An important component of research using animal models is ensuring rigor and reproducibility.,This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy.,Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC).,Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically ‘cold’ tumors, has not been well studied.,The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation.,We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank.,When tumors reached 190-230 mm3, they were grouped into a ‘wave’ and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine).,Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia.,Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors.,SC tumors reached the target tumor volume significantly faster compared with ID tumors.,Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not.,Further, the ‘mobile’ or ‘fixed’ phenotype of tumors predicted response to therapy, regardless of intended implantation depth.,These findings were then extended to additional immunotherapy regimens in four separate tumor models.,These data indicate that the physical ‘fixed’ versus ‘mobile’ characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment.,Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments.
Melanoma, the most dangerous type of cutaneous neoplasia, contributes to about 75% of all skin cancer-related deaths.,Thus, searching for new melanoma treatment options is an important field of study.,The current study was designed to assess whether the condition of mild and low-dose UVA radiation augments the lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effect of the drug in melanoma cancer cells through excessive oxidative stress generation.,C32 amelanotic and COLO829 melanotic (BRAF-mutant) melanoma cell lines were used as an experimental model system.,The combined exposure of cells to both lomefloxacin and UVA irradiation caused higher alterations of redox signalling pathways, as shown by intracellular reactive oxygen species overproduction and endogenous glutathione depletion when compared to non-irradiated but lomefloxacin-treated melanoma cells.,The obtained results also showed that lomefloxacin decreased both C32 and COLO829 cells’ viability in a concentration-dependent manner.,This effect significantly intensified when melanoma cells were exposed to UVA irradiation and the drug.,For melanoma cells exposed to lomefloxacin or lomefloxacin co-treatment with UVA irradiation, the concentrations of the drug that decreased the cells’ viability by 50% (EC50) were found to be 0.97, 0.17, 1.01, 0.18 mM, respectively.,Moreover, we found that the redox imbalance, mitochondrial membrane potential breakdown, induction of DNA fragmentation, and changes in the melanoma cells’ cell cycle distribution (including G2/M, S as well as Sub-G1-phase blockade) were lomefloxacin in a dose-dependent manner and were significantly augmented by UVA radiation.,This is the first experimental work that assesses the impact of excessive reactive oxygen species generation upon UVA radiation exposure on lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effects towards human melanoma cells, indicating the possibility of the usage of this drug in the photochemotherapy of malignant melanoma as an innovative medical treatment option which could improve the effectiveness of therapy.,The obtained results also revealed that the redox imbalance intensification mediated by the phototoxic potential of fluoroquinolones may be considered as a more efficient treatment model of malignant melanoma and may constitute the basis for the development of new compounds with a high ability to excessive oxidative stress generation upon UVA radiation in cancer cells.