Tumor environment dictates medulloblastoma cancer stem cell expression and invasive phenotype.

Abstract

The neural precursor surface marker CD133 is thought to be enriched in brain cancer stem cells and in radioresistant DAOY medulloblastoma-derived tumor cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP) expression is a hallmark of highly invasive, radioresistant, and hypoxic brain tumor cells, we sought to determine whether MT1-MMP and other MMPs could regulate the invasive phenotype of CD133(+) DAOY cells. We found that when DAOY medulloblastoma or U87 glioblastoma cells were implanted in nude mice, only those cells specifically implanted in the brain environment generated CD133(+) brain tumors. Vascular endothelial growth factor and basic fibroblast growth factor gene expression increases in correlation with CD133 expression in those tumors. When DAOY cultures were induced to generate in vitro neurosphere-like cells, gene expression of CD133, MT1-MMP, MMP-9, and MDR-1 was induced and correlated with an increase in neurosphere invasiveness. Specific small interfering RNA gene silencing of either MT1-MMP or MMP-9 reduced the capacity of the DAOY monolayers to generate neurospheres and concomitantly abrogated their invasive capacity. On the other hand, overexpression of MT1-MMP in DAOY triggered neurosphere-like formation which was further amplified when cells were cultured in neurosphere medium. Collectively, we show that both MT1-MMP and MMP-9 contribute to the invasive phenotype during CD133(+) neurosphere-like formation in medulloblastoma cells. Increases in MMP-9 may contribute to the opening of the blood-brain barrier, whereas increased MT1-MMP would promote brain tumor infiltration. Our study suggests that MMP-9 or MT1-MMP targeting may reduce the formation of brain tumor stem cells.

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Tumor Environment Dictates Medulloblastoma Cancer Stem
Cell Expression and Invasive Phenotype
Borhane Annabi,1Shanti Rojas-Sutterlin,2Carl Laflamme,1Marie-Paule Lachambre,2
Yanne `ve Rolland,2Herve ´ Sartelet,3and Richard Be ´liveau2
1Laboratoire d’Oncologie Mole ´culaire, De ´partement de Chimie, Universite ´ du Que ´bec a ` Montre ´al,2Centre de
Cance ´rologie Charles-Bruneau, Ho ˆpital Sainte-Justine-UQAM, and3Department of Pathology,
Ho ˆpital Sainte-Justine, Montreal, Quebec, Canada
Abstract
The neural precursor surface marker CD133 is thought
to be enriched in brain cancer stem cells and in
radioresistant DAOY medulloblastoma-derived
tumor cells. Given that membrane type-1 matrix
metalloproteinase (MT1-MMP) expression is a hallmark
of highly invasive, radioresistant, and hypoxic brain
tumor cells, we sought to determine whether MT1-MMP
and other MMPs could regulate the invasive phenotype
of CD133(+) DAOY cells. We found that when DAOY
medulloblastoma or U87 glioblastoma cells were
implanted in nude mice, only those cells specifically
implanted in the brain environment generated CD133(+)
brain tumors. Vascular endothelial growth factor and
basic fibroblast growth factor gene expression
increases in correlation with CD133 expression in those
tumors. When DAOY cultures were induced to generate
in vitro neurosphere-like cells, gene expression of
CD133, MT1-MMP, MMP-9, and MDR-1 was induced
and correlated with an increase in neurosphere
invasiveness. Specific small interfering RNA gene
silencing of either MT1-MMP or MMP-9 reduced the
capacity of the DAOY monolayers to generate
neurospheres and concomitantly abrogated their
invasive capacity. On the other hand, overexpression of
MT1-MMP in DAOY triggered neurosphere-like formation
which was further amplified when cells were cultured
in neurosphere medium. Collectively, we show that
both MT1-MMP and MMP-9 contribute to the invasive
phenotype during CD133(+) neurosphere-like formation
in medulloblastoma cells. Increases in MMP-9 may
contribute to the opening of the blood-brain barrier,
whereas increased MT1-MMP would promote brain
tumor infiltration. Our study suggests that MMP-9 or
MT1-MMP targeting may reduce the formation of brain
tumor stem cells. (Mol Cancer Res 2008;6(6):907–16)
Introduction
Recently, a small population of cancer stem cells (CSC) in
adult and pediatric brain tumors has been identified (1, 2).
These CSC, once isolated from tumor tissues, form neuro-
spheres when cultured in vitro and possess the capacity for cell
renewal. Based on their high expression of the neural precursor
cell surface marker CD133 (prominin-1), these CSC have been
further hypothesized to bear properties such as resistance to
apoptosis and resistance to drugs and to ionizing radiation
(3, 4). Interestingly, among primary human medulloblastoma
and glioblastoma multiforme–derived cells, only those cells
expressing CD133 on their surfaces can initiate tumors in the
brains of nonobese diabetic/severe combined immunodeficient
mice (5). In fact, the injection of as few as 100 CD133(+) cells
produces a tumor that could be serially transplanted and that is a
phenocopy of the patient’s original tumor, whereas injection of
CD133(?) cells did not cause a tumor. Such evidence
highlights the fact that in brain tumors, and in other
malignancies, the tumor clone is functionally heterogeneous,
existing, however, in a cellular hierarchy based on small
subpopulations of stem cells (6).
Given that CD133 has been identified as a powerful CSC
marker, and that its gene expression is significantly higher in
the recurrent brain tumor tissue, we examined whether other
molecular players may also contribute to the invasive potential
of medulloblastomas. Matrix metalloproteinases (MMP) have
been shown to be associated with glioblastoma (7, 8) and
medulloblastoma cell invasiveness (9, 10). In particular, levels
of MMP-9 and membrane type-1 (MT1)–MMP expression
were increased in >75% of medulloblastoma tumor tissues (11).
Whether the CD133(+)-CSC subpopulation of cells was also
targeted remains unknown. When MMP-9 gene expression was
silenced in DAOY medulloblastoma-derived cells, experimen-
tal in vivo tumor growth was inhibited in an intracranial animal
model (12). Moreover, in line with the CD133(+) tumor cell
phenotype that accounts for glioma stem cells’ radioresistance
(13), the expression of MT1-MMP expression was also
increased in several cell line models that escaped radiation-
induced apoptosis (14, 15). Previous reports from our
laboratory have documented the contribution of MT1-MMP
to the infiltrative and invasive phenotype of brain tumor–
derived cells (16, 17), and strategies which inhibit either
Received 12/3/07; revised 1/26/08; accepted 2/3/08.
Grant support: B. Annabi holds a Canada Research Chair in Molecular
Oncology from the Canadian Institutes of Health Research. This study was funded
by grants from the Natural Sciences and Engineering Research Council of Canada,
the Canadian Institutes of Health Research, the Charles-Bruneau Foundation, and
the Claude Bertrand Chair in Neurosurgery (R. Be ´liveau), and from the Natural
Sciences and Engineering Research Council of Canada (B. Annabi).
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.
Requests for reprints: Richard Be ´liveau, Laboratoire de Me ´decine Mole ´culaire,
Universite ´ du Que ´bec a ` Montre ´al, C.P. 8888, Succursale Centre-ville, Montreal,
Quebec, Canada H3C 3P8. Phone: 514-987-3000, ext. 8551; Fax: 514-987-0246.
E-mail: oncomol@nobel.si.uqam.ca
Copyright D 2008 American Association for Cancer Research.
doi:10.1158/1541-7786.MCR-07-2184
Mol Cancer Res 2008;6(6). June 2008
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MMP-9 or MT1-MMP activities and/or expression may,
furthermore, be envisioned to reduce tumorigenicity (17-19).
Whether expression of MMP-9 or MT1-MMP affects the
acquisition of an invasive phenotype during neurosphere-like
formation of CSC is currently unknown.
In this study, we sought to examine the effect of tumor tissue
environment and the contribution of CD133(+) brain tumor–
derived CSC to tumor development in vivo. We also
investigated the in vitro expression of MMP-9 and MT1-
MMP, two MMPs which contribute to the blood-brain barrier
opening and to the radioresistance phenotype of brain tumor
cells in the neurosphere formation capacity of DAOY
medulloblastoma cells.
Results
The Tissue Environment of Cell Implantation Dictates the
Expression of CD133(+) Brain Tumor–Derived Stem
Cells
In an attempt to assess the contribution of CD133(+) CSC
to experimental brain tumor growth, monolayer cultures of
DAOY medulloblastoma cells were trypsinized and cell
suspensions were implanted either subcutaneously or by
stereotaxis within the brain and left to develop for several
weeks as described in Materials and Methods. Histopathologic
analysis of intracerebrally implanted human DAOY medullo-
blastoma cells shows well-defined but not encapsulated large
malignant proliferation composed of a majority of small
undifferentiated round cells found in normal mouse brain
(Fig. 1A1). At high magnification, hematoxylin-phloxin-
saffron staining shows that the tumor was highly cellular with
numerous mitoses (Fig. 1A2), and presents an aggressive
pattern with a limited area of necrosis (Fig. 1A3). The nuclei
are often densely hyperchromatic, and are associated with poor
cytoplasm. No fibrillar material and no rosette could be
identified. Stromal elements were scant, consisting of small
blood vessels. Moreover, some tumoral cells were found to
exhibit membranous expression of CD133 (Fig. 1A4, arrows).
Although subcutaneous tumor growth was only observed with
implanted U87 glioblastoma cells, we found that U87 and
DAOY cells implanted within the cerebral environment readily
developed into tumors. Intracerebral growth of U87 tumors
was routinely found to occur within the first 2 to 3 weeks,
whereas significant intracerebral growth of DAOY tumors only
occurred after 9 to 11 weeks. Homogenates were then generated
from the individual tumors and CD133 immunodetection was
done. We found that CD133 protein expression was only
observed when U87 or DAOY cells were implanted within the
cerebral environment (Fig. 1B). The origin of CD133 expression
was further examined when homogenates from DAOY cell
monolayers, control brain hemisphere, and intracerebral DAOY-
derived tumors were probed for CD133. We found that DAOY
cell monolayers expressed low basal CD133 (Fig. 1C, in vitro),
whereas CD133 expression was enriched in the brain hemi-
sphere in which DAOY cells had been implanted (Fig. 1C,
in vivo). Quantitative PCR was also done with the control and
DAOY-derived tumors and confirmed the angiogenic phenotype
of DAOY-derived tumors as vascular endothelial growth factor
and basic fibroblast growth factor gene expression were
significantly increased in those tumors (Fig. 1D).
Increased CD133, MDR-1, MT1-MMP, and MMP-9
Expression Characterize the Invasive Phenotype of
Neurosphere-Like DAOY Cells
Neurosphere-like brain CSC are thought to contribute to a
subpopulation of CD133(+) brain CSC (4). Neurosphere
induction of DAOY cells was done according to established
protocols (20-22), and this process promoted the transition of
adherent DAOY monolayer cells to neurosphere-like cells
(Fig. 2A). Total RNA was isolated from monolayers and from
neurosphere-like DAOY cells and semiquantitative reverse
transcription-PCR (RT-PCR) was done. We found that gene
expression of CD133, MDR-1, HuR, MMP-9, and MT1-MMP
all increased in neurosphere-like DAOY cells (Fig. 2B).
Although the increase in CD133 cell surface expression was
confirmed in cells cultured in neurospheres by flow cytometry
(data not shown), the increases in MMP-9 and in MT1-MMP
were both confirmed when conditioned media were isolated
from serum-starved monolayers and from neurosphere-like
DAOY cells and we observed a massive increase in
extracellular proMMP-9 gelatinolytic activity, as assessed by
gelatin zymography (Fig. 2C) from the latter medium. Although
the latent proMMP-2 extracellular levels remained unchanged
between monolayers and neurosphere-like cells, the active
forms of MMP-2 were exclusively produced in neurospheres,
most likely due to an increased MT1-MMP activity (Fig. 2C).
Cell migration was also assessed by seeding monolayers and
neurosphere-like DAOY cells on top of modified Boyden
chambers. We found that the invasive capacity of neurosphere-
like DAOY cells was significantly increased when compared
with DAOY cells grown into monolayers (Fig. 2D).
MT1-MMP and MMP-9 Contribute to the Invasive Pheno-
type and Contribute to the Neurosphere-Like Formation
Process in DAOY Cells
We next assessed the effects of MMP-9 and MT1-MMP
activities in promoting neurosphere-like formation and invasion
using RNA interference technology. DAOY monolayer cells
were transiently transfected with small interfering RNA to
specifically down-regulate either MMP-9 or MT1-MMP gene
expression as described in Materials and Methods. Transfection
efficiencies were monitored either through the protein expres-
sion of MMP-9 or through the MT1-MMP–mediated activation
of proMMP-2 using gelatin zymography. We found that basal
levels of proMMP-9 in DAOY monolayers (Fig. 3A, monolayers)
as well as the proMMP-9 induced in neurosphere-like DAOY cells
(Fig. 3A, neurospheres) were significantly decreased in siMMP-
9–transfected cells when compared with mock-transfected
cells. MT1-MMP was also efficiently silenced and this was
reflected by the complete inability of neurosphere-like DAOY
cells to activate latent proMMP-2 into its active MMP-2 form
(Fig. 3A, neurospheres). MT1-MMP pro- and active forms of
protein expression are further shown to be efficiently and
specifically diminished using immunoblotting in siMT1-
MMP–transfected cells but not in siMMP-9–transfected cells
(Fig. 3B, exposition 30 s). Moreover, the increased proMMP-2
activation observed in neurospheres by zymography (Fig. 3A)
also correlates with MT1-MMP proteolytic activation as seen
by the appearance of the well-documented 45 kDa MT1-MMP
immunoreactive inactive form (Fig. 3B, exposition 2 min).
Annabi et al.
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Glyceraldehyde-3-phosphate dehydrogenase protein expression
was used as an internal loading control and remained unaffected
throughout the conditions. When neurosphere-like formation
was monitored, we found a significant decrease in neurosphere-
like formation in cells whose MT1-MMP or MMP-9 had been
silenced, as can be seen in images recorded at two different
magnifications (?10 versus ?4; Fig. 3C). Cell migration was
also assessed with mock-transfected cells as well as with
DAOY monolayers and neurosphere-like DAOY cells that were
transfected with MT1-MMP and MMP-9 silencers (Fig. 3D).
The induced migration observed in neurosphere-like DAOY
cells was completely abolished in those cells when either MT1-
MMP or MMP-9 gene expression was silenced (Fig. 3D,
neurospheres). No significant decreases were observed in
siMT1-MMP or in siMMP-9 DAOY cells from monolayer
cultures (Fig. 3D, monolayers).
Overexpression of MT1-MMP Triggers Neurosphere-Like
DAOY Differentiation
We further assessed the specific contribution of MT1-MMP
toward the control of neurosphere-like DAOY formation. We
first transiently transfected DAOY monolayers with cDNA
encoding a recombinant MT1-MMP protein fused to green
FIGURE 1.
were trypsinized and 106cells injected either subcutaneously or intracerebrally into nude mice. Tumors were left to develop as described in Materials and
Methods. A. Histopathologic analysis of paraffin sections (3 Am) of intracerebrally implanted human DAOY medulloblastoma cells was done with
hematoxylin-phloxin-saffron staining and shows (1) malignant proliferation composed of a majority of small round cells (arrow) found in normal mouse brain
(*; magnification, ?100). At high magnification (?400), hematoxylin-phloxin-saffron staining shows (2) that tumor is highly cellular with numerous mitoses,
and presents an aggressive pattern (3) with limited area of necrosis (magnification, ?200). Some tumoral cells (4) had a membranous expression of CD133
(arrows). B. Immunoblotting was used to assess CD133 protein expression in lysates (20 Ag proteins) generated from subcutaneous and intracerebral U87
glioblastoma-implanted or DAOY medulloblastoma-implanted cells as described in Materials and Methods. C. Lysates (5 Ag protein) from in vitro cultured
DAOY cell monolayers (DAOY), contralateral healthy brain (Ctrl), and from intracerebrally implanted DAOY cells (Tumor) were assessed for CD133
expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as an internal loading control. D. Relative gene expression levels
of CD133 (CD133), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) were assessed by quantitative PCR using total
RNA extracted from contralateral healthy brain (white columns) or from intracerebrally implanted DAOY tumors (black columns).
The tissue environment of cell implantation dictates the expression of CD133(+) brain tumor–derived stem cells. Cultured DAOY monolayers
CD133(+) Induction of Medulloblastoma Neurospheres
Mol Cancer Res 2008;6(6). June 2008
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fluorescent protein (GFP; ref. 23). Transfection efficiency was
monitored using fluorescence microscopy and was confirmed
by the expression of fluorescent cells (Fig. 4A). Moreover,
recombinant MT1-MMP was also found to be active as its
expression led to proMMP-2 activation and to an increase in
proMMP-9 secretion as assessed by gelatin zymography
(Fig. 4B). Mock- and MT1-MMP–transfected DAOY mono-
layers were then trypsinized and cultured either in monolayer
medium or in neurosphere medium for 24 hours. Phase contrast
pictures were taken in order to monitor for neurosphere-like
formation in each culture condition. We found that mock-
transfected cells were, as expected, still able to adhere and grow
as monolayers when cultured in monolayer medium, although
they formed neurosphere-like cells when cultured under neuro-
sphere medium (Fig. 4C, phase contrast pictures). In contrast,
when MT1-MMP–transfected DAOY monolayer cells were
seeded in monolayer medium, we observed that very few GFP-
positive cells adhered to the culture dishes but rather formed
neurosphere-like cells (Fig. 4C, phase contrast pictures). This
was also evident through fluorescence visualization. Recombi-
nant MT1-MMP overexpression was thus found to enhance
neurosphere-like DAOY formation as shown through phase
contrast and fluorescent microscopy imaging (Fig. 4C).
Discussion
Among brain tumors in children, the highly metastatic
medulloblastomas represent f25% of all pediatric intracranial
neoplasms (24). Although modern neurosurgical and radiother-
apy techniques combined with chemotherapy regimens result in
prolonged free survival rates, the long-term prognosis is poor
and the quality of life of these patients is generally poor (25).
Recently, an isolated CD133(+) cell subpopulation from human
brain tumors was shown to exhibit stem cell properties (5) and
is thought to play a pivotal role in brain tumor initiation,
growth, and recurrence (5, 26). Consequently, a better
understanding of the potential contribution of brain tumor–
derived CSC will help develop new clinical approaches in order
FIGURE 2.
Representative phase contrast pictures of DAOY monolayers and neurosphere-like cells were taken after 48 h of culture. B. Total RNA was extracted from
DAOY monolayers and neurosphere-like cells as described in Materials and Methods. Semiquantitative RT-PCR was done and cDNA amplicons for MMP-9,
HuR, CD133, MDR-1, MT1-MMP, and glyceraldehyde-3-phosphate dehydrogenase were revealed by electrophoresis using agarose gels as described in
Materials and Methods. C. Conditioned medium from DAOY monolayers and neurosphere-like cells were assessed for their MMP-9 and MMP-2 secretion
and activation levels as described in Materials and Methods. D. DAOY monolayers and neurosphere-like cells were trypsinized and seeded (5?104cells) on
gelatin-coated filters in modified Boyden chambers. Migration was allowed to proceed for 16 h at 37jC. Cells on filters were then fixed and stained; one out of
five representative stainings for each condition. Columns, mean of a representative experiment in which five random fields per filter were counted for each
condition; bars, SD.
Increased CD133, MDR-1, MT1-MMP, and MMP-9 expression characterize the invasive phenotype of neurosphere-like DAOY cells. A.
Annabi et al.
Mol Cancer Res 2008;6(6). June 2008
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to improve the efficiency of current treatment. In the present
study, we identified and evaluated new biological and
molecular features regulating the invasive phenotype associated
with the formation of neurosphere-like CD133(+) medulloblas-
toma-derived CSC. Using RNA interference technology, we
provided evidence for crucial roles for MMP-9 and for MT1-
MMP, two major actors in cell invasion, metastasis, and
resistance to radiation, in CD133(+) neurosphere-like formation
in DAOY medulloblastoma cells.
It is noteworthy that as few as 100 CD133(+) cells from a
brain tumor fraction were found to be capable of tumor
initiation in nonobese diabetic/severe combined immunodefi-
ciency mouse brains (5). The use of an intracerebral
implantation model versus a flank implantation model in our
current study, further added to the stringency of our experi-
ments, proving that CD133 expression only arose from
implanted brain tumor cells within a cerebral environment,
possibly attributable to increased angiogenesis as reflected by
high vascular endothelial growth factor and basic fibroblast
growth factor transcript levels. Gene expression profiles of
experimental brain tumors grown as subcutaneous flank
xenografts, intracerebral xenografts, and cells grown in vitro
have been shown to be different suggesting that the tissue
environment dictates the experimental tumor phenotype
(27, 28). Intracerebral models can more closely mimic the
actual environment of glioma- and medulloblastoma-derived
CD133(+) CSC growth (29). The demonstration that
CD133(+) cells express increased MDR-1 transcript levels
(this study; ref. 30) also reveals issues important to drug
delivery and to the blood-brain barrier environment that can
now be addressed in terms of chemoresistance or angiogenic
phenotype.
The CD133(+) fraction among medulloblastomas, which
initiated tumors upon intracranial transplantation into the
nonobese diabetic/severe combined immunodeficiency mouse
forebrain, also correlated closely with an in vitro primary
FIGURE 3.
silencing using siRNA was used to down-regulate the expression of MT1-MMP and MMP-9 in DAOY cell monolayers (top) or DAOY neurospheres (bottom)
as described in Materials and Methods. Conditioned medium from each respective condition was harvested 48 h posttransfection and the extent of either
MT1-MMP or MMP-9 silencing assessed using gelatin zymography. B. MT1-MMP immunoblotting was done with cell lysates isolated from A.
Autoradiograms are shown at 30-s vs. 2-min expositions in order to appreciate MT1-MMP down-regulation (30 s exposition), and the appearance of the 43 to
45 kDa inactive immunoreactive MT1-MMP that is generated during proMMP-2 activation (2 min exposition). Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as an internal loading control. C. Phase contrast (magnification, ?10 and ?4) microscopy enabled the visualization of the decrease
in neurosphere-like formation in cells from which MT1-MMP or MMP-9 had been silenced. D. Cell migration was also assessed 48 h posttransfection in
modified Boyden chambers as described in Materials and Methods for control (white columns) monolayers and neurosphere-like DAOY cells, and cells in
which MMP-9 (black columns) and MT1-MMP (gray columns) gene expression was silenced. Columns, mean of a representative experiment in which five
random fields per filter were counted for each condition; bars, SD.
MT1-MMP and MMP-9 control the neurosphere-like formation process in DAOY cells and contribute to their invasive phenotype. A. Gene
CD133(+) Induction of Medulloblastoma Neurospheres
Mol Cancer Res 2008;6(6). June 2008
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