Efficacy of 5-aminolevulinic acid-based photodynamic therapy in different subtypes of canine mammary gland cancer cells

Canine mammary gland tumors (CMGTs) are heterogeneous disease and subclassified [sarcomas (S), carcinomas (C), and carcinosarcomas (CS)] according to histopathological differentiation. Photodynamic therapy (PDT) is a promising treatment strategy based on the use of a photosensitizer (PS) activated by light. However, the therapeutic potential of PDT in the treatment of CMGTs has not been investigated, yet. Therefore, the aim of this study was to determine the in vitro protocol of 5-ALA-based-PDT for the treatment of three subtypes of CMGTs, for the first time. The intracellular PpIX florescence intensity was measured for 5-ALA (0.5 and 1 mM). After irradiation with different light doses (6, 9, 12, 18, and 24 J/cm 2 ) for two different modes [continuous wave (CW) and pulse radiation (PR)], the cytotoxic effects of 5-ALA (0.5 and 1 mM) on the subtypes (C, S, and CS) of CMGTs were analyzed by WST-1. Finally, the optimal PDT treatment protocol was validated through Annexin V and AO/EtBr staining. Our results showed that 1 mM 5-ALA for 4-h incubation was the best treatment condition in all subtypes of CMGTs due to higher intracellular PpIX level. After irradiation with different light doses, PR mode was more effective in S primary cells at 9 J/cm 2 . However, a significant decrease in the viability of C and CS cells was detected at 12 /cm 2 in CW mode ( p < 0.05). Additionally, 1 mM 5-ALA induced apoptotic cell death in each subtype of CMGTs. Our preliminary findings suggest that (i) each subtype of CMGTs differentially responds to PDT and (ii) the light dose and mode could play an important role in the effective PDT treatment. However, further studies are needed to investigate the role of the different light sources and PDT-based apoptotic cell death in CMGTs cells.