Stefania Ricci1, Ian Postuma2, Claretta Guidi3, Patrizia Sommi2,4, Agostina Vitali2,4, Cinzia Ferrari2,5, Laura Cansolino2,5, Silva Bortolussi2,3, Federica Riva1
1Department of Experimental Medicine, Histology and Embryology Unit, University of Pavia, Pavia, Italy;
2National Institute of Nuclear Physics (INFN), Unit, Pavia, Italy;
3Department of Physics, University of Pavia, Pavia, Italy;
4Department of Molecular Medicine, Human Physiology Unit, University of Pavia, Pavia, Italy;
5Laboratory of Experimental Surgery, Department of Clinical-Surgical, Diagnostic and Paediatric Sciences, University of Pavia, Pavia, Italy
Correspondence to: Stefania Ricci, PhD student. Department of Experimental Medicine, Histology and Embryology Unit, University of Pavia, via Forlanini 10, 27100 Pavia, Italy. Email: stefania.ricci01@universitadipavia.it; stefania.ricci@hotmail.com.
Background: Boron neutron capture therapy (BNCT) is an innovative radiotherapy to treat solid tumors that hardly respond to traditional therapies. The administration of a compound labelled with boron-10 (10B; non-radioactive) is a pre-treatment, followed by irradiation with low-energy neutrons. The neutron beam penetrates the patient across the skin, making it a limiting tissue; it’s thus important to study the dose-effect relation in skin. To this aim, the skin substitute SkinEthicTM was used: an in-vitro reconstructed human epidermal model from normal human keratinocytes seeded on an inert polycarbonate filter. Several studies confirm the reliability of the response of the constructs to what observed on the native tissue. This study aims to understand how healthy skin responds to BNCT is crucial for optimizing safety and efficacy of the treatment. It focuses on assessing the impact of neutron radiation on the reconstructed human epidermal model in presence and absence of boron.
Methods: Samples were incubated with boronophenylalanine (BPA; 10B delivery agent). Thereafter, they were irradiated at three different irradiation powers. Models were subsequently fixed at different observation times, embedded in paraffine, and sliced. The percentage of proliferating cells [bromo-deoxy-uridine (BrdU) incorporation assay] and the percentage of positive cells to the expression of the proliferation-cell nuclear antigen (PCNA) were evaluated. Same analyses were performed both on constructs irradiated with the neutron beam without BPA, and with the photon beam, to investigate the effectiveness of BNCT.
Results: Morphological and immunohistochemical analyses showed a progressive degradation of the constructs over time and following different radiation doses. The anti-BrdU immunolabeling showed that the cell-proliferation rate was variable. However, on the second observation day, the samples treated with BNCT showed lower positivity. Variable positivity trends were also observed in the anti-PCNA assays. The results show peaks of positivity on the second day in culture.
Conclusions: On the second day after BNCT treatment, the reduced proliferative activity is associated with a high presence of proteins involved in reparative mechanisms, according to what has already been observed in previous in-vitro studies on cell cultures. These preliminary experiments aim to optimize the study protocol, improve the constructs characterization and effects in response to radiation. The high variability makes it difficult to standardize the results, so it would be desirable to increase the number of samples to obtain statistically significant results: observations at shorter times and lower radiation doses would complete this dose-effect study.
Keywords: Human epidermal model; healthy human epidermis; radiobiological effects; mixed-field radiation; dose-effect relation
