Contrast-enhanced, conebeam CT-based, fractionated radiotherapy and follow-up monitoring of orthotopic mouse glioblastoma: a proof-of-concept study

Abstract

Background: Despite aggressive treatment regimens comprising surgery and radiochemotherapy, glioblastoma (GBM) remains a cancer entity with very poor prognosis. The development of novel, combined modality approaches necessitates adequate preclinical model systems and therapy regimens that closely reflect the clinical situation. So far, image-guided, fractionated radiotherapy of orthotopic GBM models represents a major limitation in this regard. Methods: GL261 mouse GBM cells were inoculated into the right hemispheres of C57BL/6 mice. Tumor growth was monitored by contrast-enhanced conebeam CT (CBCT) scans. When reaching an average volume of approximately 7 mm 3 , GBM tumors were irradiated with daily fractions of 2 Gy up to a cumulative dose of 20 Gy in different beam collimation settings. For treatment planning and tumor volume follow-up, contrast-enhanced CBCT scans were performed twice per week. Daily repositioning of animals was achieved by alignment of bony structures in native CBCT scans. When showing neurological symptoms, mice were sacrificed by cardiac perfusion. Brains, livers, and kidneys were processed into histologic sections. Potential toxic effects of contrast agent administration were assessed by measurement of liver enzyme and creatinine serum levels and by histologic examination. Results: Tumors were successfully visualized by contrast-enhanced CBCT scans with a detection limit of approximately 2 mm 3 , and treatment planning could be performed. For daily repositioning of the animals, alignment of bony structures in native CT scans was well feasible. Fractionated irradiation caused a significant delay in tumor growth translating into significantly prolonged survival in clear dependence of the beam collimation setting and margin size. Brain sections revealed tumors of similar appearance and volume on the day of euthanasia. Importantly, the repeated contrast agent injections were well tolerated, as liver enzyme and creatinine serum levels were only subclinically elevated, and liver and kidney sections displayed normal histomorphology.

Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond

“…We made use of a syngeneic orthotopic mouse GBM transplantation model with i.p. injection of an in vivo NW457 formulation and contrast-enhanced, conebeam (CB)CT-based, fractionated radiotherapy ( 43 ) ( Figure 2 ). Importantly, we first recapitulated the basic in vitro experiments with GL261 cells, the murine cell line that was used for transplantation.…”

“…Criteria for immediate sacrifice comprised the following: Strongly altered hygiene behavior, flattened breathing, body weight loss of ≥ 20%, ulcerating wounds, epileptic seizures or spasms, paralysis of extremities, bloody diarrhea, apathy, hunchbacked posture, self-mutilation, isolation from the group. In milder occurrence of these criteria, mice were sacrificed within 48 h. Intracranial implantation of GL261 cells was performed as described recently ( 43 ). Briefly, mice were medicated with 200 µg/g metamizol (WDT, Garbsen, Germany) and anesthetized by intraperitoneal injection of 100 µg/g ketamine and 10 µg/g xylazine (both from WDT).…”

“…NW457 was administered intraperitoneally at 10 µg/g or 50 µg/g 24 h before irradiation. Tumor volumes were determined by Lx Wx H measurements of the 3 longest orthogonal axes as described ( 43 ), and 3D reconstructions were generated in 3D-Slicer ( ).…”

“…For histological analyses, mice were anesthetized by intraperitoneal injection of 50 µg/g pentobarbital (WDT), followed by cardial perfusion with 3.5% paraformaldehyde (Sigma-Aldrich) as described ( 43 ). Then, brains were explanted and fixed with 3.5% paraformaldehyde for 48 h at 4°C.…”

DataSheet_1.pdf

“…1 ml (10,000 cells) was used for inoculation. Implantation was performed as previously described (22). In brief, after premedication and anesthesia (200 µg/g metamizol, 100 µg/g ketamine and 10 µg/g xylazine, all WDT, Garbsen, Germany) heads were mounted on a stereotactic frame (David Kopf Instruments, Tujanga, CA, USA).…”