Purpose: RECA (Radiotherapy Enhanced by Cherenkov photo-Activation) is a novel treatment with potential to add an anti-cancer immunogenic component through Cherenkov activation of a photo-chemotherapeutic (psoralen). Here we investigate RECA in a novel tissue representative in-vitro model consisting of 4T1 murine cancer cells grown on thin slices of viable rat-brain tissue.
Methods: Six, 12-well-plates, each containing 1cm of agarose supporting a 400 µm thick coronal slice of viable rat brain tissue were created. Each plate corresponded to an arm of an experiment incorporating the psoralen derivative 4’-aminomethyl trioxsalen (AMT): MV control, MV+AMT, no-irradiation control, and AMT-alone control. 20,000 4T1 cells expressing both mCherry-flourescent and firefly luciferase-luminescent reporter proteins were plated on each rat brain slice hemisphere. The MV arms received ~4 Gy from a 15 MV Varian linear accelerator beam. Images were taken of the plates each day for 5 days, including filtered for mCherry signal. An optimized CellProfiler pipeline quantified cell colony survival and growth. A luciferase assay was done on day 5 as another measure of relative viability.
Results: The brain slice model was assessed daily, and found viable for supporting 4T1 cell colony growth for up to 5 days. The optimized CellProfiler colony counting pipeline was validated in simulations, and found to have excellent performance, also enabling uncertainty estimation. Integrated intensity analysis of mCherry signal revealed a significant decrease in tumor proliferation by day 5 between the MV control (5.65±0.78-fold growth) and MV+AMT (3.49±-0.52-fold growth) arms. This result is consistent with psoralen activation in the MV+AMT arm. The growth observed in the Dark control arm was consistent with the 13.6±1.5 hour doubling time for 4T1 cells.
Conclusion: The early results from ongoing experiments are consistent with psoralen activation and cell kill in RECA treated cells (MV+AMT arm). Further work will be presented to fully quantify the effect.