Radiosensitivity enhancement of radioresistant glioblastoma by epidermal growth factor receptor
antibody-conjugated iron-oxide nanoparticles
Journal of Neuro-Oncology
Alexandros Bouras, 1 Milota Kaluzova, 1 and Costas G. Hadjipanayis 1, 2*
*to whom correspondence should be addressed
Address for Correspondence/Reprints:
Costas G. Hadjipanayis, MD, Ph.D.
Dept. of Neurosurgery
Emory University School of Medicine
1365B Clifton Rd. NE, Suite 6200
Atlanta, GA 30322, USA
Email: chadjip@emory.edu
Phone: 404-778-3091
Fax:
404-778-4491
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Supplementary experimental procedures
Cell lines
U87MGEGFRvIII human GBM cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM,
Cellgro USA) supplemented with 10% bovine serum (FBS HyClone), 2 mM glutamine, sodium pyruvate,
and penicillin/streptomycin (HyClone) at 37 °C and 5% CO2. Human astrocytes were obtained from
ScienCell Research Laboratories and maintained according to the supplier instructions. Early passage was
used for both cell lines and no Mycoplasma infection was identified during the entire experiment period.
IONPs, EGFR antibody, bioconjugation
Water soluble IONPs with amphiphilic polymer coating were purchased by Ocean NanoTech, (Arkansas,
USA, SHP-10-5) with a core size of 10nm and a concentration of 5mg/ml. Cetuximab (erbitux; Imclone
LLC;) was kindly provided by the pharmacy of the Winship Cancer Institute of Emory University. For
covalent conjugation of cetuximab to IONPs, the Carboxyl Magnetic Iron Oxide Nanocrystal Conjugation
kit (Ocean NanoTech, Arkansas, USA) was used. Bioconjugation was initiated with activation of IONPs
carboxyl groups by an activation buffer consisting of ethyl dimethylaminoprolyl carbodiimide (EDC) and
sulfo-N-hydroxysuccinimide (NHS). After vigorous mixing of the IONPs with the activation buffer at
room temperature for 20 minutes, cetuximab along with coupling buffer were added to the activated
IONPs and the components were mixed for 2 hours at room temperature. Excess antibody, unbound to
IONPs, was effectively washed and removed with a wash/storage buffer (provided by the conjugation kit)
by centrifugation with 300K MWCO OMEGA membranes, three times, followed by resuspension of
IONPs into phosphate-buffered saline (PBS). The quality of the conjugation was tested by visualization of
mobility shift in 1% agarose gel compared to the free IONPs and the conjugation reaction mixtures were
then stored at 40C.
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Cell viability/toxicity studies
For both cell viability and toxicity studies, an MTT Cell Proliferation Assay (CellTiter 96 Aqueous One
Solution, Promega WI USA) was used. Human GBM cells (5 x 103 cells/well in a total volume of
100μl/well) and human astrocytes (15 x 103 cells/well in a total volume of 100μl/well) were seeded in 96well flat-bottomed plates in triplicate and incubated at 370C overnight. Cells were then treated with PBS
(control), IONPs (0.3mg/ml), cetuximab (0.3mg/ml), and cetuximab-IONPs (0.3mg/ml) for 24 h at 370C,
followed by a subsequent single IR dose of 10Gy or fractionated IR of 10Gy x 2 every 24 hours. The
cytotoxicity analysis was performed at three different time points of 24, 48, and 72 h after application of
the last IR dose. For reading of the absorbance values, which assessed viable cells, 20 μl of the MTT
reagent was added to each well and each plate was then incubated at 370C for 1 hour. The plates were
then analyzed using a microtiter plate reader (Synergy HT BioTek Instruments USA) and the readings
were taken at a wavelength of 490nm. All the measured absorbance values were within the range of the
linear response curve for the 490nm wavelength provided by the manufacturer and corrected for
background.
Cell apoptosis studies
U87MGEGFRvIII cells were seeded in 6-well flat-bottomed plates at a density of 5 x 105/well and
incubated at 370C overnight, followed by treatment with PBS (control), IONPs (0.3mg/ml), cetuximab
(0.3mg/ml), and cetuximab-IONPs (0.3mg/ml) for 24 h at 370C. Subsequent fractionated IR of 10Gy x 2
was administered with a 24 h interval between each IR treatment. Cell lysates were harvested 24 h postIR in radioimmunoprecipitation assay (RIPA) buffer (50mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA
and 1% NP40, with protease and phosphatase inhibitors). Protein levels were determined by Western blot
analysis with primary antibodies against cleaved caspase-3 and caspase-3 antibody (Cell Signaling,
1:1000) according to the manufacturer’s recommendations.
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Cellular DNA damage levels
U87MGEGFRvIII cells (7 x 103 cells/chamber in a total volume of 200μl) were cultured in 8 chamber
polystyrene vessel tissue culture treated glass slides and intubated overnight at 370C. Cells underwent
four different treatments with PBS (control), IONPs (0.3mg/ml), cetuximab (0.3mg/ml), and cetuximabIONPs (0.3mg/ml) for 24 h at 370C, followed by a single IR dose of 2Gy. Development of γH2AX foci
was evaluated 30 minutes post IR. The cells were fixed in 4% formaldehyde at room temperature for 10
min and permeabilized with 0.2% tritron X-100 in PBS for another 10 minutes. After washing with PBS,
the cells were blocked by the addition of 10% normal goat serum for 1 h at room temperature before
incubation with the anti-phospho H2AX (Ser139) clone JBW301 (Millipore), at a dilution of 1:1000 at
room temperature for 2 h and Alexa Fluor 488 (Life technologies-Molecular Probes) at a dilution of 1:500
at room temperature for 1 h. The cells were washed three times with PBS, nuclei were counterstained
with addition of mounting solution with DAPI (ProLong Gold antifade reagent with DAPI, Molecular
probes-Invitrogen), and finally the slides were mounted with coverslips. Slides were examined under the
fluorescent microscope (Axio Observer A1; Zeiss) with a green filter for detection of γH2AX foci. The
images were captured with a camera (AxioCam MRm). The quantification of γ-H2AX foci formation was
performed by semi-automated measurement of the foci density (number of foci per square inch of
nucleus). Image J software analysis was used for the semi-automated quantification and approximately 30
nuclei were evaluated for each treatment group in three separate measurements.
Cellular oxidative stress levels
The Image-iT LIVE Green Reactive Oxygen Species Detection Kit (Molecular probes-invitrogen by life
technologies I36007) was used to measure the ROS levels in live human GBM cells. The assay was based
on 5-(and-6) - carboxy-2′, 7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA), a peroxidesensitive fluorescent probe and a marker for ROS detection in live cells. Carboxy-H2DCFDA permeates
live cells and is deacetylated by nonspecific intracellular esterases to a non-fluorescent compound. In the
presence of nonspecific ROS produced particularly during oxidative stress, the reduced fluorescein
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compound is oxidized and emits bright green fluorescence. ROS generation was assessed in
U87MGEGFRvIII cells after treatment with PBS, IONPs, cetuximab, and cetuximab-IONPs with
subsequent IR. Tert-butyl hydroperoxide (TBHP), an inducer of ROS production, was used as a positive
control and Hoechst 33342, a blue-fluorescent, as a cell-permeant nucleic acid stain. The cells (7 x 103
cells/chamber in a total volume of 200μl) were seeded in 8 chamber polystyrene vessel tissue culture
treated glass slides and incubated overnight at 370C. Cells underwent four different treatments with PBS
(control), IONPs (0.3mg/ml), cetuximab (0.3mg/ml), and cetuximab-IONPs (0.3mg/ml) for 24 h at 370C,
followed by a single IR dose of 10Gy. The intracellular extent of ROS generation was evaluated 3 h postIR. The cells were washed once with Hanks’ Balanced Salt Solution (HBSS) 1X containing calcium and
magnesium chloride, loaded with 25μM Carboxy-H2DCFDA (200μl/chamber), and incubated at 370C for
30 min in the dark. During the last 5 minutes of incubation, 1μM Hoechst 33342 (0.2μl/chamber) was
added. The cells were then washed with HBSS 1X containing calcium and magnesium chloride three
times, the slides were covered with coverslips, and immediately observed for fluorescence intensity under
a fluorescent microscope (Axio Observer A1; Zeiss) with a green filter. Images were acquired using the
AxioCam MRm camera.
Animals, orthotopic xenografts, and CED treatment
All the in vivo procedures were initially approved by the Institutional Animal Care and Use Committee
(IACUC) of Emory University and performed on 6 - to 8 - week old athymic nude (nu/nu) mice. After
general anesthesia application, all animals were temporarily restrained in a small animal stereotaxic
frame. A Hamilton syringe attached to the frame was used to stereotactically inoculate 2 x 105
U87MGEGFRvIII human GBM cells into the right striatum of each animal, 2 mm below the brain
surface. Five days post-tumor implantation, the animals were randomly assigned to CED of HBSS
(control group), cetuximab, or cetuximab-IONPs at a concentration of 0.3mg/ml (7 animals were assigned
in each group). CED was performed in each animal by utilizing a Hamilton syringe attached to a pump
connected to a microinfusor apparatus and the stereotaxic frame in order to infuse a 10μl volume at a rate
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0.5μl/min over a 20 minute total infusion period. The same coordinates used for tumor inoculation were
used for CED infusion in all animals. Subsequent fractionated whole brain IR of 10Gy x 2 was performed
in all treated animals 24 and 72 h after CED.
IR administration procedure
All IR therapies were administered using the irradiator X-RAD 320kV, 10mA (Precision X-Ray, INC., N.
Branford, CT, USA). For the in vitro radiosensitization experiments, the 96-well plates and the tissue
culture glass slides were placed into the irradiator under the adjustable collimator and the cells were
irradiated at a dose rate of 1.2Gy/min with a filter made of 2 mm Al.
For the in vivo experiments, the anesthetized animals were positioned in a small rectangular holder with a
1x1 inch marked area used for head position. The animals underwent whole brain IR by using a filter
consisting of 1.5mm Al + 0.25mm Cu + 0.75mm Sn at a dose rate of 0.5Gy/min. In all the IR
experiments, the field collimator was adjusted to cover the area of interest and setting of appropriate
machine parameters was repeatedly performed.
Imaging
Imaging was done on a 4.7-T animal MRI scanner with a dedicated mouse coil (Varian Unity).
Anesthetized animals were positioned into a molded plastic restraint supporting the animal for imaging
and the required sensors were also routinely placed for measuring physiologic animal parameters during
the entire imaging procedure. The sequences used for imaging of the xenografts and IONPs in the brain
were T2 weighted fast spin echo sequences with TR/TE=6500/70ms. A total of 11 slices with a slice
thickness of 1 mm axial images covering the entire brain region were collected for each scanned animal.
Histology
The brains of all animals implanted with tumor cells were harvested and fixed with 10% neutral buffered
formalin. After proper identification of the needle tract on the brain surface, coronal sections were made
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through the injection tract in order to section the deeply located tumor xenografts. Afterwards, tissue
blocks were initially embedded in paraffin, then sectioned at a thickness of 5 micrometers, and finally
mounted on slides for observation under the microscope. Slides of formalin-fixed brain tissue underwent
Prussian blue staining with a 1:1 mixture of 5% potassium ferrocyanide and 5 % HCl acid for 30 min at
37°C, followed by a rinse with distilled water. For EGFRvIII immunohistochemistry, initial
deparaffination of tissue sections was followed by incubation with the primary rabbit polyclonal
EGFRvIII antibody (GenScript Corp., Piscataway, NJ) and the secondary anti-rabbit antibody provided in
a rabbit ABC staining system (Santa Cruz Biotechnology, Santa Cruz, CA).
Animal survival studies
All animals were monitored daily for neurological deficits such as paresis and seizures, behavior changes
such as aggression, isolation and lack of eating/drinking, as well as for difficulty ambulating, hunching,
and weight loss. As soon as the first sign of an adverse appeared, the animals were sacrificed and the all
brains were removed and fixed in 10% formalin.
Statistical analysis
Cell viability and toxicity data acquired from three independent experiments were all carried out in
triplicate for each treatment. The results are expressed as the average value of three independent
absorbance values (triplicate) and the standard deviation values are indicated as error bars in the relevant
plots. Statistical differentiations between treatment groups were made by the 2-tailed unpaired Student’s t
test assuming unequal variance. Animal survival statistical analysis was performed by using the MedCalc
statistical software and data were plotted onto a Kaplan-Meier survival curve. A probability value less
than 0.05 was considered as statistically significant.
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