LEGENDS TO SUPPLEMENTARY TABLES
The supplementary tables below summarize the data concerning the effect of
saturating concentration of BMP4 (100 ng/ml) on cells isolated from human GBMs.
The cell preparations shown here were established as follows:
1. GBSC-1: specimen from an adult GBM, sorted for their positive staining with
the anti-CD133 antibody and briefly cultured (48 hours) in the presence of
EGF and FGF2 1. Data from this preparation are also presented in the main
manuscript.
2. GBSC-2: specimen from an adult GBM, sorted for their positive staining with
the anti-CD133 antibody and briefly cultured (48 hours) in the presence of
EGF and FGF2 1.
3. GBSC-3: cells isolated from a specimen of adult GBM, unsorted and briefly
cultured (48 hours) in the presence of EGF and FGF2 2.
4. Acutely Dissociated Cells-1: Cells analyzed soon after the same
enzymatic/mechanical dissociation used for GBSCs above.
5. Acutely Dissociated Cells-2: Cells analyzed soon after the same
enzymatic/mechanical dissociation used for GBSCs above.
Supplementary table 1a – analysis of the phosphorylation of Smad proteins by
BMP4
The data show a significant increase in the levels of phosphorylation of smad 1,5,8
proteins (as shown in figure 1, panels h through l) upon exposure of the different cell
preparations to BMP4 for 90’ minutes as evaluated by cytofluorimetric analysis (* p<
0.01, n=3, two-tailed Student’s t-test).
Supplementary table 1b, c and d – analysis of apoptosis, cell death and
proliferation in BMP4-treated GBM cells.
Upon 48-hour exposure to BMP4 neither cell death (table 1b) nor apoptosis (table 1c)
were significantly changed. Conversely, the same treatment triggered significant loss
of proliferation capacity as indicated by a significant decrease in the KI67 labelling
index in all cell preparations (table 1d) (* p< 0.05, n=3, two-tailed Student’s t-test).
Supplementary table 1e – Analysis of the cell cycle following exposure of GBM
cells to BMP4
In all cell preparations, a 48-hour exposure to BMP4 consistently elicited an increase
in the percentage of cells in the G1 phase of the cell cycle, accompanied by a
concomitant decrease of the percentage of cells in S phase (*p<0.05; **p<0.001, n=3,
two-tailed Student’s t-test).
Supplementary table 1f and 1g – BMP4 decreases both the clonogenic index and
percentage of CD133+ cells.
A 48-hours exposure to BMP4 significantly decreased both the clonogenic frequency
(table 1f) and the size of the CD133+ population (table 1g) in all of the GBM
preparations tested (* p<0.05, ** p<0.01 n=3, for briefly cultured cells and *p<0.05,
** p<0.01 n=1 in triplicate, for acutely dissociated cells, two-tailed Student’s t-test).
Supplementary table 1h - phenotype analysis on GBM cells
Similar to the data shown in fig. 3 panels m through r, the table shows the results
from quantitative flow cytometry analysis of the changes in the levels of expression of
neural lineage specific markers in cells from human GBMs following a 48 hours
exposure to BMP4 (in the continued presence of mitogens). A major increase is
observed in the expression of the astroglial marker GFAP in all cell preparations,
together with a consistent increase in the neuronal III-tubulin (TUJ1) or TAU1
antigens and the oligodendroglial marker GalC. Data are from one representative
experiment, out of three, giving similar results.
Supplementary table 1i - phenotype analysis on GBM cells
A 48-hour exposure to BMP4 caused a significant increase in the number of MAP5immunoreative (IR) cells displaying a mature phenotype as established by counting
all MAP5-IR cells with processes at least three times longer that the diameter of the
soma. (* p< 0.05, n=3, two-tailed Student’s t-test).
LEGENDS TO SUPPLEMENTARY FIGURES
Supplementary Figure 1 – The figure shows a quantitative analysis (Real Time PCR
(RT-PCR)) of the expression of the BMPR type 1A, 1B, and 2 genes in the different
GBM cell preparations investigated in this study – acutely dissociated or briefly
cultured (GBSC) cells. The analysis confirmed a lower, though variable level of
expression for all the BMPRs in cells from GBMs with respect to normal human
foetal neural stem cells (HFNSCc). Values shown are representative of three different
experiments (*p<0.001 vs HFNSCs, n=3, two-tailed Student’s t-test).
Supplementary Figure 2 – a. Dose response curves showing the ability of BMP4 to
inhibit the proliferation of two representative GBM cell preparations (one established
from CD133+ sorted (blue line) and one from unsorted cultures (purple line)) in a
dose-dependent fashion, at a saturating concentration of 100ng/ml. b.
Cytofluorimetric analysis (one representative plot of three independent experiments
yielding similar results) showing the phosphorylation and nuclear translocation of
phospho Smad 1,5,8 as elicited 1.5 hours post BMP4 treatment in cultured GBM cells
(left panel). No activation of the p38 MAPK pathway (right panel) was seen 1.5 hours
after BMP4 treatment (similar plots were seen at 15 minute intervals, up to 2 hours;
control-red, BMP4-green, dotted line-isotype control). See also supplementary table
1a for complete, quantitative data.
Supplementary Figure 3 – Effects of various BMPs (all at 100 ng/ml) on the growth
of GBM cells. BMP2, -4,-5,-6,-7,-8b inhibit cell growth, whereas BMP1, -3 and -3b
appear to be ineffective, similar to TGF1 and 2 and to TGF1.2 (a chimerical TGF
agonist polypeptide) (all at 100 ng/ml), which were also ineffective.* p<0.005 BMPs
vs control, mean ± SE n=3, two-tailed Student’s t-test; ** p<0.001 BMP4 vs control,
mean ± SE n=3, two-tailed Student’s t-test.
Supplementary Figure 4 – a. The figure shows the effect of BMP4 on the expansion
rate of briefly cultured GBM cells (GBSC-2, pre-sorted for CD133+, black lines;
GBSC-3, unsorted, red lines) expanding in the presence of mitogens. BMP4 inhibits
cell expansion in both preparations (mean±SE, n=3; p<0.005) b. Representative plots
portraying the relative percentage of cells engaged in the various phases of the cell
cycle in control and BMP4-treated GBM cells. This was investigated by analyses of
the DNA content per cell, using propidium iodide incorporation followed by flow
cytometry. The data demonstrate that BMP4 treatment (right) results in a significant
increase of the proportion of cells in G0/G1 with a corresponding decrease in the
percentage of cells in S phase as compared to control cells (left). Complete
quantitative analysis in supplementary table 1e. c. Relative to control (left panel) a
more differentiated phenotype is seen upon exposure to BMP4 (second panel from
left) of GBM cells plated onto Matrigel. Consistent with the concept that BMP4
induces a more differentiated phenotype, a subjective more differentiated morphology
and an increase in the expression of MAP5-immunoreactivity is seen in BMP4-treated
(right panel) as compared to control (second panel from right) GBM cells (see also
supplementary table 1i for quantitative analysis).
Supplementary Figure 5 – The figure shows one example of the dot plot graph
concerning the quantitative analysis of the expression of the CD133 antigen. This was
performed by flow cytometry on control (b and isotype control in a) and BMP4-
treated GBM cells (d and isotype control in c). It is clear that BMP4 triggers a
significant decrease in the actual overall percentage of CD133-immunoreactive cells.
Supplementary Figure 6 – Untreated GBM cells constitutively express neuronal
markers such as MAP2 (a) and NF200 (c) as well as the intermediate filament nestin,
normally found in immature neural precursor, (e). These markers are still expressed
after 48-hour exposure to BMP4 (b, d and f, respectively). a-f. Scale 15µm, as in f.
Supplementary Figure 7 – BMP4 inhibits the tumorigenicity of GBM cells.
Transient (48 hours) exposure of cultured GBM cells to BMP4 in vitro, prior to
transplantation into the striatum of adult SCID/bg mice, caused a dramatic reduction
in the ability of the implanted cells to form tumors (b,d,f,h,j,l) as compared to
controls (a,c,e,g,i,k). Hematoxylin and eosin staining showed typical glioblastoma
masses five weeks after injection of control cells (a,e,g), whereas BMP4-treated cells
generated very small grafts (b,f,h). Tumors established from control cells displayed a
much higher mitotic index, as revealed by immunocytochemistry for Ki67 (4.3 ±
0.3% control vs. 0.76 ± 0.5% BMP treated, mean ± SE, n=3, Student’s t-test, p<0.05)
(c, high power in i), than BMP4-treated cells (d, high power in j). k,l. Hematoxylin
and eosin staining showing invasion of the lumen of the right lateral ventricle at
bregma point (k, arrow) five weeks post-injection) following implantation of control
cells. This phenomenon was never observed with BMP4-pretreated GBM cells (l,
arrow). Three months post-injection, all control animals had died, whereas all animals
receiving BMP4 pre-treated cells survived well beyond 5 months. m,n. Following a
48 hours incubation in control conditions (control), CD133+ cells were purified by
FACS (purity 97.3%) and the ability of 3 x 105 of these cells to establish GBMs
intracerebrally was compared to that of the same number of CD133+ cells, purified
from sister cultures (purity 98.7%) after incubation for 48 hours in the presence of
BMP4 (treated). Mice transplanted with CD133+ cells from control cultures
developed large neoplastic formations by 5-7 weeks post transplant (m; n=8),
whereas none of the animals (n=8) receiving CD133+ cells sorted from BMP4-treated
cells developed any tumor (n). By ninety days post-injection all of the control animals
had died whereas those receiving CD133+ cells from BMP4-treated cultures were
alive. This shows that the tumorigenic ability of the residual fraction of CD133+ cells
found in BMP4-treated cultures is virtually abolished by BMP4. o,p. When CD133+
cells, that were acutely isolated from primary tumors, established in mice transplanted
with CD133+ acutely isolated control cells, were re-transplanted into the brain of
secondary recipients they established secondary tumors in less than 4 months
(example in o). These same cells could also be cultured and expanded with mitogens
(see main text). Neither culturing nor establishment of secondary tumors could be
accomplished with cells extracted from mice, which were initially transplanted with
BMP4-treated, acutely isolated GBM cells. Similar serial transplantation experiments
yielded equivalent results when the primary tumor was generated by transplantation
of 3 x 105 briefly cultured CD133+ cells (p; example of secondary tumor established
from CD133+ briefly cultured cells). Magnification: a-h and k-n, 5X; i,j and o,p,
10X.
Supplementary Figure 8 – Ex vivo pre-treatment of GBM cells with BMP4 inhibits
ventricular invasion following orthotopic injection. Five weeks after transplantation
into the adult brain of scid/bg mice, tumors from control GBM cells have invaded the
ventricular system (see also supplementary fig.7) including the fourth ventricle (a).
Cells from cultures pretreated with BMP4 for 48hours showed no signs of ventricular
invasion (b). Magnification: 5X.
Supplementary Figure 9 – a,c. Phenotype analysis confirmed that, as shown
previously 2, tumors induced by transplanting briefly cultured GBM cells into the
brain of scid/bg animals generated astroglial-like cells, (shown by GFAP-IR (green
and anti-human mitochondria [red in a] or anti-human nuclei immunofluorescence
staining [red in c]). Within the tumor mass, numerous cells were also nestin-IR
(intermediate filament found in immature neural precursors; e, magnified in g, brown
staining). No labelling with neuronal markers, such as III-tubulin or NF200, or
oligodendroglial antigens, such as GC or O4 was observed. Scarce GFAP- (b,d) and
nestin-IR (f,h) were also observed in BMP4-treated GBM cell implants. When, as in
the case of BMP4 co-treatment of GBM cells during transplantation (b,f), tumor
development was poor, the staining for GFAP revealed scattered reactive astroglial
cells (green in b) surrounding the transplantation area (red, anti-human mitochondria
in b), wherein a few surviving GBM cells were weakly stained with the anti-nestin
antibody (f,h). In all the BMP4 treatment paradigms, the tumors contained GFAPimmunoreactive cells (d), but no labelling with neuronal or oligodendroglial marker
was observed. Magnification: a,b and e,f 5X; c and d 20X; g and h, 40X.
Supplementary Figure 10 – a. Quantitative RT-PCR analysis of the expression of
thirteen different BMPs genes in briefly cultured GBM cells. Data are the mean ± se
samples in triplicate from one out of three independent experiments yielding
overlapping results. b. When GBM cells, cultured with mitogens, were exposed to a
neutralizing antibody (nAb-BMP4; 10µg/ml, R&D Systems) that specifically blocks
BMP4, the cell growth rate increased significantly as compared to control conditions
(*p<0.01 nAb-BMP4 vs control). This indicates that endogenous BMP4 (RT-PCR
and western blot in Fig. 1) physiologically restricts the proliferation of human GBM
cells. c. The normal levels of phosphorylation of smad 1,5,8 proteins observed in
control cells is almost halved following a 3 hours exposure to 10µg/ml of
neutralizing anti-BMP4 antibody, whereas exposure to BMP4 produces the usual
increase, as described earlier on Fig.1. Evaluation by cytofluorimetric analysis (* p<
0.01, n=3, two-tailed Student’s t-test).
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