A new valid rhabdomyosarcoma spheroid culture model for in vitro evaluation of hypericin-based photodynamic therapy.

Abstract

Advanced stages of pediatric alveolar rhabdomyosarcoma (RMA) are associated with an unfavorable outcome at established therapeutic strategies, accentuating the need for novel treatment options. Photodynamic therapy (PDT) with hypericin (HYP) has shown strong cytotoxic effects in two-dimensional (2D) cell culture. In order to more accurately mimic in vivo tissue architecture and better predict pharmaceutical response, the aim of this study was to establish a spheroid culture model by which PDT efficacy could be assessed in a three-dimensional (3D) context. 3D multicellular tumor spheroids were generated using various scaffold-based and scaffold-free techniques. On two reproducible methods, HYP-PDT was performed varying spheroid sizes, photosensitizer concentrations, and illumination times. The ability for HYP uptake within the spheroid was analyzed assessing the substrate's autofluorescence. Antitumorigenic treatment effects were evaluated investigating cell viability, spheroid morphology, proliferative activity, and induction of apoptosis. Magnetic spheroid printing and orbital shaking methods were established as reproducible culturing systems producing uniform spheroids. Within assessed incubation times, HYP showed good penetration depth in spheroids containing 50,000cells. PDT was causing metabolic and molecular impairment of RMA cells, resulting in viability decrease, reduction of cell proliferation, and induction of apoptosis. Assessing HYP-based PDT in a 3D culture model, we were able to gain an insight on how parameters like photosensitizer, oxygen, and light distribution contribute to the phototoxic effect. Compared to 2D cell culture, a higher treatment resistance was detected, which can be related to spheroid structure and mechanisms of intercellular communication, signal transduction, and gene expression.