Evaluation of radiation therapy produced Cherenkov light emissions used for photo-activation of psoralen (AMT)
Purpose: Radiotherapy Enhanced by Cherenkov photo-Activation (RECA) is a novel radiation treatment method that seeks an anti-cancer effect with the introduction of a psoralen compound administered for treatment. The goal of the RECA method is to enhance standard radiation therapy treatments with the addition of psoralen being photo-activated by Cherenkov radiation that is generated during radiotherapy. The purpose of this work is to investigate the effectiveness of RECA on 4T1 mCherry FLuc breast cancer cells seeded on a psoralen-baked-agarose-based rat brain slice.Methods: A previously established CellProfiler pipeline, developed in our lab by Holden et al., was used to assess tumor burden on rat brain slices used for a tissue-equivalent medium for cell culturing. The CellProfiler pipeline was implemented on images of 4T1 breast cancer cells growing over the course of four to five days post-treatment to measure the average intensity of fluorescing cells. Prior to the RECA experiment, multiple preparatory experiments were conducted to refine and optimize experimental techniques. The first preparatory experiment tested the possibility of a plate reader bias effect, i.e., signal from nearby wells contributing to signal of other wells, seen during measurements of cell luminescence within individual wells of a clear-bottom 96-well plate. A CellTiter-Glo endpoint readout was taken 48-hours post-treatment for an endpoint measure to assess the if there was any added signal from nearby wells in the clear-bottom plates. The next experiment tested whether fractionation of dose was feasible and preferrable to single dose treatment by irradiating 4T1 mCherry Fluc cells with 2 Gy and 4 Gy of kV radiation with and without fractionation. An endpoint CellTiter-Glo readout was conducted 72 hours post-treatment to assess cell viability between the treatment plans. Additional preparatory experiments investigated whether psoralen-doped agarose was an effective method for cell loading. A 30 µM AMT-baked agar base was placed in half of the wells in plates with 4T1 mCherry Fluc cells seeded on brain slices on top of the agar. One plate received no treatment and one plate received treatment of 365 nm UVA, and an endpoint Firefly Luciferase reporter assay was conducted 48 hours post-treatment to assess cell viability between the conditions. For the RECA experiment, five 12-well plates, each containing 1 cm of agar with a 400 µm thick coronal slice of rat brain tissue, were given one of five conditions of treatment: no treatment, 4.95 Gy of fractionated kV or MV treatment, or 4.95 Gy of whole kV of MV treatment. Each plate condition consisted of six wells containing AMT-baked agar and six wells containing a standard agar base. After irradiation, images were taken of each of the plates for each day over the course of five days five days with a Zeiss Lumar microscope. The microscope was equipped with a rhodamine filter to analyze the luminescence readings from each well for assessment of cell viability. Results: The preparatory experiments all yielded results that allowed for development of the RECA experiment procedure. Investigation of the plate reader effect showed that background signal from nearby wells was not leaking into well signal readout, with all wells having nearly consistent signal throughout all the wells. Fractionating the dose was found to be preferable because it decreased cell viability less than delivering all dose at once, which floored cell viability. Testing psoralen-doped agar demonstrated that this is an effective delivery method for psoralen to intercalate with cells. The RECA experiment utilizing kV and MV whole dose conditions allowed comparison between irradiations with and without a fractionation scheme. The Firefly Luciferase reporter assay signal for the MV treatment conditions showed less cell viability than the Dark control conditions for both AMT and DMSO. Additionally, the whole dose MV conditions demonstrated a more pronounced decrease in cell viability than the fractionated MV conditions, as expected. The CellProfiler analysis demonstrated the same trends with the whole dose MV AMT condition (8.54 ± 0.99-fold increase) and whole dose MV DMSO condition (11.80 ± 0.70-fold increase) demonstrating less cell viability than the Dark AMT (13.41 ± 0.83-fold increase) and Dark DMSO (14.11 ± 0.62-fold increase). Interestingly, there was not a significant difference in cell viability seen between the fractionated and whole dose conditions. Conclusions: The procedural techniques developed for the analysis of the RECA effect during the preparatory experiments ruled out a plate reader effect and demonstrated that introducing fractionation and psoralen-baked agar is effective. The testing of the fractionation scheme used for kV irradiations proved to be sufficient for decreasing cell viability without killing all the cells. Additionally, the testing of the psoralen-baked agarose slabs proved to be an adequate psoralen delivery method when compared to methods that used cells suspended in psoralen treated media in prior studies. When these changes to the procedure were introduced together during MV irradiations, the RECA effect did not clearly replicate the results demonstrated during kV irradiations in the preparatory experiments. Further investigation is required to confirm and validate the RECA effect generated during radiotherapy.
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