Title

 

Authors

E. Mastrantoni¹ , S. Chen², D. Melchiorri<sup>1

1</sup>Dept. of Human Physiology and Pharmacology, University of Rome “Sapienza”, Rome, Italy
² Dept. of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ, USA.

 

Abstract

Human malignant melanoma is a highly aggressive and drug-resistant cancer. Detection of tumor heterogeneity, undifferentiated molecular signatures, and increased tumorigenicity of melanoma subsets with embryonic-like differentiation plasticity, strongly suggests the presence and involvement of malignant melanoma stem cells (MMSCs) in the initiation and propagation of this malignancy. Parallel to the role that normal stem cells play in organogenesis, cancer stem cells, CSCs, are thought to be crucial for tumorigenesis. They may play a pivotal role in tumor initiation, growth, and recurrence. Many investigators, indeed, propose that a subset of MMSCs may be responsible for melanoma chemoresistance, tumor invasiveness, and neoplastic progression and that targeted abrogation of a MMSCs population could therefore ultimately lead to stable remissions and perhaps cure of metastatic melanoma. We were able to isolate a highly tumorigenic, drug resistant, slow-cycling cell population from the melanoma cell line C8161, using the spheres formation assay. Melanoma cells were cultured in a medium optimized for the selection of neural stem cells (i.e. enriched in growth factors and deprived of serum) (Di Giorgi-Gerevini et al., 2005). Under these culture conditions, cells proliferated and aggregated into floating spheres, similar to the neurospheres and mammospheres generated from brain and breast cancer tumors, respectively. Interestingly, cells growing as floating spheres were slower-cycling compared to their monolayer counterpart, a characteristic shared with stem cells. Although no difference in the expression of the classical stem cell markers, Nestin and CD133, was detected between cells of the floating spheres and their monolayer counterpart, cultured in RPMI with 10% of FBS, cells aggregated into floating spheres showed an increased tumorigenity in vivo. In vivo tumorigenity was assessed by implanting s.c. both types of cells (floating and monolayer cells) into nude mice, at a dose, 10⁴ cells/site, that usually does not induce tumors. Following visual examination, tumour masses were consistently detected in mice injected with cells deriving from floating spheres whereas no tumour mass was evident in mice injected with monolayer culture-derived cells. We then tested the sensitivity of C8161 melanoma spheres to antineoplastic drugs (Etoposide, Temozolomide) and Riluzole (Glutamate release inhibitor; phase II trial in advanced melanoma currently undergoing study) in comparison with cells grown in monolayer. Using the MTT assay to test cell viability, the C8161 melanoma spheres showed a higher viability when challenged with any of the listed drugs than cells grown in monolayer. Our data show that it is possible to isolate, from melanoma cell lines, a population of slow-cycling floating melanoma cells with enhanced tumogenicity that could represent a good cell model to study Melanoma resistance and recurrence both in vitro and in vivo.