Abstract B67: Cytotoxic effect of isothiouronium salt MF08 in tumorsphere model
Authors: Marin Marjorie G. P. , Rode Michele P. , Assunção Laura S. , Kretzer Iara F. , Ferreira Misael , Sá Marcus Mandolesi , Creczynski-Pasa Tânia B. 
Published: May 2019

Abstract
Skin cancer is the most common type of cancer in Brazil and accounts for 25% of all registered cases. Within this group, melanoma is considered the most severe and aggressive due to the possibility of metastasis. Melanoma is responsible for 79% of skin cancer deaths. This cancer type develops in melanocytes, cells responsible for the production of melanin, and can be found in many tissues and organs such as epidermis, nasal cavity epithelium, oropharynx, oral mucosa, and eyes. Some of the treatments for melanoma include interleukin-2 or interferon-α and chemotherapy with dacarbazine or temozolomide, but these drugs induce tumor regression in only 5-20% of patients. Chemoresistance and relapses found in patients with melanoma could be explained by the survival of a subpopulation of cells called cancer stem cells (CSC). The CSC are highly proliferative, can self-renew, initiate tumors, and differentiate to a heterogeneous population. As relapses could be caused by ineffective treatments against cancer stem cells, the evaluation of the activity of new antitumor agents on this specific population is strategic to prevent the disease recurrence. Thus, the evaluation of cytotoxic effect of new antitumor agents on this subpopulation becomes necessary for the development of effective treatment approaches. In a previous in vitro work, the isothiouronium salt MF08 (IS-MF08) presented antitumoral activity by G2/M cell cycle arrest, DNA fragmentation, and cell death by extrinsic apoptosis pathway. Furthermore, IS-MF08 presented remarkable tumor growth inhibition and increased the survival rate in melanoma-bearing mice model. Therefore, the aim of this study was to define the best condition to tumorspheres formation and evaluate the potential activity of the IS-MF08 on cancer stem cell population of murine melanoma B16F10. Tumorspheres formation assay was used to enrich melanoma CSCs from B16F10 cells. Therefore, spheres were obtained through a nonadhesive 96-well plate coated with agarose 2% tested in three different volumes (30, 40, and 50 μL) with cells plated at low density (300 cells/cm2). Spheres were successfully formed within 7 days of culture in all agarose volumes tested. Plates coated with 30 μL of agarose presented spheroids with the highest area on the 4th day (34.0 ± 7.2 µm2) and 7th day (146.4 ± 38.2 μm2) and cells survived in all conditions (cell viability of 86-96%). Moreover, scanning electron microscopy showed different features among the conditions. The spheroids formed at the lowest volume showed a more defined and smooth surface when compared to the other volumes. Based on these results, the lower agarose volume was used in cells incubation with MF08. The IS-MF08 was added in two moments: at the same day cells were plated or after spheres formation (4th day after), and at two different concentrations: 2.5 μM and 5.0 μM. After adding the IS-MF08 the spheroids' area decreased significantly; on the 7th day 64.7% and 77.2% for 2.5 and 5.0 μM, respectively, as well as after sphere formation (17% for both concentrations). Next step is the characterization and quantification of the CSCs in the tumorspheres regarding cell viability to evaluate specifically the effect of IS-MF08 in this cell group. In conclusion, IS-MF08 seems to inhibit the tumorspheres growth as previously observed in tumor growth in in vivo model. Furthermore, the concentration of IS-MF08 used in this work was ~6 times lower than the inhibitory concentration needed to inhibit 50% of cells in monolayer culture of B16F10 (IC50 35 μM). This work could provide new insights for the in vitro analysis of new anticancer agents, as well as in the therapeutics of melanoma treatment.
Citation Format: Marjorie G. P. Marin, Michele P. Rode, Laura S. Assunção, Iara F. Kretzer, Misael Ferreira, Marcus Mandolesi Sá, Tânia B. Creczynski-Pasa. Cytotoxic effect of isothiouronium salt MF08 in tumorsphere model [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B67.