No abstract

Background 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. Materials and methods 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. Results 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 50,000 cells. PDT was causing metabolic and molecular impairment of RMA cells, resulting in viability decrease, reduction of cell proliferation, and induction of apoptosis. Conclusion 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.

Background Cytoreductive surgery (CRS) in combination with hyperthermic intraperitoneal chemotherapy (HIPEC) is an option in advanced peritoneal sarcomatosis. Nevertheless, CRS and HIPEC are not successful in all patients. An enhancement of HIPEC using photodynamic therapy (PDT) might be beneficial. Therefore, a combination of the photosensitizer hypericin (HYP) with HIPEC was evaluated in an animal model. Procedure An established HIPEC animal model for rhabdomyosarcoma (NOD/LtSz‐scid IL2Rγnullmice, n = 80) was used. All groups received HYP (100 μg/200 μl) intraperitoneally with and without cisplatin‐based (30 or 60 mg/m2) HIPEC (37°C or 42°C, for 60 minutes) (five groups, each n = 16). Peritoneal cancer index (PCI) was documented visually and by HYP‐based photodynamic diagnosis (PDD). HYP‐based PDT of the tumor was performed. Tissue samples were evaluated regarding proliferation (Ki‐67) and apoptosis (TUNEL). Results HYP uptake was detected even in smallest tumor nodes (<1 mm) with improved tumor detection during PDD (PCI with PDD vs. PCI without PDD: 8.5 vs. 7, p < .001***). Apoptotic effects after PDT without HIPEC were limited to the tumor surface, whereas PDT after HIPEC (60 mg/m2, 42°C) showed additional reduction of tumor proliferation in the top nine to 11 cell layers (50 μm). Conclusion HYP as fluorescent photosensitizer offers an intraoperative diagnostic advantage detecting intraperitoneal tumor dissemination. The combination of HYP and cisplatin‐based HIPEC was feasible in vivo, showing enhanced effects on tumor proliferation and apoptosis induction across the tumor surface. Further studies combining HYP and HIPEC will follow to establish a clinical application.

Background: Cytoreductive surgery (CRS) in combination with hyperthermic intraperitoneal chemotherapy (HIPEC) is an option in advanced peritoneal sarcomatosis. Nevertheless, CRS and HIPEC are not successful in all patients. An enhancement of HIPEC using photodynamic therapy might be beneficial. Therefore, a combination of the photosensitizer Hypericin (HYP) with HIPEC was evaluated in an animal model. Procedure: An established HIPEC animal model for rhabdomyosarcoma (NOD/LtSz-scid IL2Rγnullmice, n=80) was used. All groups received HYP (100 µg/200 µl) intraperitoneally with and without cisplatin-based (30 or 60 mg/m2) HIPEC (37 or 42 °C, for 60 min) (five groups, each n=16). Tumor dissemination was documented visually and by using HYP-based fluorescence guidance. HYP-based photodynamic therapy (PDT) of the tumor was performed. Finally, tissue samples were evaluated regarding proliferation (Ki-67) and apoptosis (TUNEL). Results: HYP uptake even in smallest tumor nodes (< 1 mm) was found. HYP-based fluorescence guidance allowed a better tumor detection in comparison to visual inspection. Immunohistochemistry revealed HYP penetration across the tumor surface. HYP-based PDT without HIPEC induced marginal apoptotic effects at the tumor surface. Combining HYP with HIPEC revealed cisplatin concentration dependent decrease in proliferation capacity and induction of apoptosis across determined cell layers of the tumor surfaces. Conclusion: HYP as fluorescent photosensitizer offers an intraoperative diagnostic advantage detecting intraperitoneal tumor dissemination. The combination of HYP and cisplatin-based HIPEC was feasible in vivo showing enhanced effects on tumor proliferation and apoptosis induction across the tumor surface. Further studies combining HYP and HIPEC will follow to establish a clinical application.