Effect of Neutron Radiation on ¹⁰BPA-Loaded Melanoma Spheroids and Melanocytes

Melanoma is an aggressive disease that arises from mutations in the cells that produce the pigment melanin, melanocytes. Melanoma is characterized by a high mortality rate, due to avoidance of applied therapies and metastasis to other organs. The peculiar features of boron neutron capture therapy (BNCT), particularly its cell-level selectivity, make BNCT a promising modality for melanoma treatment. However, appropriate cellular models should be used to study new therapies or improve the efficacy of existing therapies. Spheroids, which have been used for years for in vitro studies of the efficacy of anti-cancer therapies, have many characteristics shared with tumors through which they can increase the accuracy of the cellular response compared to 2D culture in vitro studies and reduce the use of animals for research in the future. To the best of our knowledge, when we started researching the use of spheroids in BNCT in vitro, there was no publication showing such use. Our study aimed to evaluate the efficacy of a 3D cellular model (spheroids) for testing BNCT on melanoma cells. We assessed boronophenylalanine (¹⁰BPA) uptake using inductively coupled plasma mass spectrometry in both spheroids and 2D cultures of melanoma and melanocytes. DNA damage, Ki67 protein expression, and spheroid growth were analyzed. The experimental groups included: (1) IR_B (neutron flux + 50 µg ¹⁰B/mL), (2) IR (neutron flux alone), (3) C_B (no irradiation, 50 µg ¹⁰B/mL), and (4) C (no irradiation and no treatment with boron). The total absorbed doses were estimated to be 2.1-3.1 Gy for IR_B cells and spheroids as well as 8.3-9.4 Gy for IR_B spheroids, while estimated doses for IR cells were 0.5-1.9 Gy. The results indicated that IR_B spheroids might exhibit a reduced diameter. Melanoma cells in the 3D model showed that their DNA damage levels may be higher than those in the 2D model. Moreover, the Ki67 assay revealed differences in the expression of this marker between irradiated melanoma cell lines. In conclusion, preincubation with ¹⁰BPA enhances BNCT efficacy, leading to cell growth inhibition and increased DNA fragmentation. Differences in DNA damage between 2D and 3D models may be due to dissimilarities in cell metabolism caused by a changed cell architecture.