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In the absence of serum, CSCs/CS-LCs have the ability to grow as lung tumorspheres (LTSs), and this system is routinely used for isolation and characterization of putative CSCs/CS-LCs.

Methods to isolate LTSs are usually performed in serum-free media supplemented with specific additives such as epidermal growth factor and basic fibroblast growth factor.

Despite the incidence of melanoma has been growing faster than other human cancers during last decades among Caucasian populations (4), most of melanoma cases is diagnosed at early stages of the disease.

From the practical point of view, increasing evidences indicate that some differences in biological and clinical behaviors within the traditional subgroups of melanomas defined by conventional diagnostic procedures are due to the existence of different “molecular subtypes” of the disease (2).

Actually, the criteria commonly used to classify melanomas are based on: (a) relationship between the degree of sun exposition and the site of primary tumor [according to such criteria, melanomas are classified into four groups: melanoma on skin with or without chronic sun-damage (CSD or non-CSD melanoma); melanoma on palms, soles, and nail bed (acral melanoma); and melanoma on mucous membrane (mucosal melanoma)] (1) or (b) evaluation of the tumor growth pattern [according to this criterion, four histological types of melanoma have been described: superficial spreading melanoma (SSM), lentigo maligna melanoma (LMM), nodular melanoma (NM), and acral lentiginous melanoma (ALM)] (3).

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In this study, we report the generation of LTSs without the addition of any external mitogenic stimulation.

LTSs generated in this manner demonstrated several traits usually associated with increased stemness such as elevated expression of the stemness-associated marker Sox2 and increased chemoresistance to conventional anticancer drugs.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular mechanisms involved in pathogenesis of malignant melanoma have been widely studied and novel therapeutic treatments developed in recent past years.

In addition, we report that the FDA-approved drug Digitoxin, at concentration close to its therapeutic level, decreased the viability of LTSs and downregulated Sox2 independent of the PI3K/AKT pathway.

The potential use of LTSs generated without the addition of any external mitogenic stimulation to study the role of specific factor(s) associated with stemness properties is also discussed.

Rcho-1 trophoblast stem cells can be effectively used as a model for investigating trophoblast cell differentiation.