A new anti-glioma therapy, AG119: pre-clinical assessment in a mouse GL261 glioma model

Orthotopic HGG model

Procedures for preclinical assessment of anti-cancer therapeutics in an orthotopic
GL261 mouse glioma model were approved by the Institutional Animal Care and Use Committees
(IACUC) at the Oklahoma Medical Research Foundation (OMRF) and the University of Oklahoma
Health Sciences Center (OUHSC). Anesthetized C57BL6 mice (Harlan Laboratories) were
implanted with GL261 mouse glioma cells (1?×?10
⁴
cells in 6 ?L PBS) (ATCC) as previously described for other glioma cells 16]–18]. There were 5 treatment groups (untreated, anti-VEGF antibody, anti c-Met antibody,
TMZ or AG119), which had 5–7 mice per group. Treatments were started once tumors were
10–20 mm
³
as measured by MRI. Antibody therapies were administered at a dose of 1 mg/kg body
weight i.v. via a tail-vein catheter every 3 days for up to 21 days 19]. TMZ and AG119 were administered at a dose of 30 mg/kg, i.p., twice weekly for 2 weeks.
AG119 was dissolved in 5 % N-methylpyrrolidine (Pharmasolve; Sigma-Aldrich), 5 % solutol-15
(BASF, Bern, Switzerland) in sterile normal saline. TMZ was dissolved in 5 % DMSO
and 5 % solutol-15 in sterile saline. Antibody therapies (anti-c-Met (Met (B-2): sc-8057;
Santa Cruz Biotechnology Inc., Santa Cruz, CA) and anti-VEGF (anti-mouse VEGF-A; Biolegend
Inc., San Diego, CA) were prepared in sterile saline. Control untreated tumor-bearing
mice received the same solvent as for those that were treated with AG119 (vehicle
control).

MRI assessment of tumor volumes

MRI experiments were performed on a Bruker Bio-spec 7.0 Tesla/ 30-cm horizontal-bore
magnet imaging system. Animals were immobilized with 1.5–2.5 % isoflurane and 0.8 L/min
O
₂
and placed in a 72-mm quadrature volume coil for signal transmission, and a surface
mouse-head coil was used for signal reception. Morphological T
₁
and T
₂
-weighted MRI were used to assess tumor growth and calculate tumor volumes, as previously
described 17], 18], 20], over a 25–35 day time period at 5–7 day intervals. Tumor volumes were calculated
from multiple MRI slice datasets. Percent survival [Kaplan-Meier plots generated in
Prism (GraphPad Software)] were also obtained from time-points when mice were euthanized
1–2 days prior to expected disease-initiated deaths. All animals were humanely euthanized
(CO
₂
asphyxiation) when they met tumor burden criteria (tumors???150 mm
³
) and/or showed signs of illness, weight loss, poor body condition, porphyria, hypoactivity,
restlessness, aggressiveness, ataxia, shallow, rapid and/or labored breathing, cachexia,
failure to respond to stimuli, lack of inquisitiveness, vocalization, seizures, hunched
posture and ruffled fur. Two animals died due to anesthesia complications, but were
included in the survival data.

Perfusion imaging (ASL)

Arterial spin label (ASL) perfusion images were obtained to calculate relative cerebral
blood flow (rCBF) rates in tumors, as previously described 21]. Perfusion maps were obtained on a single axial slice of the brain located on the
point of the rostro-caudal axis where the tumor had the largest cross-section 21]. The imaging geometry was a 3.5?×?3.5 mm
²
slice, of 1.5 mm in thickness, with a single shot echo-planar encoding over a 64?×?64
matrix. An echo time of 20 ms and a repetition time of 18 s were used. To obtain perfusion
contrast, the flow alternating inversion recovery scheme was used. Briefly, inversion
recovery images were acquired using a slice-selective (SS) inversion of the same geometry
as the imaging slice or a non-selective (NS) inversion slice concentric with the imaging
slice.

Recovery curves obtained from each pixel of non-selective (S _NS
(TI)?=?A – B • e^-TI/T1*
) or selective (S _SS
(TI)?=?A – B • e^-TI/T1*
), with 1/T ₁
*?=?1/T ₁
?+?CBF/?, inversion images were numerically fitted to derive the pixelwise T ₁
and T ₁ * values, respectively 22]. The results were stored as maps for further analysis. These longitudinal recovery
rates were then used to calculate the cerebral blood flow, CBF (ml/(100 g?·?min)) on a pixelwise basis using the following relationship: CBF?=?? • [(1/T ₁
*) – (1/T ₁
)] 22]. The partition coefficient, ?, was scaled by assigning the generally adopted value
of 0.9 ml/g 22]. Regions of interest (ROIs) were manually outlined around the tumor and a copy will
be positioned onto the contralateral side of the brain for comparison purposes.

Viability assay

U251 (TMZ-sensitive; low level of methyl guanine transferase (MGMT)) and T98G (TMZ-resistant;
high level of MGMT) cells 23] (American Type Culture Collection, Manassas, VA) were maintained at Dulbecco’s minimal
essential medium (DMEM) (Thermo Fisher Scientific) with 10 % Cosmic Calf Serum (CCS,
Hyclone, Logan, UT) and added glutamine/pyruvate (HyClone) at 37 °C with 5 % CO
₂
. Cells were treated with AG119 or TMZ (Sigma, St. Louis, MO) dissolved in Opti-MEM
(Invitrogen, Carlsbad, CA) from 50 mM DMSO stock solutions. After 4 h of treatment,
10 % CCS was added and cells incubated for an additional 44 h. To determine viability,
PrestoBlue (Invitrogen) was added as per manufacturer’s protocol and read on a microplate
reader (BioTek, Winooski, VT). IC
₅₀
values were determined by nonlinear regression analysis in Prism 6.0 software (GraphPad,
San Diego, CA).

Statistical analyses

Statistical analyses were performed by using One-way ANOVA with a post Tukey’s multiple
comparison test for evaluating differences in tumor volumes between untreated and
treated groups. Data is represented as mean?±?S.D., and p-values??0.05 (*), 0.01
(**), 0.001 (***) were considered statistically significant. For survival curves,
statistical differences were determined using a Log-rank (Mantel-Cox) test and a Gehan-Breslow-Wilcoxon
test.