2663 - Enhancing the Therapeutic Effect of Radiodynamic Therapy Using Carbamide Peroxide: Validation Against Immunohistochemical Analysis

Purpose/Objective(s): 5-aminolevulinic acid (5-ALA)-mediated radiodynamic therapy (RDT), which integrates principles of radiotherapy and photodynamic therapy, exploits the effective tumoricidal capabilities of x-ray irradiation and the activation of protoporphyrin IX (PpIX) metabolized from 5-ALA. During x-ray irradiation in-vitro, PpIX catalyzes carbamide peroxide (PRX) to generate singlet oxygen (¹O₂), thereby increasing the catalytic yield of reactive oxygen species (ROS). Moreover, the emission of Cherenkov light that activates PpIX increases with higher photon energy in ex-vivo tissues. Therefore, our study aimed to investigate tumor response and the impact of PRX as a coenzyme catalyst in 5-ALA RDT using 6–45 MV photon radiation in an in-vivo mouse model and validating with immunohistochemical (IHC) analysis.

Materials/Methods: A preclinical in-vivo murine model of small-cell lung cancer in immunocompetent C57BL/6 mice was examined. The tumors (n=320) were randomized into 16 groups (20 tumors/group), consisting of individual and possible combinations of control (untreated), 5-ALA, PRX, and radiation treatment (RT) with 6, 18, or 45 MV photons. A single fraction of 4 Gy was delivered to the tumors. 5-ALA was administered intravenously at 100 mg/kg 4 hours before irradiation for endogenous PpIX accumulation in the tumor. PRX was injected intratumorally at 60 mg/kg approximately 5 minutes before irradiation. Tumor volumes were monitored with a 1.5T MRI scanner over 14 days. Two-way repeated ANOVA with Bonferroni correction was used to compare treatment groups and determine statistically significant differences in tumor growth. Radiation-treated tumors were sampled from additional mice (2 tumors per treatment per tissue sampling day) on the day of treatment and 3 days post-treatment to validate each treatment with IHC analysis of Ki-67 (proliferation marker), Caspase-3 (apoptosis marker), ?H2AX (DNA double-strand breaks biomarker), and Cytochrome C (mitochondrial dysfunction marker).

Results: Exceptional properties of RDT (5-ALA+RT) were observed compared to the conventional RT. An additional decrease in tumor growth was observed in RDT as the photon energy used increased. Tumor growth has been reduced by about 50% using 45MV RDT compared to 45MV RT (P < 0.001). Moreover, when PRX was introduced into RDT, tumor growth delay was prolonged, with a significant effect observed after 7 days post-treatment. 45MV RDT with PRX showed an additional decrease in tumor growth of 14-15% compared with 45MV RDT without PRX. The IHC analysis showed that cytochrome-C expression increased in 45MV RDT overall by 41-42% on 3 days post-treatment, and ?H2AX expression increased only in the 45MV RDT with PRX by 22%.

Conclusion: The catalytic effect of photon irradiation, PpIX, and carbamide peroxide was observed in 5-ALA RDT using both 1.5T MRI monitoring and IHC analysis. The enhancement results suggest that the additional ROS produced by PRX prolongs the tumor growth delay in 5-ALA radiodynamic therapy.