Characterization and in Vitro Radiation Response Analysis of Patient‐Derived Heterogeneous Cells From Canine Oral Melanoma

ABSTRACT Conventional cancer cell lines often fail to capture tumour heterogeneity, limiting their translational relevance for radiation response studies. This study aimed to establish a low‐passage, patient‐derived heterogeneous culture and to evaluate its radiation response characteristics. Tumour tissue obtained from a canine oral melanoma patient was used to generate the culture. Cellular heterogeneity was confirmed by morphological features and the expression of stem cell‐associated markers (CD133 + CD44 + : 50.53% ± 5.05%, CD133 − CD44 + : 30.93% ± 7.21%). Tumorigenicity was validated using a xenograft model. Radiation responses to 2 and 10 Gy were assessed using clonogenic survival, viability, proliferation, apoptosis, DNA damage, migration, and invasion assays. The culture demonstrated morphological heterogeneity, expression of stem cell‐associated markers, and tumorigenic potential in vivo. Compared with the 0 Gy control, 10 Gy irradiation significantly reduced cell viability, proliferative activity, and migration, while increasing early apoptosis and DNA damage (all p < 0.006). These effects were generally more pronounced after 10 Gy than after 2 Gy. Notably, clonogenic survival curves deviated from the linear‐quadratic model, indicating non‐classical radiation response behaviour in heterogeneous cell populations. This proof‐of‐concept study suggests that a low‐passage, patient‐derived heterogeneous culture may serve as a practical and biologically informative in vitro platform for investigating radiation responses in canine oral melanoma. Further validation using additional patient‐derived cultures and comparisons with corresponding clinical radiation responses is needed.