The U87 (American Type Culture Collection, Rockville, MD, USA, ATCC HTB-14) human glioblastoma cell line was maintained in DMEM medium supplemented with 10 % fetal bovine serum, 3.2 % non-essential amino acids, 100 units/ml Penicillin/Streptomycin, 400 mol/l L-glutamine (all Sigma-Aldrich, St.Lous, MO, USA) and 0.005 mg/ml Plasmocin (InvivoGen, San Diego, CA, USA), at 37 °C and 5 % CO2.
Cells from serially passaged xenograft spheroids (P3) were maintained as a monolayer in NB medium (Thermo Fisher Scientific Corporation, Carlsbad, CA, USA) with the addition of 32 IE/ml heparin, 20 ng/ml bFGF and 20 ng/ml EGF (Millipore Corporation, Billerica, MA, USA).
For in vitro assessment of dactolisib efficacy, a 10 mM stock solution was prepared by dissolving dactolisib (kindly provided by Novartis (Basel, Switzerland): Also, dactolisib was obtained from Selleckchem (Houston, TX, USA) in 100 % DMSO (Sigma Aldrich, St. Louis, MO, USA). Further dilution was done in cell culture medium.
Patient tumour material
In our study, we used a GBM xenograft model (P3) previously described . This model reflects the growth pattern of human tumours in situ, including extensive infiltration into the brain parenchyma, prominent angiogenesis, and necrosis. In short, tumour biopsy tissue was obtained from the operating theatre, Haukeland University Hospital, Bergen, after approval from the regional Ethical Board and consent from patient. Tumour material was then cut into smaller pieces and maintained in medium to make spheroids , which again were serially passaged in rodents as described by Wang and colleagues . In our experiment, the spheroids were enzymatically dissociated at 37 °C by trypsin-EDTA (Sigma-Aldrich, St. Louis, MO, USA) and DNase (Roche, Basel, Switzerland) for implantation in rats. The cells were resuspended in sterile PBS with 25 mM glucose (both Sigma Aldrich, St. Louis, MO, USA) and kept on ice until implantation of 100 000 cells in each animal. Three spheroids ranging in size between 500 and 600 μm in diameter were used for implantation in each mouse. The spheroids were kept in sterile, ice cold PBS with 25 mM glucose until implantation.
Cell viability (MTS assay)
1000 U87 cells or 5000 P3 xenograft cells were seeded in 96-well plates 24 h prior to dactolisib exposure at following concentrations: 0, 1, 10, 20, 30, 40, 50 and 250 nM. After 72 h, the cells were analyzed using the MTS viability assay according to the manufacturer’s protocol (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA), and absorption was measured at 490 nm using a plate reader (Asys UVM340, Biochrom, Cambridge, UK). Viability was determined relative to untreated controls. These data were used to make dose response curves for determination of IC50 values in GraphPad 6 Prism (GraphPad Software Inc., La Jolla, CA, USA).
Cells exposed to 0, 10, 50, 250 and 1000 nM dactolisib for 72 h, were treated with 10 μM BrdU (Sigma-Aldrich, St. Louis, MO, USA) in medium for 45 min at 37 °C. They were detached using a cell scraper, washed once with 1xPBS and resuspended to a concentration of 1 × 105 cells/ml. Cell suspensions were kept on ice and processed within minutes. One hundred microliter cell suspension from each sample was loaded into individual sample chambers and centrifuged in a Shandon CytoSpin centrifuge (Thermo Fisher Scientific, Wilmington, DE, US) at 800 rpm for 3 min. Immobilized cells were fixed (described in the ICC-section below), and subsequently subjected to immunocytochemistry, imaging and quantification. For each slide, three randomly picked areas (832 μm × 665.6 μm, 554 mm2) were selected for quantification. The FITC stained cells and the total number of cells was manually counted, and the proportion of FITC positive cells was calculated.
Cells on coverslips in 24-well plates were fixed in 4 % paraformaldehyde (Thermo Fisher Scientific Corporation, Carlsbad, CA, USA) for 10 min, permeabilized by 0.5 % Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 4 min and incubated with blocking buffer (0.5 % BSA (Sigma-Aldrich, St. Louis, MO, USA) in PBS) for 15 min. All steps were performed at room temperature. Cells were incubated with primary antibodies overnight at 4 °C, in a humid atmosphere. The primary antibodies used were total Akt, pAkt S473, pAkt T308 (All Cell Signaling Technology, Danvers, MA, USA), and BrdU (Abcam, Cambridge, UK) together with DNAse (Roche, Basel, Switzerland). Following incubation, cells were washed in PBS and incubated with secondary antibodies for 45 min at 37 °C in a humid atmosphere. The secondary antibodies used were FITC-conjugated goat anti-mouse IgG1 and FITC-conjugated goat anti-rabbit (both from Southern Biotechnologies Associates Inc., Birmingham, AL, USA). After sequential washing with PBS and deionized water, cells were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were obtained with a Nikon TE2000-E microscope (Nikon Corporation, Tokyo, Japan).
Cell number quantitation
Cells were seeded in 96-well plates 24 h prior to exposure to dactolisib for 72 h at the following concentrations: 0, 10, 50 and 250 nM. The cells were detached enzymatically by Trypsin-EDTA solution, transferred to a Burker chamber haemocytometer and manually counted using a light microscope.
Immunoblotting (Western blot)
Cell lysates were prepared by resuspending mechanically harvested cells or finely minced tissue in kinexus buffer (20 mM MOPS, 5 mM EDTA, 2 mM EGTA, protease- and phosphatase inhibitor tablets (Roche, Basel, Switzerland)), followed by Vibra-Cell sonication (Sonics & Materials Inc, Newton, CT, USA) for 3 × 5 s. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Corporation, Carlsbad, CA, USA). 20 μg lysate was mixed with NuPAGE LDS sample loading buffer and NuPAGE sample reducing agent (both Thermo Fisher Scientific Corporation, Carlsbad, CA, USA) and incubated at 70 °C for 10 min. Samples were run on a pre-cast SDS-gel (NuPage, Invitrogen, Thermo Fisher Scientific Corporation, Carlsbad, CA, USA) at 200 V for 60 min. Transfer to a nitrocellulose membrane was done at 30 V for 80 min. Following blocking in 5 % (w/w) Difco Skim milk powder (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), in TBST (50 mM Tris, 150 mM NaCl, 0.05 % Tween20) for 1 h at room temperature, the membrane was incubated with primary antibody (total Akt, pAkt S473, pAkt T308 (All Cell Signaling Technology, Danvers, MA, USA) and β-actin (Santa Cruz Biotechnology Inc, Dallas, TX, USA) or GAPDH (Abcam, Cambridge, UK) at 4 °C O/N. The membrane was washed with TBST before incubation with the secondary antibodies goat anti-mouse IgG-HRP (Santa Cruz Biotechnology Inc, Dallas, TX, USA) and goat anti-Rabbit IgG (H + L) Cross Adsorbed Secondary Antibody, HRP conjugate (Thermo Fisher Scientific Corporation, Carlsbad, CA, USA) for 1.5 h. For detection, the Supersignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Rockford, IL, USA) was used, and chemiluminescent detection was obtained by a Fuji LAS 3000 Imager (Fuji Photo Film, Tokyo, Japan). Densitometric quantification of the bands was done using ImageJ software (National Institutes of Health, Bethesda, MA, USA).
Annexin V / Propidium Iodide (PI) apoptosis assay
Cells were stained with the Annexin V apoptosis assay according to the manufacturer’s protocol (Thermo Fisher Scientific Corporation, Carlsbad, CA, USA). Briefly, cells were detached and washed twice by PBS (without calcium and magnesium) and once in Annexin V binding buffer. Samples were resuspended in 100 μl Annexin V binding buffer and 5 μl Annexin V Alexa Fluor 488 and 1 μl PI was added before incubation in the dark for 15 min at RT. Four hundred microliter Annexin V binding buffer was then added to each sample and the samples were kept on ice and analysed immediately on the Accuri C6 (BD Biosciences) flow cytometer.
The in vivo studies were performed on a total number of 33 athymic homozygous nude rats (Han: nru/nru Rowett) and 32 NOD/SCID mice (NOD.CB17-PrkdcScid). Animals were bred and maintained in animal facility at University of Bergen, certified by AAALAC international. The animals were provided a standard pellet diet and tap water ad libitum. They were kept in a pathogen free environment at a constant temperature and humidity and standard 12/12 h light and dark cycle.
Prior to tumour implantation, all animals were anaesthetized with isoflurane gas (Abbott Laboratories, Abbot Park, IL, USA) (3 % mixed with 50 % air and 50 % O2) and given Marcaine (AstraZeneca, London, England) subcutaneously. The head was secured in a stereotactic frame (Benchmark, Neurolab, St Louis, MO, USA) before a longitudinal incision was made in the scalp. Through a burr-hole obtained with a micro-drill, the tumour material was slowly inserted via a Hamilton syringe with an inner diameter of 810 μm, at the following coordinates for rats: 1 mm posterior of the bregma suture, 2 mm right of the sagittal suture and 3 mm below the brain surface. For mice, the coordinates were 0.5 mm posterior of the bregma suture, 1.5 mm right of the sagittal suture and 1.5 mm below the brain surface. The skin incision was closed using an Ethilon 3-0 suture. Animals were weighed five times a week on a PGW 2502e weight (Adam Equipment, Danbury CT, USA), inspected daily and euthanized by CO2 inhalation at the onset of symptoms such as passiveness, neurological deficits or other signs of illness. The brains were harvested, snap frozen in liquid nitrogen and stored at −80 °C. All procedures and experiments involving animals in this study were approved by The Norwegian Animal Research Authority (Bergen, Norway) and is in accordance with Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council. Washington, DC: National Academy Press, 1996).
Tumour bearing animals were randomly assigned to two different groups: 1) untreated controls and 2) dactolisib treatment. The medication started by the time tumour engraftment was confirmed by MRI. Dactolisib was administered by oral gavage, using malleable oral dosing needles with silicone tips (Scanbur, Karlslunde, Denmark). Dactolisib was delivered as a suspension in 0.5 % methyl cellulose and 0.5 % Tween20 (both Sigma Aldrich, St. Louis, MO, USA), once daily, 5 days a week. Vehicle (0.5 % methyl cellulose and 0.5 % Tween 20) was equally given per os(p.o.) to the animals in the control group. Both groups received 10 ml/kg solution each treatment day.
After longitudinal observation of healthy, non-implanted animals, the dose was set to 10 mg/kg for rats. The dose 45 mg/kg for mice was determined from studies published by other groups [15, 16], but was rapidly adjusted to 25 mg/kg in the study of tumour bearing mice.
During the dactolisib-exposure of non-tumour bearing animals, dactolisib was delivered as a solution in 1 volume NMP and 9 volumes PEG300 (Both Sigma Aldrich, St. Louis, MO, USA).
Assessment of side effects
During the daily inspection of the animals, any changes and possible side effects observed (gavage reluctance, skin rash, diarrhoea and excessive salivation) were registered. To allow for semi-quantitative comparison between the groups, the animals were scored for the presence of side effects. The total number of events for each possible side effect was summed and displayed as a histogram.
Blood collection and analysis
Immediately post mortem, blood was collected from the aorta. The blood was transferred to an Eppendorf tube, allowed to coagulate at room temperature for 30 min and centrifuged for 10 min at room temperature, 1300 rcf. The plasma was then collected and stored at −80 °C until analysis by Sentrallaboratoriet NMBU Veterinærhøgskolen (Oslo, Norway). Blood glucose levels were measured by Accu-Chek Aviva blood glucose meter (Roche, Basel, Switzerland) by one drop of freshly collected blood.
Magnetic resonance imaging (MRI)
The animals were anaesthetized with 3 % isoflurane, in a mixture of 50 % N2O and 50 % O2, and brain images were obtained, using a Bruker Pharmascan 7 T MR scanner (Bruker Biospin MRI GmbH, Ettlingen, Germany). For rats, a coronal T2 weighted TurboRARE sequence was acquired (TR 3500 ms and TE 36 ms), in addition to an axial T1 weighted RARE sequence (TR 1000 ms and TE 9 ms), after subcutaneous injection of contrast agent (1–2 ml of Dotarem, 279.3 mg/ml, Guerbet LLC, Bloomington IN, USA). Common for both sequences for rats was slice thickness 1 mm, FOV 3.2 cm, matrix size 256 × 256, 20 slices.
Similarly, for mice, a coronal T2 weighted TurboRARE sequence was acquired (TR 4300 ms and TE 36 ms), in addition to a coronal T1 weighted RARE sequence (TR 1000 ms and TE 9 ms), after subcutaneous injection of contrast agent (0.2 ml of Dotarem). Common for both sequences for mice was slice thickness 1 mm, FOV 2 cm, matrix size 256 × 256 and 15 slices. The tumour volumes at treatment start and on follow-up MRI were calculated in Gamma Plan (Elekta Instrument AB, Stockholm, Sweden).
In vitro experiments were repeated three times and assessed by ANOVA with Tukey’s multiple comparion test, with a p-value <0.05 considered significant. Kaplan-Meier survival curves were generated in GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA). Median survival times for the treatment groups were compared using the log-rank test.