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Introduction: Melanoma, an aggressive and highly immunogenic cancer, arises from uncontrolled melanocyte growth. FLASH radiotherapy, a breakthrough technique, delivers ultra-high radiation doses, offering the potential for improved cancer treatment while minimizing harm to healthy tissue. Material and Methods: To study the short-term response of spheroids to FLASH radiotherapy, 3D cultures of melanocytes and melanoma were used. Spheroids were irradiated using the FLASH method with the total doses of 3, 20, and 40 Gy, and conventionally with a dose of 3 Gy. After 8 days from irradiation, the measurements were taken using an imaging cytometer, FTIR and colorimetric microscopy (C-Microscopy). Results: Studies conducted on melanocytes showed that doses of 20 and 40 Gy are toxic to them and cause cell necrosis. In contrast, for melanoma, these two doses resulted in tumor growth inhibition. IR measurements revealed spectral changes in lipids, proteins, and DNA/RNA, indicating similarities between the effects of the FLASH method and conventional radiotherapy for both spheroid models (i.e., cancerous and normal). The spheroid quantitative color analysis allowed for the differentiation between different irradiated and control groups. Conclusion: Both colorimetric and infrared microscopy can be used to analyse the response of tumors to radiation.
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