2471 - Preclinical Evaluation of Tumor Treating Fields (TTFields) Applied with Radiation Therapy in Glioblastoma: Impact of Treatment Sequencing

Purpose/Objective(s): Tumor Treating Fields (TTFields) therapy delivers low-intensity, alternating electric fields that disrupt processes essential for mitosis and is approved for newly diagnosed and recurrent glioblastoma (GBM). In addition to their antimitotic activity, TTFields have been shown to impair DNA damage repair pathways and to enhance the activity of DNA-damaging agents. We investigated preclinically whether brief TTFields exposure enhances the response of GBM to radiation therapy (RT) and whether treatment sequencing influences this interaction.

Materials/Methods: U87-MG and LN229 GBM cells were irradiated with 2 Gy, and concurrent TTFields (200 kHz, 24 h) with different treatment schedules: before RT, after RT, or both before and after RT. Clonogenic survival and ?H2AX staining were assessed. RNA and protein from control and TTFields-treated cells were analyzed by PCR and western blot for DNA damage response markers. In vivo, C57BL/6 mice bearing intracranial GL261-mCherry tumors were randomized into 6 groups: control, TTFields, RT (6 Gy), TTFields before RT, TTFields after RT, or TTFields both before and after RT. The study included two predefined endpoints: an early mechanistic endpoint, in which tumors were excised, dissociated, and ?H2AX and Ki-67 expression were analyzed by flow cytometry; and a late efficacy endpoint, in which tumor volumes were quantified by MRI-based segmentation, followed by tumor excision and weighing.

Results: In vitro, brief TTFields exposure decreased expression of DNA repair genes/proteins. While TTFields alone did not impair clonogenicity, their administration prior to RT reduced colony formation and increased ?H2AX levels relative to RT alone. In vivo, applying TTFields both before and after RT decreased tumor volume and weight compared with controls and with TTFields post-RT. TTFields exposure only before or both before and after RT increased DNA damage in tumors (without increasing DNA damage in infiltrating immune cells and normal brain tissue), whereas TTFields only after RT had minimal impact. Tumor expression of Ki-67 was significantly elevated by RT application, a phenomenon alleviated by concurrent TTFields treatment, irrespective of the treatment sequencing.

Conclusion: Brief TTFields exposure augments radiation response in GBM models, and therapeutic effect is dependent on treatment timing and sequencing. This effect may be associated with a mechanism involving downregulation of DNA damage response proteins and anti-proliferative effect of TTFields in this model.