181 | Exploring boron neutron capture therapy effects on healthy skin: from cells to tissues

Stefania Ricci^1|2|3, Silva Bortolussi^3|4, Ricardo Luis Ramos^3|4, Ian Postuma³, Daniele Dondi^3|5, Cinzia Ferrari^3|7, Laura Cansolino^3|7, Tea Maverna², Federica Riva^2|3 | ¹Dept of Internal Medicine and Medical Therapy, University of Pavia, Italy; ²Dept of Experimental Medicine, Histology and Embryology Unit, University of Pavia, Italy; ³National Institute of Nuclear Physics INFN, Unit of Pavia, Italy; ⁴Dept of Physics, University of Pavia, Italy; ⁵Dept of Chemistry, University of Pavia, Italy; ⁶Dept of Molecular Medicine, Human Physiology Unit, University of Pavia, Italy; ⁷Laboratory of Experimental Surgery, Department of Clinical-Surgical, Diagnostic and Paediatric Sciences, University of Pavia, Italy.

Boron Neutron Capture Therapy (BNCT) is an innovative binary radiotherapy for the treatment of tumors non responsive to conventional radiotherapy. BNCT is based on the administration of a 10B-labeled compound (the most used is boronophenylalanine, BPA) followed by thermal neutron irradiation: 10B undergoes a nuclear reaction that releases energy over a very short path length, causing DNA damage leading to apoptotic events. As the neutron beam must cross the skin to reach deep tumor cells, healthy epidermis represents a critical dose-limiting tissue. This study aims to characterize the radiobiological effects of BNCT and photon radiations on healthy skin using 2D and 3D static and dynamic models: 2D model: HaCaT keratinocytes photon-irradiated (at 0.5 Gy, 1 Gy and 2 Gy) in proliferating conditions and after induction of suprabasal differentiation; 3D models: static culture: reconstructed human epidermis, SkinEthic™, exposed to photons (6, 12, 24, 36 and 108 Gy) and neutrons (35.7, 32.5 and 160 Gy with BPA; 4.5, 11.4 and 22.7 Gy without BPA); dynamic culture: EpiDerm™ tissues maintained in microfluidic bioreactors to better recreate physiological conditions. Radiobiological effects were assessed through clonogenic assays, morphological analyses (H&E), immunohistochemistry (BrdU, PCNA, Pan-CK, filaggrin) and immunocytochemical analyses (CLDN-1, Zo1, foci). In HaCaT cells, photon irradiation induced a dose-dependent decrease in clonogenic survival and alterations in the expression and localization of several markers: CLDN-1 showed progressive mislocalization at higher doses; Pan-CK intensity increased in both proliferating and differentiated irradiated cells; ZO1 intensity decreased proportionally with dose. DNA damage analysis confirmed a proportional increase in γH2AX foci and 53BP1 complex, reflecting dose-dependent induction of DNA damage and activation of repair mechanisms. In the 3D SkinEthic™ model photon-irradiated, morphological and immunohistochemical analyses revealed a dose-dependent reduction in proliferative activity (BrdU) and fluctuating PCNA levels, with a peak on 2nd day post-irradiation. CK10 expression decreased and became progressively less homogeneous with increasing dose, indicating early alterations in epidermal differentiation. On the 2^nd day after BNCT treatment, a reduction in proliferative activity was observed together with an increased presence of proteins involved in repair mechanisms, in line with previous in-vitro findings. In addition, filaggrin and pan-cytokeratin level decreased after radiation. The EpiDerm™ model allowed the irradiation of a larger number of samples maintained in microfluidic bioreactors. Tissue organization remained stable up to day 7 in culture, for better reproducing physiological conditions. This approach is currently being refined. These results support the use of both 2D and 3D epidermal models to characterize radiobiological effects at different levels of tissue organization. Ongoing experiments include additional clonogenic assays and more immunohistochemical analyses to improve the dose-effect relationships in healthy skin and optimize BNCT protocols.