The effect of PDT based on ALA and its derivatives in 3D ovary cancer models

CRISTIAN GABRIEL ORLANDO,GUSTAVO CALVO, GABRIELA DI VENOSA, PABLO VALLECORSA, GABRIELA CERVINI, DANIEL SÁENZ, ADRIANA CASAS

Encuentro; XIII Encuentro Latinoamericano de Fotoquímica y Fotobiología; XIII ELAFOT; 2017

Over the last decade, the employment of 3D models in cancer assays has grown. Spheroids have been shown to be much less sensitive than monolayer cells in treatment response studies, where physical variables such as penetration reflect a more realistic scenario [1]. On the other hand, it has been determined that 3D oncospheres are enriched in stem-like cells, which are often related to the development of metastasis and persistence after chemotherapy [2]. Thereby, 3D models allow us to study keys of photodynamic therapy (PDT) efficiency and its importance as a synergic treatment.PDT is a treatment for tumors of easy access by endoscopic route, like the ovarian cancer, based on the preferential accumulation of the photosensitizers in the malignant tissue after administration and the subsequent exposure to light [3]. This combination promotes reactions mediated by reactive oxygen species, which destroy the tumor cells. Since ovary cancer in most cases presents peritoneal dissemination at the time of diagnosis [4], the use of 3D models mimicking peritoneal metastasis, constitutes an interesting approach.Our group is specialized in PDT with the photosensitizer Protoporphyrin IX biosynthesized from its precursor, 5-aminolevulinic acid (ALA) and its derivatives synthesized to increase its penetration [5]. Among the latter, we used the 89-ALA and 95-ALA conjugates, as they synthesized more porphyrins at lower concentrations than those generated by ALA and thus giving better effectiveness.In the present work, we tested the response to PDT based on ALA and its derivatives in 3D spheroids as compared with monolayer cultures of IGROV-1 and SKOV-3 ovary cancer. We also analyzed the PDT response of oncospheres of the same cell lines enriched in stem cells.Analysis of viability through the MTT assay was performed. In addition, use of imaging metrics (area, circularity, sphericity and density) allowed us to evaluate certain effects that cannot be achieved through traditional methods.Spheroids showed a decrease in cell survival after PDT with all the photosensitizer, being 95-ALA the most efficient. However, a degree of resistance to the treatment was observed as compared to monolayer cultures of the same line, and this may be due to a lower degree of penetration of either light or photosensitizer to the inner layers of the spheroids. In addition, the analysis of images prior and after treatment indicated an increase of diameter, associated with a clear disruption of the micronodular structure. Finally, oncospheres enriched in stem cells exhibited a greater resistance to PDT than monolayers.In conclusion, the observations detailed herein highlight the potential value of the use of PDT in the treatment of ovary cancer dissemination, in combination with antineoplastics. In addition, the use of ALA derivatives improves ALA performance, thus reinforcing the importance of photosensitizer penetration in 3D models.[1] J. C. Finlay, S. Mitra, M. S. Patterson and T. H. Foster. Photobleaching kinetics of Photofrin in vivo and in multicell tumour spheroids indicate two simultaneous bleaching mechanisms, Phys. Med. Biol. 2004, 49, 4837.[2] S. Condello, C. A. Morgan, S. Nagdas, L. Cao, J. Turek, T. D. Hurley, et al. Beta-Catenin Regulated ALDH1A1 is a Target in Ovarian Cancer Spheroids, Oncogene. 2015, 34, 2297.[3] P. Agostinis, K. Berg and K. A. Cengel. Photodynamic therapy of cancer: an update, CA:Cancer. J. Clin. 2011, 61, 250.[4] W. F. Sindelar, F. J. Sullivan, E. Abraham, P. D. DeLaney, W. S. Smith, G. F. Friauf, et al. Intraperitoneal photodynamic therapy shows efficacy in phase I trial, Proc. Am. Soc. Clin. Oncol. 1995,14,1550.[5] H. Fukuda, A. Casas and A. Batlle. Use of ALA and ALA derivatives for optimizing ALA-based photodynamic therapy: a review of our experience, J. Environ. Pathol. Toxicol.Oncol. 2006, 25, 127.