## 4 Preclinical Studies
### In Vitro Studies  
Although animal models are appropriate for human and veterinary oncology studies, in vitro tests were used to replace animals to evaluate the photosensitizer outcomes initially. These studies mainly used cell cultures, photosensitizing molecules, and light sources. In this section, in vitro studies using cells of canine and feline origin are described. Canine glioma cells were cloned and subjected to photosensitizer BPD-MA. After three hours of incubation in the dark, the cells were exposed to a LED light (100 J/cm\({}^{2}\)). Approximately 24 h after PDT, cell viability was evaluated through 3H-thymidine incorporation into the DNA. A 50% growth inhibition was achieved with 10 and 4 ng/mL [160]. The hematoporphyrin monomethyl ether was administered to canine mammary carcinoma cell lines [161; 162; 163]. Apoptosis plays an important role in the reduction induced by PDT (20 mg/mL; He-Ne laser, 2.8 J/cm\({}^{2}\), 632.8 nm) in the viability of canine mammary carcinoma cells [161]. Treatment lead to loss of mitochondrial membrane potential [162]. Additionally, significant changes in cell morphology were observed, such as the formation of cytoplasmic vacuoles and the gradual rounding of cells, together with decreased size and detachment [163]. Aluminum-chloride-phthalocyanine encapsulated in liposomes induced cytotoxicity in a primary culture of female dog mammary gland carcinoma cells. Nevertheless, photosensitizer or laser irradiation per se did not induce cytotoxicity or morphological changes, indicating the safety and efficacy of PDT (2.5 M; LED, 10 J/cm\({}^{2}\), 660 \(\pm\) 40 nm) [164]. Canine mammary carcinoma cells were treated with different concentrations (0.16, 0.80, 4.0, and 20 uM) of maltotriose\(-\)chlorin e6 conjugate (Mal\({}_{3}\)-TEG-Ce6). The cells exposed to 5 J/cm\({}^{2}\) of 671 nm red light suggested the antitumor activity of this photosensitizer is extremely high. Cell viability was reduced to 50% when 0.16 uM was used [165]. The viability of the canine mammary carcinoma SNP cells was evaluated after PDT with NPe6 and a glucose conjugate of this photosensitizer. The half-maximal inhibitory concentration (IC50) of NPe6 against SNP cells exposed to light doses of 1, 5, and 15 J/cm\({}^{2}\) (650 nm) were 75.2, 30.4, and 30.6 u/mL, respectively. The glucose conjugate was even more effective with IC50 values of 33.4, 10.4, and 1.7 ug/mL, respectively [159]. To assess the efficacy of 5-ALA-PDT in several subtypes of canine mammary gland tumors, mammary gland cancer cells were obtained from three dogs diagnosed with carcinoma, sarcoma, and carcinomas, respectively. Cancer cells were then plated and treated with 0.5 and 1 mM of 5-ALA. From 6 to 24 J/cm\({}^{2}\), several light doses were used to activate 5-ALA through a continuous wave or pulse radiation. The intracellular PpIX peak of fluorescence was achieved after 4 h of 1 mM 5-ALA administration. The cytotoxic effect of 5-ALA was dose-dependent. Indeed, carcinoma and carcinomas cells treated with a continuous wave at 12 J/cm\({}^{2}\) presented a substantial reduction in the cell viability and DNA damage. Nevertheless, 1 mM 5-ALA promoted apoptosis in all canine mammary gland tumor cells independently of the light dose [166].