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

Nodal protein intracellular distribution depends on GBM differentitation status

Nodal immunostaining was detected symmetrically distributed in the cytoplasm of OB1
stem cells (Fig. 1a), appearing as vesicle-like subcellular particles (Fig. 1b, arrow). Additionally, we noticed that the presence of Nodal in these cells changed
along the development of the oncospheres. While small spheres contained only cells
that show a symmetrical Nodal distribution (Additional file 1: Figure S1a), large ones were comprised by cells with distinct Nodal distributions
(Additional file 1: Figure S1b). Nodal-positive cells were found on top of the oncospheres and presented
Nodal immunostaining mainly localized to points of cell–cell contact (Additional file
1: Figure S1c–e). 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). The lack of the initially seen Nodal protein symmetrical distribution
in some cells was most likely was due to the beginning of their differentiation in
the interior of the oncospheres. Thus we conclude that Nodal is localized to vesicles-like
particles that are symmetrically distributed in the cytoplasm of undifferentiated
OB1 stem cells.

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)

In GBM011 cells, Nodal immunostaining was present in an asymmetric pattern, characterized
by a perinuclear distribution, with no evidence of association to the plasma membrane
(Fig. 1c). Nodal positive vesicle-like particles were observed, and unlike OB1 stem cells,
presented a perinuclear localization (Fig. 1c, arrow).

To test whether Nodal asymmetric distribution is restricted to mdGBM cells or if it
is a broader marker of differentiated cancer cells, we also tested and found the same
asymmetry in the well-described prostate cell line DU145 that has been shown to transduce
the Nodal signaling pathway (Additional file 2: Figure S2). Thus we conclude that Nodal immunostaining is found in asymmetric perinuclear
localization in more differentiated GBM cells during interphase.

Nodal asymmetric cytoplasmic distribution shifts to a symmetric distribution in dedifferentiated
GBM cells

We next tested whether the Nodal asymmetric and symmetric distributions represent
markers of the differentiation state of GBM cells. We reasoned that the asymmetric
distribution, found in mdGBM cells, would shift to a symmetric distribution, if these
cells were induced to a less differentiated status. To test this hypothesis, we have
used the U87MG cell line induced with a dedifferentiation protocol. We verified the
differentiation status through Nanog Western Blot, in OB1, differentiated OB1, U87MG
and U87MG dedifferentiated cells (Additional file 3: Figure S3). In the original differentiated cells, Nodal immunostaining was asymmetrically
located in the perinuclear area (Additional file 4: Figure S4, arrows). Conversely, when U87MG cells were induced to dedifferentiate
(U87MG-O), the Nodal asymmetric distribution shifted to a symmetric pattern (Additional
file 4: Figure S4b–d, arrows), similar to that observed in OB1 stem cells. We also found
that U87MG-O cells upregulate Nodal protein intracellular levels, reinforcing our
data obtained on OB1 cells. This finding indicates that Nodal cytoplasmic distribution
and levels are dynamically regulated and strongly correlate with the differentiation
status of GBM cells.

Nodal protein intra and extracellular levels are reduced upon GBM differentiation

To better understand the dynamics of Nodal protein during the transition between a
stem cell like to a more differentiated cell behavior of GBM cells, we have quantified
the abundance of intracellular Nodal in OB1 stem cells induced to differentiate. To
confirm the differentiation of the cells, we evaluated cell morphology and the distribution
of Nestin, a marker for differentiating progenitor cells 29]. OB1 stem cells grown as spheres, in the absence of FBS, were Nestin-negative and
Nodal positive (Fig. 2a, b). Differentiated OB1 cells presented a spread out morphology, were Nestin-positive
and mostly negative for Nodal (69 %, n = 66 cells; Fig. 2c, d). As expected, in OB1 stem cells subjected to differentiation there was a decreased
in Nodal protein levels (Fig. 2e; n = 3).

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
?-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)

As Nodal is a member of the TGF? superfamily, we asked whether the extracellular levels
of Nodal are also dynamically regulated during GBM tumorigenesis. Protein quantification
of the conditioned medium showed that Nodal extracellular levels were downregulated
as GBMsc were induced to differentiate (Fig. 2f). These results show that not only Nodal distribution is altered but also its intra
and extracellular availability are reduced upon GBMsc differentiation.

Nodal protein co-localizes with different endosomal vesicles depending on the differentiation
status of GBM cells

Previous studies have shown that Nodal was found in endosomal vesicles 30]. Therefore, to elucidate whether the vesicle-like particles observed in stem and
differentiated GBM cells correspond to the same subcellular compartments or not or
even whether they may change according to the differentiation status of GBM cells,
we performed immuno co-localization of Nodal and different endosomal proteins in OB1
and differentiated OB1 cells (Figs. 3, 4). Nodal immunostaining in OB1 stem cells was found to co-localize with both early
(EEA1 and Rab5) and late (Rab7 and Rab11) endosomes (Figs. 3, 4), suggesting that OB1 stem cells are continuously recycling and degrading Nodal.
In contrast, in differentiated OB1 cells, Nodal immunostaining was mostly co-localized
with late (Rab7 and Rab11) endosomal vesicles (Figs. 3, 4). These results are in agreement with a decrease in the levels of secreted Nodal
in mdGBM cells as shown in Fig. 2f. It was possible to verify the same pattern and rates on U87MG and U87MG-O cells
(Additional file 5: Figure S5, Additional file 6: Figure S6), suggesting that the Nodal endocytic processing is not specific of a
cell line, relating to the phenotypic state. Also, our analysis of Nodal/endosomal
markers co-localization in GBM011 cells reveal that the GBM primary cultures reproduced
the pattern observed in mdGBM cells (Additional file 6: Figure S6). Thus, we conclude that Nodal availability might be controlled by an
endocytic pathway, in a differentiation status dependent manner.

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

Based on the results obtained in this study, it is possible to summarize in an illustration
the dynamics of Nodal distribution and availability during differentiation of GBM
stem cells, as well as the endocytic mechanisms that may regulate Nodal during GBM
tumorigenesis (Fig. 5). Briefly, the GBM stem cells present a large amount of Nodal symmetrically distributed
in their cytoplasm and presenting similar co-localization with both early (EEA1 and
Rab5) and late (Rab7 and Rab11) endosomal vesicles. After differentiation, an asymmetric
distribution of Nodal is seen in the cytoplasm, mostly limited to the perinuclear
region. Moreover, the levels of Nodal in the cells are reduced and its co-localization
with endosomal vesicles changes, showing a decrease in its association with early
(EEA1 and Rab5) endosomes and increase in its association with late (Rab7 and Rab11)
endosomes. The dedifferentiation of the cells can return these characteristics back
those seen in the stem cells.

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