Brain tumor specimen
Primary pediatric DIPG cells (HSJD-DIPG-007) were derived from autopsy of a 6-year-old female and were transferred as neurospheres, exponentially growing in suspension in tumorsphere media (TSM) supplemented with corresponding growth factors (15, 16), and checked for mycoplasma contamination.
Mouse DIPG model
For the DIPG mouse model, HSJD-DIPG-007 neurospheres were implanted into the fourth ventricle of 6-8 week old, male, NOD/SCID (immunocompromised) mice as described previously (14, 15, 17, 18). All mice were sedated with isoflurane. Each mouse was immobilized using the stereotaxic device and placed on a tooth bar to assure a level, stable field. The surgical field was sterilized using povidone-iodine antiseptic swabs and a 2 cm vertical incision was created. A 1mm burr hole was drilled after identifying coordinates, 1.4 mm posterior to the lambda and 1.0 mm lateral to midline. A glass gas-tight sterile Hamilton syringe loaded with 5 x 105 DIPG neurospheres mixed with matrigel was suspended in 4 µl volume and further advanced 3.5 mm beneath the skull to reach the fourth ventricle. Cells were injected slowly over the course of 3 minutes. Following injection, sterile bone cement was applied to the skull and the skin was closed with silk 4.0 surgical sutures. Intraperitoneal buprenex was administered post-operatively for pain relief during recovery. A single dose of intraperitoneal dexamethasone 2mg/kg was also administered post-operatively to reduce intracerebral edema.
Treatment
The animals were divided into three treatment groups: Untreated (UT) (n=4), OKN-007 treated (n=4), LDN-193189 treated (n=5) mice. Initially, we had planned on having 5 mice in each treatment arm but unfortunately, several mice had to be terminated for neurological deficits noted a few days post-operatively. Both OKN-007 and LDN-193189 treatments were initiated at day 28 after surgery. Treatment with both LDN-193189 and OKN-007 continued daily for 28 days, and mice were terminated for histological analysis on day 58.
OKN-007 (Ryss Laboratories, Union City, CA; 99.8% purity via HPLC) was dissolved in drinking water at a concentration of 150 mg/kg/day, and treatment bottles were changed every 3 days. Water bottles were weighed prior to bottle changes, and the amount of OKN-007 consumed per mouse was readily determined, as each OKN-007 treated mouse was housed individually. The average consumption of OKN-007 ranged from 130-150 mg/kg/day in all treated mice.
LDN-193189 (Thomas Scientific, Swedesboro, NJ; 98% purity via HPLC) was dosed at 25 mg/kg/day and administered as daily gavage treatments. Plastic tubing oral gavage needles were used. Mice were anesthetized shortly with isoflurane to help decrease agitation during daily gavage treatments.
Magnetic resonance techniques
Morphological imaging. MRI scans were obtained using a 30-cm horizontal Bruker Biospin magnet operating at 7 Tesla (T; Bruker Biospin GmbH, Karsruhe, Germany), with a S116 gradient set to perform all MRI experiments. An EPI (echo planar imaging) transceiver 1H 50W coil with a 38.0 mm inner diameter was used for signal transmission and detection. NOD/SCID mice underwent MR imaging investigations while under anesthesia (isoflurane 1.5%- 2.0% and 0.8 L/min O2) and restrained in a cradle, which was inserted into the MRI scanner. All animals have their body temperature maintained at 37 degrees Celsius with a water bath driven heating pad placed under the animal in the MRI cradle. Respiratory rates were also monitored for the entire duration of the MRI scan. Fluid attenuated inversion recovery (FLAIR) imaging (FOV = 2.0 x 2.0 cm2, TR = 10,000 ms, TE = 37.0 ms, matrix size = 256 x 256, averages = 4, slices = 14, slice thickness = 0.7 mm) was used to calculate tumor volumes and to inspect tumor morphology. In-vivo diagnostic contrast-enhanced MRI approach with injection of gadolinium contrast via an intravenous tail-vein catheter was performed at different time intervals to assess for an intact BBB. This assessment used multi-slice spin echo T1-weighted images (TR = 1000.0 ms, TE = 14 ms, FOV = 2.50 x 2.50 cm2, averages = 2, slices = 16, matrix size = 256 x 256) attained before and 15 minutes after tail vein gadolinium contrast agent injection (Gd-DTPA, Magnevist, Bayer Inc., Wayne, NY, USA; 0.4 mmol/kg).
MRI scans were initially performed two weeks after surgical implantation of tumor cells, followed by weekly imaging until termination. During scanning procedures, physiological monitoring for respiration was conducted to assess the animal’s status. Survival of mice following therapeutic protocols were monitored routinely with MRI scans and an MRI were obtained as the last time-point when the mouse is euthanized.
Diffusion imaging. Diffusion-weighted imaging (DWI) was also used to characterize the mobility of water in a clinical setting with diffuse tumor involvement. Diffusion data is also valuable in documenting cellular response to drug therapy. A coronal axial multi-slice DWI sequence covering the entire tumor was performed with the following parameters: TR = 3000 ms, TE = 32.5 ms, matrix size = 96 x 96, slice thickness = 0.75 mm. Apparent diffusion coefficient (ADC) values were acquired by placing four circular regions-of-interest (ROIs) within the tumor bed and these values were averaged, and then normalized to the contralateral normal brain.
Histology and immunohistochemistry
Histology Staining. Four micron thick histological sections, embedded in paraffin and mounted on HistoBondÒPlus slides (Statlab Medical Products, Lewisville, TX) were rehydrated and washed in Tris Buffered Saline (TBS). Rabbit antibodies for anti-Histone H3K27M (cat# 31-1175-00,1 µg/ml, RevMAB Biosciences, San Francisco, CA), ALK2/ACVR1 (cat# LS-B11761, 1:100, 5µg/ml, LSBio, Seattle, WA), CD34 (cat# ab81289, 5µg/ml, abcam, Cambridge, MA), Met (cat# sc-10, 1:50, 4 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), Caspase-3 (cat# sc-7148, 1:50, 4µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA). Nuclear Antigen antibody was raised in mouse against Human (cat# LS-C277289, 1:100, LSBio, Seattle, WA). Slides were processed for Immunohistochemistry using ImmPRESS VR horse anti rabbit IgG Polymer kit (cat# MP-6401, Burlingame, CA) or M.O.M. ImmPRESS™ Peroxidase Polymer kit (cat# MP-2400, Vector Labs Inc., Burlingame, CA). Antigen retrieval (pH 6 Citrate Antigen Unmasking Solution (cat# H-3300,Vector Labs Inc., Burlingame CA) was accomplished via twenty minutes in a steamer followed by thirty minutes cooling at room temperature for all antibodies except CD34 which was ten minutes in a steamer followed by twenty minutes in ice cold pH 6. Sections were treated with a peroxidase blocking reagent (Bloxall, cat# SP-6000, Vector Laboratories, Inc, Burlingame, CA) to inhibit endogenous peroxidase activity, followed by 2.5% normal horse serum or mouse IgG blocking reagent to inhibit nonspecific binding. Appropriate washes were in TBS. Antibodies were applied to each section and following incubation overnight at 4°C in a humidified chamber, sections were washed in TBS and reagents were applied according to the manufacturer’s directions. Slides were incubated with NovaRed® ( Vector Laboratories, Inc., Burlingame, CA) chromogen for visualization. Counterstaining was carried out with Hematoxylin QS Nuclear Counterstain (Vector laboratories, Burlingame, CA). Appropriate positive and negative tissue controls were used.
Immunohistochemistry (IHC). Aperio ScanScope Image Analysis System was used to analyze all the IHCs. H3.K27M, ACVR1, and human nuclear antigen IHCs were evaluated using a Positive Pixel Count procedure with the Aperio ImageScope viewer. Although the DIPG were diffuse, five ROIs were placed in heavily stained areas along the brainstem to analyze IHC expression. ROI placement was avoided in areas of necrosis or substantial artifact. The number of positive pixels from ROIs were divided by the total number of pixels (negative and positive) in the examined area, and multiplied by 100, in order to determine the percentage of positivity.
Aperio microvessel analysis algorithm using CD34 as a marker was also utilized to determine microvascular density (MVD, number of vessels per mm2). Again, ROIs were arbitrarily selected, and the MVD was calculated for untreated, OKN-007 treated, and LDN-193189 treated groups.
Statistics. Graph Pad Prism 8 was the software used for statistical analysis. Statistical significance was assigned only for p values < 0.05. For statistical analysis, ANOVA testing (one-way ANOVA, multiple comparisons) was used for both tumor volume, diffusion data, and IHC analysis to compare the differences of untreated, OKN-007 treated, and LDN-193189 treated pediatric HSJD-DIPG-007 mice. A two-tailed students t-test was used to compare CD34 expression on IHC of OKN-007 and LDN-193189 treated mice.