Heat Shock Protein 90 Targeted Radiation Therapy Enhanced with Cherenkov Light photo-Activation

Abstract

Introduction: RECA (Radiation Therapy Enhanced with Cherenkov light photo-Activation) is a treatment method that utilizes the Cherenkov light generated in the body during external beam radiation therapy (EBRT) to activate photodynamic molecules, thereby increasing the efficacy of external beam therapy. Previous work into RECA has utilized non-targeted photodynamic molecules and has relied on the local generation of Cherenkov production in the body to reduce normal tissue effects. In this study, we explore the use of a new photodynamic molecule, HS583, that targets extracellular Heat Shock Protein 90 (eHSP90), as the first exploration of the targeted RECA approach.Methods: HS583, a novel photodynamic therapy molecule consisting of an HSP90 inhibitor bound to a verteporfin (VP) molecule, was synthesized in house. MCF10A and MDA-MB-231 cell lines were cultured and treated with either VP alone or HS583. Uptake for both drugs in both cell lines was analyzed qualitatively by confocal microscopy and quantitatively by High Performance Liquid Chromatography (HPLC). Photodynamic cytotoxicity from visible light was measured for both drugs by WST-1 assay using a ML660 Modulight laser. Inhibitory Concentrations (IC50s) were determined for different laser doses. Silver stains and western blots were performed to determine whether unbound VP and HSP90-bound HS583 produce different effects on the cellular protein composition when activated by laser light. Simulations of laser light photodynamic therapy and Linac-generated RECA were conducted in TOPAS Monte Carlo to assess whether VP absorbs equivalent amounts of energy from Cherenkov light during EBRT as it does from standard laser light setups. Radiation damage and the RECA effect were explored experimentally using a clinical Linac and measured via WST-1 cell proliferation assay.Results: Equivalent cellular uptake was observed for both HS583 and VP after a 90-minute incubation. However, six hours post-incubation, Verteporfin levels decreased 49-fold, while HS583 levels decreased only 7-fold, demonstrating a significantly greater retention of HS583 within cells. IC50 values ranged from 0.0045±0.0008 µM at 30 J/cm^2 to 2.329±0.6645 µM at a dose of 0.01 J/cm^2. Proteomic analysis showed that in MDA-MB-231 cells treated with HS583 and activated by light, the HSP90 signal was reduced 70% in Western blot, indicating degradation of HSP90 by activated HS583. In contrast, HSP90 remained intact in cells treated with HS583 but not activated by light and in all MCF10A cells treated with HS583. Laser Simulations demonstrated that for a 1 J/cm^2 688 nm incident beam, 6.1E12±0.23E12 eV is absorbed by 1 µM HS583 solution. Linac simulations showed that for a 1 Gy irradiation of a 6 cm water phantom, 7.678E12±1.081E12 eV was absorbed by a 1 µM HS583 solution. Preliminary Linac experimental results indicate that cells incubated with 10 µM HS583 exhibited extensive cytotoxicity, with near-complete cell death observed at 2 Gy.Conclusion: HS583 represents the continued advancement of RECA. HS583 shows specific targeting to cancer cells and is retained within a cell for significantly longer than standard photodynamic therapy drugs. Simulations and experimental studies demonstrate the feasibility of increasing cell cytotoxicity in external beam radiation therapy. RECA therefore has the potential to increase the therapeutic ratio and allow for better treatments and clinical outcomes. The even higher intensity of Cherenkov Light generated in FLASH beams may increase this effect even further.