The availability of the embryonic TGF-β protein Nodal is dynamically regulated during glioblastoma multiforme tumorigenesis

Abstract

Background: 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. 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.

Methods: We 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.

Results: The 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.

Conclusions: Our 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.

Keywords: Cancer stem-cell; Endocytosis; Glioblastoma; Nodal; Tumorigenesis.

Fig. 1

Nodal protein intracellular distribution depends on GBM differentitation status. a Nodal immunostaining was symmetrically distributed in the cytoplasm of OB1 stem cells. b Optical slices projected on the YZ axis showing a virtual reconstruction of the cell seen in “a” (asterisk). Nodal was localized to vesicle-like particles (arrow). c In GBM011 cells, Nodal immunostaining was found in a rare population of cells and was asymmetrically localized to a perinuclear region. Vesicle-like particle were also observed (arrow)

Fig. 2

Nodal protein levels are dowregulated during GBM differentiation. a, b OB1 stem cells forming oncospheres were immunostained for Nodal (red) and Nestin (green). DAPI (blue). OB1 stem cells present Nodal immunostaining localized to vesicles-like particles symmetrically distributed in the cytoplasm of GBM stem cells that are Nestin-negative (c, d). Upon differentiation, less differentiated cells were found positive for both Nodal and Nestin. More differentiated GBM cells were found negative for Nodal and positive for Nestin (69 % of cells; d, asterisk). Cells presenting a less elongated morphology were found Nodal-positive and Nestin-negative (26 % of cells). e Quantification of intracellular Nodal protein in OB1 stem cells and upon differentiation by Western blot. Nodal protein levels decrease in 50 % upon differentiation (quantification of average across three separate experiments). Nodal protein normalization through α-Tubulin immunoblotting. f Quantification of extracellular Nodal protein in protein precipitation of proteins present in conditioned medium by Western blot, presenting visible decrease in Nodal levels in OB1 cells upon differentiation. Nodal protein normalization through Coomassie Blue staining of gel. Data are mean ± SD. ***P < 0.001 by unpaired t test, n = 3)

Fig. 3

Nodal protein co-localizes with different endosomal vesicles depending on the differentiation status of GBM cells. Representative images of Nodal immunostaining with endosomal markers. In OB1 stem cells, Nodal co-localized with both early (EEA1 and Rab5) and late (Rab7 and Rab11) endosomes. In contrast, in differentiated OB1 cells, Nodal immunostaining was mostly co-localized with late (Rab7 and Rab11) endosomal vesicles

Fig. 4

Nodal mostly co-localizes to Rab7 and Rab11 in more differentiated GBM cells. Pearson’s coefficient of relative amount of co-localization of endosomal markers/Nodal in OB1 and differentiated OB1 cell cultures (quantification of average across three separate fields, each containing an average of three to four spheroids—OB1 cells—or 20–30 cells—differentiated OB1 cells). Data means are ± SD. ***P < 0.001 by two-way ANOVA for repeated measures followed by Tukey’s test for correction of the P value

Fig. 5

Illustration of the dynamics of Nodal distribution and availability during differentiation of GBM stem cells and of the endocytic mechanisms that may regulate Nodal during GBM tumorigenesis. The GBM stem cells shows a large amount of Nodal symmetrically distributed in their cytoplasm. The presence of Nodal in these cells is co-localization with both early (EEA1 and Rab5) and late (Rab7 and Rab11) endosomes. Upon differentiation, an asymmetric distribution of Nodal is found in the perinuclear region of the cells. In these cells, the intra and extracellular levels of Nodal are reduced and its co-localization with endosomes changes. There is a decrease in the association of Nodal with early (EEA1 and Rab5) endosomes and increase in its association with late (Rab7 and Rab11) endosomes. The characteristics seen in the stem cells can be returned after the dedifferentiation of the cells