Browsing by Subject "Psoralen"
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Item Open Access A New Method to Investigate RECA Therapeutic Effect(2020) Liu, XiangyuIntroduction: RECA (Radiotherapy Enhanced with Cherenkov photo- Activation) is a novel treatment that induces a synergistic therapeutic effect by combining conventional radiation therapy with phototherapy using the anti-cancer and potentially immunogenic drug, psoralen. This work presents a novel method to investigate the therapeutic effect of RECA using rat brain slices and the agarose- based tissue equivalent material. Methods: 4T1 mCherry Firefly Luciferase mouse breast cancer cells are placed on the brain slice after exposed to psoralen solution. Taking fluorescent imaging of the brain slices every day after irradiation, an independent luciferase imaging was taken after the fifth fluorescence imaging. Using different imaging processing and analysis method to identify the cells. Result: Four analyzing method give different result about the fluorescence signal or luminescence signal. The overall trend of the fluorescence signal is rising over day, reaches the lowest point at 48 hours after irradiation. Control group (no radiation and no Cherenkov lights) has the lowest signal compared with other groups. The signal of brain slices with 4T1 cells exposed to psoralen solution is lower than that of brain slices without psoralen exposition. Conclusion: This work shows that rat brain slice can be used to simulate in vivo environment in exploring the therapeutic effect of RECA. Future work should focus on improving the image analyze method to better identify cells and noises.
Item Open Access An Exploration of the Feasibility of Combining Radiation Therapy with Psoralen Phototherapy(2018) Yoon, Suk WhanRadiation therapy (RT) has been a standard-of-care treatment for many localized cancers for decades. Despite being an effective treatment modality for many clinical presentations, the efficacy of RT against cancer can be limited due to local recurrence, metastatic spread, and radiation resistance from tumor hypoxia. These limitations provide opportunity for innovative approaches to enhance the overall efficacy of RT. This thesis explores the potential novel approach to enhancing RT through the paradigm changing approach of adding a phototherapeutic component initiated simultaneously with RT. X-ray Psoralen Activated Cancer Therapy (X-PACT) is one such approach, where diagnostics-energy kilovoltage (kV) x-ray coupled with energy modulators (phosphors) converts kV photon to ultraviolet (UV) light, which in turn activates psoralen. Radiotherapy Enhanced with Cherenkov photo-Activation (RECA) is another approach, where therapeutic megavoltage (MV) x-ray generates UV light via Cherenkov phenomenon. Both approaches could increase local control in RT, increase treatment effectiveness in hypoxic tumors, and amplify anti-cancer systemic response. The overarching hypothesis that drives this dissertation is that X-PACT and RECA can activate psoralen to enhance cytotoxicity in-vitro and tumor growth control in-vivo compared to RT alone. In line with this hypothesis, this work explores the feasibility of both X-PACT and RECA via in-vitro and in-vivo verification as well as optimization of radiation techniques to maximize the therapeutic benefit of the approach.
X-PACT and RECA in-vitro / in-vivo studies indicate radiotherapy enhancement is plausible with psoralens activated by secondary UV light production from radiation, though further investigation is required to establish feasibility of RECA in-vivo. For X-PACT in-vitro, a substantial reduction in cell viability and increase in apoptosis was observed in various murine cancer cells (4T1, KP-B, and CT2A) when treated with a combination of 50µg/mL phosphor, 10µM psoralen (8-MOP), and 1Gy of 80kVp x-ray (viability < 20%), compared to any of these components alone (viability > 70%). This suggests a synergistic interaction between the components congruent with the X-PACT scheme, where x-ray induces phosphor UV emission, which in turn activates psoralen. The X-PACT in-vivo mice study showed improved survival with X-PACT versus saline control with flank 4T1 tumors (30.7 days for X-PACT vs. 21.6 days for saline) for survival criteria of 1000, 1500, and 2000mm3, respectively. For RECA, in-vitro results seem promising, where reductions in viability of 20% and 9.5% were observed for 4T1 and B16 murine cancer cell lines treated with RECA (radiation + trioxsalen, a potent psoralen derivative) versus radiation alone. A substantial increase in MHC I expression was observed for B16 cells treated with RECA versus those treated with radiation alone. A small RECA in-vivo pilot study using 8-MOP was inconclusive. Further in-vivo trials with a greater number mice per arm of are required to establish the RECA feasibility to enhance radiotherapy.
Feasibility of treatment optimization for both X-PACT and RECA were demonstrated with kV and MV beams respectively, by optimization of optical output per radiation dose delivered. It was found that in both X-PACT and RECA scheme, the energy of the photon radiation beam (i.e. tube voltage and LINAC energy settings) affected optical output the most. With kV beams for X-PACT, accurate beam delivery within the target volume to reduce normal tissue damage typically expected of kV beams was demonstrated with a 3D-printing-based preclinical irradiation scheme, which is expected to help X-PACT translation into the clinics. In addition, for X-PACT, novel MV-responding phosphors were characterized under MV radiation beam, suggesting the possibility of MV-radiation-mediated X-PACT. Immediate future studies should investigate the efficacy of the optimized X-PACT and RECA, as well as MV X-PACT in-vitro and in-vivo. Studies beyond these immediate ones should investigate X-PACT and RECA efficacy against hypoxic and metastatic tumor sites, where radiation can traditionally fail.
Item Open Access An investigation of photo-activation of psoralen (AMT) during radiation therapy in a novel tissue model.(2021) Holden, Russell PatrickPurpose: 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 agent (psoralen). This work investigates 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: Accurate estimation of viable tumor burden is of foundational importance to this work. A CellProfiler pipeline was created and optimized and validated on realistic simulated data/images where the ground truth of number of colonies and integrated intensity was known. Simulated data sets mimicked key features of real experimental data including colony spatial and size distributions, contaminant and stray light signals, colony overlap, and noise. The optimized CellProfiler pipeline was then applied to the original 4T1 tumor cell images to determine colony growth over five days. Several experiments were conducted prior to the RECA experiment to determine the best protocol. The first tested the optimal concentration of psoralen, loading technique, and type of psoralen by co-incubating 4T1 cells with psoralen for differing times and concentrations and subsequently exposing them to 365nm radiation at variable energy fluences. The plates were tested for 4T1 cell viability using Celltiter-glo and luciferase assay 48-72 hours later depending on confluence of the control plate. Another experiment tested the output of the CellProfiler image analysis for relative growth over time measuring 4T1 mCherry cells plated on rat brain slices at 10k,20k,30k,40k,50k cells per hemisphere. For the RECA experiment, 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 represented one arm of an experiment incorporating the psoralen derivative 4’-aminomethyl trioxsalen (AMT): MV control, MV+AMT, kV control, kV+AMT, no irradiation control, and AMT alone control. 20,000 4T1 cells expressing both mCherry-flourescent and firefly luciferase-luminescent reporter proteins plated on each rat brain slice hemisphere. For the AMT arms, the cells were co-incubated with 1 µM AMT for 1 hour prior to plating. The MV arms received 4 Gy from a 15 MV linear accelerator beam, and the kV arms received 4 Gy from 160 keV photons. Images were taken of the plates each day for 5 days with a Zeiss Lumar microscope with rhodamine filter for the mCherry protein signal. Results: The CellProfiler pipeline measured integrated intensity of the 10 simulated images that best approximated the images from the experiment with an accuracy of 99.23% ± 0.75%. Further analysis on images with increasing colonies, background, and noise showed the pipeline was accurate on images with variable features. These results gave confidence that the same pipeline could be used on images from this experiment. AMT was found to be a more effective psoralen (0.43 ± 0.22% cell survival after 48hr) relative to 8-MOP (31.3% ± 3.7% cell survival after 72hr). The psoralen cell loading was found to be optimal at 1µM for 1 hr prior to plating. The analysis of the cell titration images showed a significant increase in signal for each increase in cells plated on day one and for all subsequent days except for the 20k cell arm. Additionally, the growth in signal for the plates was consistent between the arms except for the 20k arm due to extra signal on the periphery of the slice likely from displaced cells. Integrated intensity analysis of the 4T1 mCherry cells 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 the hypothesis that psoralen is being activated, causing the decreased proliferation seen in MV AMT arm. The kV control and kV AMT arms had a smaller decrease in proliferation when compared to their MV counterparts (6.73±1.24 and 5.26±0.59-fold growth respectively). The growth observed in the Dark control arm was consistent with the 13.6 ± 1.5 hour doubling time for 4T1 cells. In the MV AMT arm, there were punctuated regions of increased signal in 7/12 wells not corresponding to colonies, making segmentation for this arm challenging. The viability of the brain slice was assessed each day and found to be stable over the 5 days. Conclusions: The technique of testing image analytic software on simulated images proved to be an effective tool to verify the software’s accuracy. A similar technique can be applied to images with new and challenging features. The rat brain slice model gives the opportunity to both generate Cherenkov in real tissue while providing a 3D matrix for the colonies to grow, which is an improvement to the 2D well plate culture for this experiment. This new model adds challenges of proper image analysis with cell autofluorescence as well as cell clumping. The preliminary results are consistent with psoralen activated in RECA treated cells causing decreased proliferation for the MV arm. Further work is needed to confirm and quantify the effect.
Item Open Access Evaluation of radiation therapy produced Cherenkov light emissions used for photo-activation of psoralen (AMT)(2022) Koch, Brendan DanielPurpose: 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.
Item Open Access Evaluation of UVA Emission from MV-Irradiated Tissues and Phantoms(2019) Jain, SagarikaIntroduction: RECA (Radiotherapy Enhanced with Cherenkov photo-Activation) is a novel treatment that induces a synergistic therapeutic effect by combining conventional radiation therapy with phototherapy using the anti-cancer and potentially immunogenic drug, psoralen. Psoralen is photo-activated in-situ by UVA (UltravioletA, 320-400nm) Cherenkov Light (CL), produced in tissue directly by the treatment beam. In this study, we develop methods to image and quantify relative CL production (primarily in the UVA range) from a range of tissue and phantom materials upon photon irradiation. These methods are further applied to identify a tissue-equivalent optical phantom, mimicking CL production in the UVA range, in order to facilitate further RECA experiments.
Methods: The imaging system included a deep-cooled, high-sensitivity CCD camera, equipped with either a visible range lens (sensitive to 400-700nm photons) or a UVA-compatible lens assembly and a band-pass filter (sensitive to 320-400nm photons). CL emission was quantified in bulk tissue samples, solid waters (SW brown and white), and agarose gels in a series of experiments. The samples and imaging equipment were placed in a dark, light-blocking chamber to avoid contamination from other light sources. In addition, the camera was carefully positioned with respect to the LINAC head and was also shielded using lead bricks to minimize radiation noise.The samples were then irradiated with clinical photon beams, while simultaneously being imaged by the camera.
Results: In the visible range, solid water had similar CL emission to that from bulk tissue samples (34% less than the maximum and 44% higher than the minimum UVA emitting tissue). A 25% reduction in radiation noise in the UVA spectrum was achieved using lead block shielding of the camera. In the UVA range at 15MV, white SW emitted 66±5%, 64±5% and 76±3% less UVA than chicken, pork loin and pork belly respectively. Similar under-response was observed at 6MV. Brown SW had 21±8% less UVA emission than white SW at 15MV, and no significant emission at 6MV. Agarose samples (1% by weight) doped with 250ppm India Ink exhibited equivalent UVA CL emission to chicken breast (within 8%).
Conclusion: The results confirm that for the same absorbed dose, SW emits lessUVA light than the tissue samples, indicating that prior in-vitro studies utilizing SW as the CL-generating source may have underestimated the RECA therapeutic effect. Agarose gel doped with 250ppm India Ink is a convenient tissue equivalent phantom for further work.