Abstract
BackgroundGlioblastoma (GBM) is the most common primary brain tumor presenting self-renewing cancer stem cells. The role of these cells on the development of the tumors has been proposed to recapitulate programs from embryogenesis. Recently, the embryonic transforming growth factor-β (TGF-β) protein Nodal has been shown to be reactivated upon tumor development; however, its availability in GBM cells has not been addressed so far. In this study, we investigated by an original approach the mechanisms that dynamically control both intra and extracellular Nodal availability during GBM tumorigenesis.MethodsWe characterized the dynamics of Nodal availability in both stem and more differentiated GBM cells through morphological analysis, immunofluorescence of Nodal protein and of early (EEA1 and Rab5) and late (Rab7 and Rab11) endocytic markers and Western Blot. Tukey’s test was used to analyze the prevalent correlation of Nodal with different endocytic markers inside specific differentiation states, and Sidak’s multiple comparisons test was used to compare the prevalence of Nodal/endocytic markers co-localization between two differentiation states of GBM cells. Paired t test was used to analyze the abundance of Nodal protein, in extra and intracellular media.ResultsThe cytoplasmic distribution of Nodal was dynamically regulated and strongly correlated with the differentiation status of GBM cells. While Nodal-positive vesicle-like particles were symmetrically distributed in GBM stem cells (GBMsc), they presented asymmetric perinuclear localization in more differentiated GBM cells (mdGBM). Strikingly, when subjected to dedifferentiation, the distribution of Nodal in mdGBM shifted to a symmetric pattern. Moreover, the availability of both intracellular and secreted Nodal were downregulated upon GBMsc differentiation, with cells becoming elongated, negative for Nodal and positive for Nestin. Interestingly, the co-localization of Nodal with endosomal vesicles also depended on the differentiation status of the cells, with Nodal seen more packed in EEA1/Rab5 + vesicles in GBMsc and more in Rab7/11 + vesicles in mdGBM.ConclusionsOur results show for the first time that Nodal availability relates to GBM cell differentiation status and that it is dynamically regulated by an endocytic pathway during GBM tumorigenesis, shedding new light on molecular pathways that might emerge as putative targets for Nodal signaling in GBM therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0324-3) contains supplementary material, which is available to authorized users.
Highlights
Glioblastoma (GBM) is the most common primary brain tumor presenting self-renewing cancer stem cells
In this study we have investigated by an original approach the dynamics of Nodal intracellular distribution and extracellular availability in both stem and more differentiated GBM cells
Nodal immunostaining was detected symmetrically distributed in the cytoplasm of cells located on the lateral edges, surrounding the whole oncosphere (Additional file 1: Figure S1f–h), as well as in the cytoplasm of cells that were directly attached to the substrate (Additional file 1: Figure S1i–l)
Summary
Glioblastoma (GBM) is the most common primary brain tumor presenting self-renewing cancer stem cells. The role of these cells on the development of the tumors has been proposed to recapitulate programs from embryogenesis. The embryonic transforming growth factor-β (TGF-β) protein Nodal has been shown to be reactivated upon tumor development; its availability in GBM cells has not been addressed so far. Glioblastoma (GBM; grade IV astrocytoma) is the most common primary brain tumor characterized by aggressive invasiveness, high proliferative rate, insensitivity to radio- and chemotherapy and a short survival period [16,17,18]. Sub-population of GBM cells with stem-like properties may be the source of tumors since, apparently, these stem cells are highly resistant to current cancer treatments and survive to regenerate new tumors [16, 24, 25]
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