A 4-branch survival study was carried out using adult male Fischer rats implanted with GS-9L glioma cells. Animals received either: A) no treatment; B) injection of nanoparticles with MRI; C) radiotherapy with MRI; D) injection of nanoparticles followed by radiotherapy with MRI. Figure 5 shows a schematic of the protocol. Radiotherapy was delivered in a single 10 Gy fraction on the Australian MRI-linac.
All animal experiments were approved by the Animal Care and Ethics committee of Western Sydney University (ACEC number A12431) and were conducted in accordance with the “Australian Code for the Care and Use of Animals for Scientific Purposes (2013)” and the “New South Wales Animal Research Act 1985”.
Cell culture
GS-9L glioma cells (CellBank Australia ECACC 94110705) (Schmidek et al. 1971) were cultivated in EMEM + 2 mM Glutamine + 1% Non-Essential Amino Acids (NEAA) + 10% Foetal Bovine Serum following the suppliers instructions. Harvested cells for implantation were confirmed to be more than 90% viable by trypan blue exclusion staining.
Tumour implantation
Surgery was carried out on day 0. Adult Fischer F344 rats were operated on at 13–15 weeks of age (weight range 250–300 g). As described in Verry et al. (Verry et al. 2016), using a Kopf stereotaxic frame and a Hamilton microlitre syringe, 104 GS-9L glioma cells suspended in 1 µL of serum-free cell growth medium were implanted to the right caudate nucleus, at 3.5 mm lateral to bregma and 6 mm depth. The injection was carried out slowly using the Kopf micro-injection unit (> 30 s to deliver 1 µL) and the needle allowed to dwell for 5 min before slow retraction. The hole in the skull was plugged with bone wax to prevent tumour cell growth above the skull before suturing the surgical wound. A local anaesthetic, bupivacaine, was injected under the skin prior to the first incision over the skull, and carprofen was administered as pain relief during the surgery. Animals were monitored at least once daily following surgery until euthanasia.
Histology
Haematoxylin and eosin staining of brain tumour samples at different stages of growth was carried out to confirm tumour morphology.
AGuIX nanoparticles
Nanoparticles were supplied by NH TherAguix in dry powder form in sterile vials. The nanoparticles have been previously described and characterised (Mignot et al. 2013; Lux et al. 2018; Verry et al. 2020). In brief, the nanoparticle consists of a polysiloxane core surrounded by covalently grafted cyclic ligands of gadolinium, derived from DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The hydrodynamic diameter is 3 ± 1.5 nm and the mass 10 ± 5 kDa. The nanoparticle has a zeta potential of 10 mV at pH 7.5. The biodistribution and pharmacokinetics of AGuIX have been previously investigated in several publications as summarised in Lux et al. (2018). Nanoparticles were injected intravenously via the tail vein (100 mg in 1 mL sterile water, i.e., 100 mM [Gd]eq).
Treatment protocol
Animals were treated on day 11 when the tumour was sufficiently vascularised to give good uptake of the nanoparticle as verified by T1-weighted image enhancement in an MR imaging pilot study. Four treatment branches were pursued as described above (see Fig. 5).
All animals were anaesthetised with isoflurane gas (induction 3%, maintenance 2–2.5%). Groups B and D received intravenous injection of AGuIX® nanoparticles via the tail vein (100 mg in 1 mL sterile water, i.e.. 100 mM [Gd]eq). Groups A and C were kept under anaesthetic for a representative 5 min but did not receive an injection.
Ketamine/xylazine anaesthetic was administered intraperitoneally at an initial dosage of 72 mg/kg ketamine/6 mg/kg xylazine/6 ml/kg saline. If anaesthesia was not achieved after 5 min, a top-up was administered at a dosage of 6 mg/kg ketamine/0.5 mg/kg xylazine/0.5 ml/kg saline, with up to 3 top-ups given in total. This produced deep anaesthesia for the minimum 1 h required for treatment.
Once anaesthesia was established, animals in groups B, C and D were transported from the biological resources unit to the MRI-linac bunker and positioned on the treatment couch. Body temperature was maintained by placing the animal on a towel-wrapped microwaveable gel heat pad. Imaging and radiotherapy protocol is described below, in brief: localiser, T2 and T1 scans were carried out followed by cine-MRI. If the animals received radiotherapy, this was delivered concurrent to the cine-MRI. Group B (nanoparticles only) received the full imaging sequence including cine-MRI for the same duration as the other groups but no radiotherapy was delivered. Group A (no treatment) was not transported to the bunker but was placed on the towel-wrapped gel heat pad for the equivalent length of time corresponding to transportation and treatment in the other branches. The time elapsed between injection of AGuIX nanoparticles in the biological resources unit and delivery of radiotherapy in the MRI-linac bunker was on average 31 min (range 25–40 min dependent on time taken to establish sufficient depth of ketamine/xylazine anaesthesia).
Recovery from anaesthetic took place in the biological resources unit. Animals were given supplementary oxygen until they gained the righting reflex and were able to move around the cage. A heat pad was provided under part of the cage for the first night following treatment and soft food was provided on the floor of the cage.
At the first clinical signs of disease progression (decreased appetite and movement, hunching), animals were euthanised by intraperitoneal injection of pentobarbital sodium. Final progression of the disease is very fast; it is estimated that animals were euthanised less than 24 h before natural death would have occurred.
MR imaging
MR imaging was carried out using a 6-channel receive-only RF coil specially designed to afford free passage of the radiation beam. In brief, rather than pursuing radiotranslucent RF coils, this coil utilises a physically open magnet and RF coils to avoid radiation induction effects, reduce beam scatter and allow interference-free imaging during treatment. The coil is described in detail in Liney et al. (Liney et al. 2018a). Originally designed as a head coil, the gel heat pads used to maintain the rat’s body temperature provided a filling factor sufficient to allow imaging.
The following imaging sequence (see Fig. 6) was run for all animals receiving imaging, as previously described (Liney et al. 2019). Briefly, a 30-s localiser scan was acquired prior to a T2-weighted scan (turbo spin echo, TE/TR = 86 ms/13493 ms, slice thickness 5 mm, in-plane resolution 1.3 mm). This allowed the position of the brain to be confirmed with respect to the radiation isocentre. A T1-weighted scan (fast gradient echo, TE/TR = 1.4 ms/3.75 ms, slice thickness 1.4 mm, resolution 1.3 mm) for animals which had received an injection of nanoparticles, confirmed the presence of a tumour and uptake of nanoparticles through an enhancing spot in the front right hemisphere of the brain.
A cine-MRI sequence of images (dynamic fast gradient echo, TE/TR = 4.29 ms/10 ms, single 10-mm slice, resolution 2.0 mm, temporal resolution 2 Hz) was then commenced along with radiation delivery. This allowed monitoring of the animal’s position during treatment and monitoring of breathing to assess depth of anaesthesia. This was continued for the 17-min duration of the 10-Gy radiation treatment. For Group B animals not receiving radiation, the full MRI sequence including cine-MRI was still carried out for consistency.
Radiotherapy
Radiotherapy treatment was delivered to animals in groups C (without nanoparticles) and D (with nanoparticles). The radiotherapy beam at the Australian MRI-linac is a 6 MV Varian Linatron, with treatment quality assurance and small-field dosimetry carried out as previously described in detail in Liney et al. (Liney et al. 2019). Briefly, the variable source to iso-centre distance was set at 1.8 m. After positioning the rat on the couch by aligning the eye and ear to the in-room lasers, a 1-cm grid aligned to isocenter was overlaid on the T2-weighted MR image to confirm that the rat was positioned correctly and the base of the field was aligned to the base of the brain. After the T1-weighted scan which would confirm enhanced tumour contrast if nanoparticles had been administered, a single dose of 10 Gy was delivered over approximately 17 min using a 2.25 × 2.90 cm rectangular field covering the whole brain.
ICP-MS
Brain tissue was collected from three rats in each of the four treatment branches following end-stage euthanasia (for groups B (nanoparticles only) and D (nanoparticles and radiotherapy), this was between 8 and 11 days post-treatment) and stored in formalin. Samples of tumour and normal tissue were dissected by eye and dried for 1 h 20 min in a cryo-freezer until no visible liquid remained. 400 µL of 70% ultra-pure nitric acid was added and the samples left until fully digested. One sample of tumour tissue from group B was contaminated during preparation for ICP-MS.
ICP-MS analysis was carried out on a Perkin-Elmer NexION 350X. Internal calibration standard 103-Rh was used. 158-Gd was measured in standard mode with a 1-s integration time. Each sample analysis was run in triplicate reporting the average result with standard deviation.
GLD is an employee of NH TherAguix SAS that is developing the clinical applications of the AGuIX nanoparticles.