Session Item

Glioblastoma (GBM) is the most aggressive primary brain tumour with a median survival of 15 months. Dysregulated DNA damage response (DDR) in GBM results in radiation (IR) and chemoresistance. DDR protein 53BP1 localises to DSBs and has been used to indicate DDR. Intravital imaging techniques using a murine cranial window model allows capture of live cell dynamics. This has not yet been utilised in DDR in glioma. Understanding DDR in GBM is essential in outlining mechanisms underpinning resistance.

Aims

-    Develop a DDR reporter cell for use in intravital glioma patient derived xenografts (pdx) imaging

-    Identify DDR following IR +/- DDR inhibitors (DDRi) as single agents and in combination

-    Develop methods of tracking live DDR to allow prediction of survival and early identification of response

We have transfected a primary GBM cell line (G7) with a fluorescent DSB reporter mApple-53BP1trunc plasmid facilitating interrogation of dynamic DSB response following IR in vitro. G7 pdx tumours have been shown to accurately recapitulate human GBM pathology. Cells were also transfected with a GFP-H2B marker for nuclear staining and localisation. 

In parallel, the intravital murine cranial window model was developed for IR planning feasibility to permit direct imaging of irradiated cells. IR planning optimisation was performed to determine the optimum planning technique accommodating the cranial window titanium ring.

Finally, 53BP1-engineered cells (G7-mApple) were seeded in nude mice following cranial window placement via intracranial injection and imaged at 2 weeks to assess tumour viability in vivo.

G7-mApple cells imaged on confocal immunofluorescence microscopy demonstrated a significant increase of 53BP1trunc foci and intensity at 3h after 8 Gray (p<0.001). Cells were also confirmed to colocalize with native DSB markers yH2AX and 53BP1 in vitro.

Three IR beam arrangements were considered for use with the cranial window model; parallel opposed pair, superior arc and single superior beam. All plans demonstrated effective tumour coverage and organs at risk (OAR) dose constraints, with parallel opposed arrangement demonstrating best sparing of OARs,

G7-mApple cells were successfully implanted and imaged via the murine cranial window model confirming tumour formation and maintenance of dual-marker cell populations.

Using G7-mApple to directly visualise live repair kinetics following IR gives key insights into formation of DSB foci and confirms feasibility of real-time foci tracking. G7-mApple cells have been successfully seeded in nude mice using the cranial window model imaging system. This model allows multiple temporal measurements within a single mouse permitting pre and post treatment imaging, providing a novel refinement in murine glioma work. G7-mApple murine xenografts will potentially allow real-time measurement of response to DDRi drugs and rapid pharmacodynamic assessment of blood-brain barrier penetration using novel compounds.