase activity for nuclear translocation and signaling, and
cells expressing it should be insensitive to effects of GSI-18
mediated by Notch2 inhibition. NICD2 expression in a stable
subclone we have previously described (8) rescued the negative
effects on DAOY cell mass (Fig. 1D). A second stable subclone, as
Cancer Res 2006; 66: (15). August 1, 2006
well as a pool of cells into which NICD2 was introduced by
transfection, was also resistant to the growth-inhibiting effects of
GSI-18 (data not shown), suggesting that this compound acts
through Notch and not other pathways regulated by g-secretase.
It has previously been reported that in pre-T cells, down-
regulation of Notch activity reduces cellular metabolism and cell
size (41). We also observed decreased cell size in medulloblastoma
cultures following GSI-18 exposure (Fig. 1E). To rule out the
possibility that our measurements of viable cell mass bioreductive
capacity (CellTITER assay) were being altered by metabolic affects
of Notch, we quantitated cell number using flow cytometry. The
number of live cells in DAOY, D283Med, D425Med, and PFSK
cultures exposed to 2 Amol/L GSI-18 for 48 hours was reduced as
compared with vehicle-treated controls, but the reduction was less
than that of cell mass (Fig. 1F). Thus, measures of both cell mass
and cell number show a slowing, but not an arrest, of tumor growth
under conditions of Notch blockade.
Decreased proliferation and increased neuronal differenti-
ation following Notch inhibition. Flow cytometric analysis
showed that 2 Amol/L GSI-18 increased the G1-G0cell fraction
and decreased the S-phase and G2-M fractions of DAOY and PFSK
cell lines (Fig. 1G), suggesting that cell cycle exit likely plays a role
Figure 1. Notch inhibition slowed growth of embryonal
brain tumor cells. A, Hes1 mRNA levels, measured using
quantitative RT-PCR and normalized to actin expression,
are decreased by 0.4 Amol/L or higher levels of GSI-18.
B, Hes1 protein levels are also reduced after 48-hour
exposure to GSI-18. C, growth of DAOY viable cell mass
over 2 days is slowed in a dose-dependent fashion by
GSI-18. D, NICD2 expression in a stable subclone rescues
the growth inhibition caused by GSI-18. E, DAOY cells
are less numerous and smaller after 2 days of Notch
blockade; bar, 100 Am. F, the number of viable cells,
expressed as a fraction of vehicle-treated cultures, is
reduced after 48 hours of Notch pathway blockade in all
four medulloblastoma/PNET cell lines tested. G, flow
cytometric analysis of DAOY and PFSK cultures after
48 hours of 2 Amol/L GSI-18 exposure revealed increases
in the percentage of cells in G1/G0and decreases in the
S and G2-M fractions, graphed as percent change from
cells treated with DMSO alone. H, expression of mRNAs
encoding the neuronal markers Tuj1 and GABRA6 are
induced by the g-secretase inhibitor GSI-18; nd, not
Notch Inhibition Depletes Stem-like Brain Tumor Cells
Cancer Res 2006; 66: (15). August 1, 2006
in its antigrowth effects. Notch pathway down-regulation has also
been linked to cellular differentiation in both normal development
and in neoplasms (42, 43) and we therefore examined whether
Notch inhibition caused cellular differentiation. g-Secretase
inhibition in DAOY cells increased RNA levels of two markers of
cerebellar neuronal differentiation, Tuj1 and GABRA6, in a dose-
dependent fashion (Fig. 1H). One of these neuronal markers,
GABRA6, is specifically found in cerebellar granule cells in the
brain, where its expression is induced as they mature (44). Its
induction after Notch pathway inhibition suggests that the
differentiation pathway being actuated resembles that in normal
cerebellar granule neuron precursors, and highlights the similar-
ities between the DAOY line and developing cerebellum.
Notch blockade by ;-secretase inhibitors suppresses tumor
formation. We next examined the effects of Notch pathway
blockade on tumor formation in vitro and in vivo. We first used a
clonogenic assay to determine whether cells capable of forming
anchorage-independent colonies were depleted by GSI-18. Vehicle-
treated DAOY cultures seeded in soft agar formed 50 colonies per
field on average (Fig. 2). This number dropped to 4 when an equal
number of viable cells was counted and seeded after 48 hours of
treatment with GSI-18 and increased to 119 in the presence of
constitutive Notch2 activation. Thus, whereas many tumor cells
continued to grow in 2 Amol/L GSI-18, their clonogenicity in soft
agar was suppressed by >90%.
To determine the effects of Notch blockade on the formation of
tumor xenografts, DAOY and DAOY:NICD2 cultures were treated
with either 2 Amol/L GSI-18 in DMSO or DMSO alone, and the
viable cells remaining after 48 hours were counted using trypan
blue. Five hundred thousand viable cells from each group were
then mixed with Matrigel and injected s.c. in athymic (nude) mice.
Large xenografts formed at the sites of all 12 control injections,
including vehicle-treated DAOY cells (n = 4; Fig. 3A), vehicle-
treated DAOY:NICD2 cells (n = 4), and GSI-18-treated DAOY:NICD2
cells refractory to Notch inhibition (n = 4). In contrast, only one
very small lesion formed among the four sites injected with 500,000
DAOY cells that had been pretreated with 2 Amol/L GSI-18,
suggesting that while viable, the cancer cells were no longer
Figure 2. Notch signaling is required for the formation of colonies in soft-agar.
Forty-eight hours of exposure to 2 Amol/L GSI-18 before seeding in soft agar
significantly reduced clonogenic potential of DAOY cells. This effect could be
rescued by NICD2 expression in a stable subclone (**, P < 0.001).
Figure 3. Notch pathway inhibition blocks xenograft formation. A to C, bulky
xenografts developed at all 12 sites injected with cells in which Notch signaling
was active. This included vehicle-treated DAOY (n = 4), vehicle-treated
DAOY:NICD2 (n = 4), and 2 Amol/L GSI-18–treated DAOY:NICD2 (n = 4; A).
Viable cells remaining following Notch blockade with 2 Amol/L GSI-18 formed
a tumor mass (B) at only one of four injection sites. D, after recovering for
24 hours in media containing 10% FBS, GSI-18- and vehicle-treated cultures
have identical proliferation rates as determined by flow cytometric analysis of
S-phase fraction (inset). Nevertheless, cells that had experienced Notch
pathway blockade were still unable to efficiently form tumors, with small lesions
developing at only two of four injection sites. As before, large xenografts formed
at all 12 control sites. E, D425Med cells treated with GSI-18 for 48 hours
also failed to generate xenografts (arrow) whereas vehicle-treated cells always
did (asterisk). F and G, in vivo administration of GSI-18 i.p. for 5 days after
s.c. tumor injection also blocked engraftment of DAOY (arrow) but not of
Cancer Res 2006; 66: (15). August 1, 2006
tumorigenic (Fig. 3B and C). To rule out the possibility that cells
were alive but moribund following g-secretase inhibition, the ex-
periment was repeated with cells counted after a recovery period.
After 2 days of treatment, followed by 24 hours of recovery in
medium containing 10% FBS and no g-secretase inhibitor, both
GSI-18-treated and vehicle-treated cells proliferated at similar
rates, suggesting that any overall effects on growth had normalized
(Fig. 3D). When equal numbers of these ‘‘recovered’’ cells were
injected into mice, GSI-18-treated cultures were still unable to form
tumors (n = 4) whereas bulky tumor xenografts formed in all 12
controls. D425Med and D283Med cells also failed to engraft (n = 2
animals D425Med, n = 3 animals D283Med) when pretreated for
48 hours with GSI-18, whereas vehicle-treated cells injected on the
contralateral flank formed large tumors at all but one site (Fig. 3E).
As a test of the requirement for ongoing Notch activity during
tumor engraftment, we treated animals with GSI-18 after s.c.
injections of DAOY and DAOY:NICD2 cells into their left and right
flanks. A treatment group of four animals received i.p. injections
containing 0.5 mg GSI-18 in DMSO for 5 consecutive days,
beginning on the day of xenograft initiation, whereas the control
group was treated with vehicle alone. DAOY cells failed to engraft
in three of the four GSI-18-treated animals whereas vehicle-treated
animals all developed tumors (Fig. 3F and G). Tumors also
developed in both vehicle- and GSI-18-treated animals at sites of
DAOY:NICD2 injection. No behavioral or physical changes were
noted in the animals treated with GSI-18.
Notch regulates the percentage of medulloblastoma cells
expressing stem/precursor markers. The data presented above
suggest that while viable and proliferative following Notch pathway
blockade, medulloblastoma cultures are altered in some way,
blocking their ability to form soft-agar colonies and tumor
xenografts. It has been shown that stem-like ‘‘side-populations’’ in
established brain tumor cell lines are uniquely able to form
xenografts (25, 26). Thus, cancer stem cells seem to be able to
persist in established brain tumor lines maintained for many years
in culture. Because stem-like tumor cells are thought to be critical
for xenograft formation, we sought to determine if this subpop-
ulation might be especially sensitive to Notch pathway blockade.
We first examined the requirement for ongoing Notch activity in
CD133-positive cells. Singh et al. (24, 33) have shown that only
CD133-positive medulloblastoma and glioblastoma brain tumor
cells are able to form multipotent neurosphere clones or tumor
xenografts. Another type of brain tumor, ependymoma, also seems
to contain a small fraction of CD133-positive cells uniquely capable
of engrafting in immunocompromised mice (27). In the adherent
embryonal brain tumor cell lines DAOY and PFSK, flow cytometric
analysis revealed that 9.9% and 10.8% of cells, respectively,
expressed CD133 on their surface (Fig. 4A and B). These CD133-
positive fractions fell within the 6.1% to 45.4% range (mean, 19.7%)
reported in primary medulloblastoma (33). The D283Med and
D425Med lines, which have elevated c-Myc levels and grow in
suspension, contained higher percentages of CD133-positive cells.
Sorting of DAOY and PFSK cultures generated populations
enriched f3-fold for cells expressing CD133 and negative fractions
in which <1% of cells were CD133 positive. Interestingly, mRNA
levels of Hes1, a marker of Notch pathway activity, were elevated
4-fold in the CD133-enriched fraction of DAOY and 5-fold in the
CD133-enriched fraction of PFSK (Fig. 4C). This suggests that
Notch signaling is especially active in stem-like cancer cells and
supports the possibility that Notch pathway inhibition may target
Gain or loss of Notch pathway activity modulated the size of the
CD133-expressing stem-like subpopulation (Fig. 4B). This fraction
was elevated from 9.9% to >17% in a pool of DAOYcells transfected
with a plasmid encoding the constitutively active NICD2 and in
two stable subclones derived from such a pool. In contrast,
Notch pathway inhibition using GSI-18 reduced the CD133-positive
stem-like fraction 3-fold to 3.3% (P < 0.01). As expected, NICD2
expression rescued the effects of GSI-18 on the CD133-positive cell
fraction. A 4.8-fold reduction in the percentage of CD133-positive
cells seen in PFSK cultures was also statistically significant
(P < 0.01). Decreases in the stem-like fraction were also observed
in the D283Med and D425Med lines but were less pronounced.
Side population was analyzed to obtain a second measure of the
effects of Notch on stem-like tumor cells. DAOY cultures contained
a Hoechst 33342–excreting side population of 1.9% (Fig. 5A). The
multiple drug resistance pumps mediating dye efflux are verapamil
sensitive (28), and adding verapamil to the medulloblastoma culture
abolished Hoechst excretion, verifying that this was a bona fide
side population. Consistent with our hypothesis that Notch activity
regulates stem-like cancer cells, constitutive activation of Notch2
increased the side population almost 4-fold to 7.3%, whereas Notch
inhibition essentially ablated it, with only 0.01% of remaining cells
negative for dye (Fig. 5A and B). NICD2 expression protected the
side population from GSI-18 exposure, but not from verapamil,
indicating that the effect of GSI-18 was specifically mediated
through its effects on Notch signaling. Small side populations were
also present in the PFSK, D283Med, and D425Med cell lines and
were ablated by both verapamil (data not shown) and 2 Amol/L
GSI-18 (Fig. 5B). Thus, Notch pathway blockade depletes the stem-
like subpopulation defined either by CD133 or by side population.
Figure 4. The CD133-positive population in medulloblastoma is regulated by
Notch. A, flow cytometric analysis was used to define the population of cells with
CD133 expression elevated above the highest level of background fluorescence
(dashed line). Cultures were evaluated following 48-hour exposure to either
2 Amol/L GSI-18 or vehicle. B, the CD133-positive subpopulation in all four
medulloblastoma/PNET lines examined was reduced by 2 Amol/L GSI-18
(*, P < 0.01). Constitutive activation of Notch2 in a pool of DAOY cells
transfected with NICD2 (P) as well as in two stable subclones (C1 and C2)
rendered the CD133-expressing subpopulation insensitive to g-secretase
inhibitor. C, Hes1 mRNA levels were elevated in CD133-enriched preparations
as compared with CD133-depleted ones, suggesting Notch activity is higher
in stem-like cells.
Notch Inhibition Depletes Stem-like Brain Tumor Cells
Cancer Res 2006; 66: (15). August 1, 2006
Notch blockade induces apoptosis in nestin-positive medul-
loblastoma cells. We next examined if stem or progenitor-like cells
expressing nestin might be especially prone to apoptosis following
Notch pathway blockade. Expression of the intermediate filament
nestin was used to directly visualize poorly differentiated cells,
as CD133 immunofluorescence was too dim to reliably score by eye.
Nestin mRNA levels were not significantly elevated in CD133-
enriched DAOY populations, suggesting that the two markers are
not equivalent (data not shown). This is not unexpected, as Lee
et al. (45) also found that only 20% of CD133-positive cells in the
postnatal cerebellum also expressed nestin. Nevertheless, nestin
was one of the first markers of neural stem cells to be identified
(46) and its expression has been shown in a subset of cells within
neurospheres derived from primary human medulloblastoma,
suggesting it marks stem-like cells in these tumors (32, 33). We
detected nestin protein in both established (DAOY) and low-
passage (MB2) medulloblastoma cultures. Nestin staining was
present in the cytoplasm and was variable in intensity, with only
10% of DAOY cells expressing high levels whereas almost half of
MB2 cellswere strongly nestin positive (Fig. 6A and B). We reasoned
that medulloblastoma cells expressing high levels of nestin might
represent a stem-like subpopulation and we therefore correlated
their percentage with varying levels of Notch activity. In DAOYcells,
constitutive Notch2 activation up-regulated the strongly nestin-
positive fraction of cells almost 5-fold to 47.7% (P < 0.001) whereas
Notch blockade using the g-secretase inhibitor GSI-18 reduced
this population 4-fold to 2.4% (P < 0.01; Fig. 6C). A significant
reduction was also observed in MB2 cells following Notch blockade.
We used double immunofluorescence to study cell death in
medulloblastoma cells positive or negative for nestin. The basal
apoptotic rate was measured using antibodies specific for cleaved
caspase-3. In DAOY cultures to which vehicle was added for
48 hours, the apoptotic rate was low in both nestin-negative (26 of
1,841; 1.4%) and nestin-positive (4 of 160; 2.5%) cells (Fig. 6D-F).
However, after 48 hours of treatment with 2 Amol/L GSI-18, the
apoptotic rate increased to 37% in the nestin-positive population
(23 of 62 cells) whereas apoptosis in the better-differentiated cells
lacking nestin only increased to 3.9% (40 of 1,017 cells).
Interestingly, nestin was less evenly distributed in the apoptotic
cells and was sometimes observed surrounding the degenerating
nuclei. Such cagelike perinuclear nestin staining has been
previously described in mitotic cells (47). Our immunofluorescent
assessment of cell death using cleaved caspase-3 corresponds
relatively well to apoptotic induction measured by flow cytometric
analysis of Annexin staining, suggesting that our manual counting
procedure is accurate (Fig. 6G). Taken together, these data indicate
that the survival of stem-like tumor cells is more sensitive to Notch
pathway inhibition than the survival of better-differentiated cells.
In this study, we examined the effects of Notch pathway
inhibition on the growth and xenograft formation of medulloblas-
toma. We found that Notch blockade using the g-secretase
inhibitor GSI-18 slowed the growth of tumor cells in vitro but
had a much more dramatic effect on the formation of colonies in
soft agar and tumor xenografts in nude mice. Indeed, our most
striking finding was that large numbers of viable, rapidly
proliferating cells were not able to generate bulky xenografts if
they had previously been treated with GSI-18, whereas equal
numbers of vehicle-treated cells always formed large tumors.
Systemic (i.p.) treatment of animals with GSI-18 also blocked
xenograft formation with no apparent side effects, indicating this
agent may be effective therapeutically in preventing tumor
metastasis or regrowth following debulking. We believe that these
dramatic effects on tumor propagation are mediated by depletion
of cancer stem cells, as subpopulations expressing the stem cell
marker CD133, as well as the stem-like side population, were pro-
foundly reduced by Notch blockade. These results must be
extended to primary tumors or additional low-passage lines and
might provide a first indication that stem-like cells can be success-
fully ablated from brain tumors.
The targeted depletion of stem-like cells we observe following
Notch pathway blockade contrasts sharply with prior reports of
cancer stem cell survival following standard chemotherapies. For
example, Wulf et al. (48) showed higher efflux of chemotherapeutic
agents and better survival in leukemic side population cells as
compared with non-side population cells. Based on this and on
similar studies, it has been suggested that the efflux pumps which
define side population also function to remove toxic chemotherapy
drugs from cancer stem cells (reviewed in refs. 49, 50). In support of
this concept, treatment of neuroblastoma cell lines with mitoxan-
trone actually increases the side population, suggesting that stem-
like cancer cells are relatively resistant to this chemotherapeutic
agent and accumulate as more differentiated cells are killed (51).
Thus, conventional chemotherapies effectively remove the better-
differentiated cells while leaving most stem-like cells alive. In
contrast, Notch blockade depletes stem-like cells but leaves many
better-differentiated cells capable of limited growth intact.
Figure 5. Notch blockade ablates side population. A, a verapamil-sensitive side
population is present in DAOY cultures and is ablated by GSI-18 treatment.
NICD2 expression in a stable subclone increases the size of the side population
almost 4-fold and rescues the effects of the g-secretase inhibitor. B, similar
effects are seen in PFSK, D283Med, and D425Med medulloblastoma lines.
Cancer Res 2006; 66: (15). August 1, 2006
We examined only one type of malignant brain tumor in this
study, but our findings may be applicable to other neoplasms as
well. The Notch pathway is known to regulate stem cells in a wide
variety of tissues and Notch blockade seems to affect survival
and proliferation of multiple types of cancer (52, 53). For example,
Notch is activated by translocation or mutation in >50% of
T-cell acute lymphoblastic leukemia, and anti-Notch therapies have
been shown to slow acute lymphoblastic leukemia growth in vitro
(38). Aberrantly activated Notch signaling has also been docu-
mented in lung, breast, salivary gland, and pancreatic carcinoma
(54–57). g-Secretase inhibitors may therefore be useful in targeting
stem-like cancer cells in a wide range of neoplasms.
In the future, multiagent chemotherapeutic regimens may target
both stem-like and better-differentiated cells. Drugs blocking
Notch signaling, or other pathways required in stem cells such as
Wnt and Hedgehog, will be used to deplete the cancer stem cell
population, and traditional chemotherapeutic agents can be used
at the same time to debulk the larger mass of tumor cells. This
will effect a rapid removal of both subpopulations and might
circumvent the possibility that some differentiated tumor cells
can dedifferentiate and repopulate the stem cell fraction (58).
Another potential complication of cancer stem cell–directed
therapies is that nonneoplastic stem cells may also be depleted
by such strategies. This is of particular concern in children who will
presumably require stem cells for the maintenance and repair of
a host of tissues over the course of their lives. Of note, we found
that i.p. injections of GSI-18 for 5 days blocked tumor formation
with no obvious ill effects on the animal’s health over the
subsequent months. Indeed, it has recently been shown that
inhibition of g-secretase activity using small-molecule drugs can
actually enhance long-term memory in rodents (59).
In summary, we show that in the malignant brain tumor
medulloblastoma, Notch pathway blockade depletes a population
of cells required for in vivo tumor formation by suppressing
Figure 6. Nestin-positive cells are
especially sensitive to Notch pathway
blockade. A, the fraction of DAOY cells
expressing high levels of nestin is lower
after GSI-18 exposure and higher in a
subclone expressing activated Notch2
(NICD2). B and C, similar effects are
seen following Notch blockade in the
low-passage medulloblastoma cell line
MB2. D to F, double immunolabeling was
used assess both differentiation status
(nestin) and apoptosis (cleaved caspase-3)
in DAOY cultures. Significant induction of
cleaved caspase-3–positive apoptotic
cells following Notch blockade was limited
to the nestin-positive subpopulation.
G, overall apoptotic induction in DAOY
cultures was similar when measured
by counts of cleaved caspase-3
immunofluorescent (IF) cells or flow
cytometric analysis of Annexin. *, P < 0.01;
** P, < 0.001 (t tests); ns, not significant.
Notch Inhibition Depletes Stem-like Brain Tumor Cells
Cancer Res 2006; 66: (15). August 1, 2006
proliferation and inducing apoptosis or differentiation in stem-
like cells. Our data suggest that Notch pathway blockers may be the
first of a new class of chemotherapeutic agents—those targeting
cancer stem cells.
Received 3/13/2006; revised 5/17/2006; accepted 5/25/2006.
Grant support: Alzheimer’s Association, Zenith Fellowship (Y-M. Li), NIH
grant 1R01AG026660-01, The Children’s Cancer Foundation of Baltimore, Fellowship
from the American Brain Tumor Association (X. Fan), Burroughs Wellcome
Fund Career Award in the Biomedical Sciences (C.G. Eberhart), and NIH grant K08
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Josh Copeland for assistance with Western blots, Dr. Tetsuo Sudo for his
generous gift of Hes1 antiserum, and Dr. J.C. Zuniga-Pflucker for his suggestions on
Notch inhibition and cell size.
Cancer Res 2006; 66: (15). August 1, 2006
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