2006-01-00089D
Supplementary Methods
Isolation of CD133+ and CD133- cancer cells from human glioma xenografts and
primary glioblastomas. D54MG and D456MG human glioma xenografts were freshly
dissected from nude mice and placed in neurobasal A medium (NBM) (Invitrogen).
Tumour specimens were washed in NBM, acutely dissociated to remove non-tumour
tissue and subject to enzymatic dissociation using Papain dissociation system
(Worthington Biomedical Corp.). The isolated tumour cells were briefly placed in NBM
with B27 supplement (Invitrogen) to permit recovery following enzymatic dissociation.
Cells were labeled with CD133/2(293C)-Allophycocyanin (APC) antibody kit (Miltenyi
Biotec), and the CD133+ and CD133- cells were sorted and analyzed by flow cytometry.
In some cases, CD133+ and CD133- cells were separated through magnetic cell sorting
with CD133 Cell Isolation Kit (Miltenyi Biotec) as described3,5,26. The sorted CD133+
cells were cultured in the NBM-B27 medium containing 20 ng/ml of both epidermal
growth factor (EGF) and basic fibroblast growth factor (bFGF) (Invitrogen) for a short
period before treatment and analyzed. The cells used for all experiments were analyzed
by Fluorescence-Activated Cell Sorter (FACS) immediately before each experiment to
make sure that the purity of CD133+ cells was greater than 85%, and the purity of
CD133- cells was above 99% (data not shown).
Differentiation assays. To induce cell differentiation, CD133+ cells from a single
neurosphere were cultured on coverslips coated with poly-ornithine (250 g/ml) and
laminin (5 g/ml) in -MEM medium with 10% serum (Invitrogen) for 7 days. FITC- or
Rhodamine- conjugated Donkey anti-mouse IgG or anti-rabbit IgG secondary antibodies
1
2006-01-00089D
(Jackson Immuno Research) were used for detection. Nuclei of the differentiated cells
were counterstained with DAPI/Antifade. The immunofluorescent staining was examined
under the Axiovert 200 Inverted Fluorescent Microscope (Zeiss).
Alkaline comet assay. CD133+ and CD133- glioma cells derived from glioma
xenografts or primary gliomas were cultured in NBM with EGF/bFGF, harvested at the
times indicated after IR (3 Gy) or neocarzinostatin (NCS) treatment (100 ng/ml), and
mixed with low-melting point agarose and spread on agarose pre-coated slides. Cells on
the slides were lysed for 1 hour at 4ºC and subjected to horizontal electrophoresis at 25 V
for 35 min under alkaline conditions, then neutralized and stained with propidium iodide
(2 g/ml). The presence of comet tails was assessed and quantified with a fluorescence
microscope using an automated image analysis system. For each experimental point, at
least 100 cells were evaluated.
Phosphorylated
H2AX
staining.
The
immunofluorescent
staining
for
the
phosphorylated H2AX after IR or NCS treatment was performed as described22.
In vitro cell mixing and repopulation. To assess the repopulation potential of CD133+
and CD133- cells after IR in vitro, CD133+ and CD133- cells derived from glioma
xenografts or the primary glioblastoma specimen were labeled with CellTracker CFSE
green fluorescent dye (Molecular Probes), and the CD133- cells were labeled with the
CellTracker Red CMTPX (Molecular Probes) separately according to the instructions,
and then mixed in defined ratios. Triplicate parallel cultures were left untreated or
treated with IR (5 Gy) in NBM-B27 without EGF/bFGF for 24 hours and then
subsequently cultured in zinc option media with 10% FBS.
2
The resulting growth
2006-01-00089D
patterns of each tumour cell population at the indicated time point was visualized using
fluorescent microscopy and analyzed by FACS. The percentage of cells derived from
CD133- and CD133+ cells under untreated or irradiated conditions was quantified at
sequential time points after IR treatment.
Cell colony formation assay.
Overall cell survival of CD133+ and CD133- cells in
response to irradiation was assessed by colony formation. Identical numbers of purified
matched CD133+ and CD133- cells were untreated or treated with a dose of ionizing
radiation (5 Gy) alone, 3 M DBH alone (debromohymenialdisine, a specific Chk1/Chk2
low molecular weight inhibitor; Calbiochem)23, or a combination of DBH (3 M) and IR
(5 Gy) in NBM without EGF/bFGF for 24 hours, and then cultured in the zinc option
media with 10% FBS in 6-well plates until visible colony formation. In the combination
treatment, cells were pre-treated with DBH 30 minutes before IR treatment.
Each
treatment for each cell type from each source was performed in triplicate. Cell colonies
were fixed and stained with 0.5 % Toludine Blue O in 4% PFA solution.
Intracranial tumor assays. The indicated number of tumour cells were implanted into
the right frontal lobes of BalbC athymic nude mice. Mice were maintained in HEPAfiltered isolation conditions under a protocol approved by the Duke University
Institutional Animal Care and Use Committee.
Mice were monitored until they
developed neurologic signs that significantly inhibited their quality-of-life (e.g. ataxia,
lethargy, seizures, inability to feed, etc.). Prior xenograft studies have demonstrated that
these signs develop shortly before animal death. After sacrifice, the brains of mice
3
2006-01-00089D
bearing tumours were collected, fixed, and the presence of tumour verified by systematic
histological review.
In vivo mixing and cell repopulation assays. To examine the repopulation and survival
potential of CD133+ and CD133- cells in vivo after IR treatment in combination with the
checkpoint inhibitor, CD133+ and CD133- cells were separately labeled with the
fluorescent CellTracker Red CMTPX and CellTracker CFSE green fluorescent dyes
(Molecular Probes). Labeled cells were mixed in defined ratios and implanted
orthotopically into the right frontal lobes of BalbC nude athymic mice, and then were
subjected to control conditions (0.1% DMSO), external beam ionizing radiation (5 Gy), 3
M DBH, or a combination of the two treatments. Mice were sacrificed after 8 days and
brains were assessed for the relative proportion of cells derived from the labeled CD133+
and CD133- cells by fluorescent microscopy of brain sections or FACS analysis of
disaggregated tumour-bearing brains.
Fluorescent in Situ Hybridization (FISH). Tumour cells derived from human primary
glioblastomas were subjected to FISH analysis with chromosome centromere probes.
Specifically, tumour cells isolated from primary glioma were treated with a hypotonic
solution and spread on slides, then probed with specific FISH probes according to the
altered genetic markers detected by the pathologist in each clinical case. For example,
chromosome 10 centromere green probe (SpectrumGreen, VYSIS, Inc. IL) was used to
detect the polysomy in cancer cells derived from T3359 specimen according to the
manufacturer’s instruction. Nuclear DNA was counterstained with DAPI. The
hybridization was visualized under a fluorescent microscope, and more than 100 cells
were analyzed to calculate the rate of polysomy of the chromosome 10 centromere.
4
2006-01-00089D
Annexin V-FITC Staining. Annexin V-FITC staining for detecting apoptotic cell death
was performed with a kit (BD bioscience) according to the manufacturer’s instructions.
Western blot analysis and ATM kinase assay in vitro.
Western blot analysis for
detecting the phosphorylated checkpoint proteins and total proteins was performed as
described18,19. CD133+ and CD133- cells isolated from glioma xenografts or primary
gliomas were treated with IR or the radiomimetic agent, NCS (neocarzinostatin), to
induce DNA damage. The activation state of the checkpoint response was assessed in
each line without and 1 hour after 3 Gy of ionizing radiation or NCS (100 ng/ml)
treatment. ATM in vitro kinase assay was performed as described18.
5