Galectin, anti-oxidant, angiogenesis, TIL and cancer.
Ito et al., Angiogenesis
Supplemental Data
Supplemental Methods.
Purification of recombinant galectin-1 proteins
The wild type human galectin-1 was obtained and purified as previously described [1]. In brief, BL21
DE3 E.coli. transformed with pET-3a-hgal-1 was amplified and lysed with 0.2 mg/ml of lysozyme
(Sigma) and 100 U/ml of DNase I (Roche Applied Science). Galectin-1 protein was the purified by
affinity chromatography using a lactose-Sepharose column and BioLogic LP system (BioRad).
The hexahistidine-tagged P79R galectin-1 mutant was expressed in BL21 DE3 E.coli. transformed with
pET-14b-P79R-hgal-1. The cells were lysed with 0.2 mg/ml of lysozyme and 100 U/ml of DNase I. After
1 h incubation at 4 oC, the his-tagged protein was purified using a small column of HIS-Select® HF nickel
affinity gel (Sigma) and overnight incubation at 40C. After washing with 50 mM sodium phosphate
containing 0.3 M sodium chloride and 10 mM imidazole, the P79R galectin-1 mutant protein was eluted
using 50 mM sodium phosphate containing 0.3 M sodium chloride and 250 mM imidazole. Purified
galectin-1 protein samples were dialyzed for 4 h at 40C and the buffer exchanged several times with PBS
containing 4 mM -mercaptoethanol and 5 mM EDTA. The protein samples were then analyzed for their
purity by SDS-PAGE (Supplementary Fig. 1) and dynamic light scattering to confirm the homogeneity of
the reduced galectin-1 dimer present in the preparations with low levels of other aggregated forms present
[2,1].
Flow cytometric analysis
All cell preparations used for immunostaining were first treated with anti-CD16/32 FcR blocker
before being stained with fluorophore-conjugated antibody. Tumors were harvested and prepared
as single cell suspensions. Antibodies used for staining included the Alexa Fluor® 488 antimouse CD31 IgG (BioLegend, clone; MEC13.3). TILs, purified PBLs and spleen cells were
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Galectin, anti-oxidant, angiogenesis, TIL and cancer.
Ito et al., Angiogenesis
stained with fluorescein isothiocyanate (FITC) anti-mouse CD4 IgG (clone RM4-5; BD
Biosciences), phycoerythrin (PE) anti-mouse CD8a IgG (clone 53-6.7; BD Biosciences) for 30
min at 4oC in the dark. For intracellular galectin-1 detection, cells were fixed first in 4% pformaldehyde/PBS followed by permeabilization with 90% cold methanol. After washing with
PBS, the cells were incubated with either biotinylated anti-mouse galectin-1 monoclonal IgG
(clone 201002; R&D Systems Cat No. BAF-1245) or affinity purified anti-mouse galectin-3
polyclonal antibodies (R&D Systems Cat. No. BAF-1197) for 1 h at 40C. The cells were washed
and treated with PE streptavidin for 30 min at 4oC in the dark. Appropriate isotype-matched
control IgGs were used for each fluorophore-conjugated antibody to gate the positive samples.
The samples were analyzed either by counting individual cell populations or by values for the
mean fluorescence units using a flow cytometer (FACSCalibur and CellQuest Pro software; BD
Biosciences). Cells prepared from tumors, blood and spleen were gated using the SSC-FSC plots
to identify profiles for the smaller lymphocytes, excluding large tumor or granulated cells and
the ratios of numbers of cells and selecting for each type of lymphocyte as either CD4+ (X-axis)
or CD8+ (Y-axis).
SDS-PAGE and Western blotting
Tumor cells lysates prepared using RIPA buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4),
0.25% Na Deoxycholate, 0.1% IGEPAL, 1mM NaF, 1 g/mL, Aprotinin, 1 g/mL Leupeptin,
1mM PMSF) or purified recombinant galectin-1 proteins were treated by denaturation and
heating in SDS-PAGE sample buffer, resolved on 12.5% SDS-PAGE gels. For Coomassie
brilliant blue staining, the gels were immensed in coomassie blue solution and incubated for 4 h
followed by destaining using 30% MeOH and 10% galacial acetic acid in water. For western
blotting, after electro-transferring to PVDF membranes blocked by 1% skim milk/PBS,
biotinylated anti-galectin-1 monoclonal IgG was added before detection with a HRP-conjugated
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Galectin, anti-oxidant, angiogenesis, TIL and cancer.
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streptavidin. Blots were developed with Lumigen TMA-6 and chemiluminescence intensity
recorded using the ChemiDoc XRS system (Biorad).
MTT cell growth assay
ECs were cultured in 96-well plates until 80% confluent. The media was replaced with phenol
red-free DMEM supplemented with galectin-1 protein, TDG, sucrose or 10 M H2O2 as required
and samples incubated for a further 2, 4 or 8 h. Next, 20 l of 5 mg/ml methylthiazol tetrazolium
(MTT, Sigma) was added to each well and cultures further incubated at 370C for 3 h. After
aspiration of the media, formazan precipitates were dissolved in 150 l DMSO and the
absorbance was detected at 570 nm (FLUOstar OPTIMA, BMG Labtech).
Apoptosis assay
EAhy926 cells or HUVECs were cultured in 6-well plates until 80% confluent. The media,
including galectin-1 protein, TDG, sucrose or 30 M H2O2 as required, were added and samples
incubated for 4, 8 or 12 h. The cells were then harvested and resuspended in 100 l of binding
buffer. 4 l of FITC annexin V (BD Biosciences) and propidium iodide (PI) were added and
samples incubated for 20 min at 40C in the dark. 400 l of binding buffer was added and cells
then analyzed by flow cytometry.
Immunohistochemistry
Freshly excised tumors were embedded in pre-cooled optimal cutting temperature (O.C.T.)
media on dry ice. The tissue block was kept at -800C until ready for sectioning. The frozen tissue
block was sectioned at 15 to 18 m using a CM 1850 Cryostat (Leica Microsystems) at -150C.
The thin sections were placed onto SuperFrost® slides (Lomb Scientific) and dried at room
temperature for 2-3 h, then stored at -800C. The slides were immersed in cold acetone (-200C) for
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Galectin, anti-oxidant, angiogenesis, TIL and cancer.
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10 min to fix and permeabilize the tissue. After evaporating the acetone at RT for 20 min, the
slides were washed with PBS and blocked with 3% BSA in PBS for 1 h. After washing with
PBS, the tissue sections were stained with Alexa Fluor® 488 anti-mouse CD31 in 1% BSA/ PBS
inside a humidified chamber for 1 h at RT, washed with PBS in the dark and stained with DAPI
(1:1000). The tissue sections were then observed using an Olympus IX81 microscope and
analyzed using the Cell^M imaging software. Average fluorescence values were determined
using five different images taken from three independent tumor sections and analyzed with a
rectangular box of fixed size to determine average pixel numbers per box area from selected
regions of interest (ROI) according to the manual for the Cell^M software.
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Supplementary Figure Legends
Supplemantal Figure 1. SDS-PAGE of purified wild type galectin-1 (14.5 kDa) and His-tagged
P79R galectin-1 mutant (18 kDa). A, E. coli lysates containing His-tagged P79R were applied to
HIS-Select HF nickel affinity gel for binding and then washed. Protein fractions were eluted,
collected and analyzed. B, purified galectin-1 wild type or P79R as indicated.
Supplemental Figure 2. A. TDG addition has no effect on tumor cell viability. B16F10 or 4T1
cells were incubated with TDG at 370C for 1 h. Cell viabilities of each sample were analyzed by
trypan blue staining, before cells were s.c. injected as tumor challenge (A, B16F10 and 4T1 as
shown). B. TDG addition, up to 20 mg/ml, has no effect on cell proliferation or adhesion of
B16F10 or 4T1 wild type or G1KD cancer cell lines growing in culture over several days. After
washing to remove non-adherent cells (negligible numbers), the adherent cells were detached using 0.1%
EDTA/PBS and the total number of adherent cells was counted by trypan blue staining. The numbers of
detached, non-adherent cells in these cultures was very low to negligible compared to the total numbers of
adherent cells growing on the culture surfaces.
Supplemental Figure 3. A, Wild type 4T1 cells were injected s.c. into Balb/c mice. The mice
were treated with intratumoral injections of PBS (control) or sucrose (120 mg/kg), every 3 d and
tumor volumes monitored (n=2). Sucrose treated tumors did not show any significant differences
to control tumor growth. B, Wild type, shRNA control, G1KD pooled or G1KD clone-2 cells
were injected s.c. into Balb/c mice and tumor volumes monitored (n=2). shRNA control 4T1
cells showed similar tumor growth to the wild type cells. The pooled G1KD 4T1 cells or clone-2
both showed delayed tumor growth. C, Gal-3 levels in wild type and G1KD tumor cells. Wild
type or G1KD tumor cells (B16F10 and 4T1) were fixed with 4%-paraformaldehyde and then
permeabilized with ice cold methanol (90%). After washing with PBS, biotinylated anti-mouse
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Galectin, anti-oxidant, angiogenesis, TIL and cancer.
Ito et al., Angiogenesis
galectin-3 IgG (R&D Systems Cat No. BAF1197) was added. After 1 h incubation at 4oC, PEstreptavidin was added and incubated for 30 min at 4oC. The samples were then analyzed by
FACSCalibur. The mean fluorescence was used to compare the galectin-3 expression levels in
wild type and G1KD tumor cells. The levels of galectin-3 were slightly increased in both
B16F10 and 4T1 G1KD tumor cells. These results were also confirmed by immunoblotting
(results not shown).
Supplemental Figure 4. Isotype control images from immunofluorescent staining of tumor
sections. Frozen sections of wild type B16F10 tumors were immunostained in the left panel with
Alexa Fluor 488 anti-CD31 IgG (green) and PE anti-CD8a IgG (red) or in the right panel with
Alexa Fluor 488 Rat IgG2a and PE Rat IgG2a isotype controls. Sections were counterstained
with DAPI. Very few CD8 positive cells were detected in untreated tumor (left hand panel).
Scale bar = 100 m.
Supplemental Figure 5. Relative suppression of 4T1 tumor growth by intratumoral TDG in
immunocompetent Balb/c versus syngeneic nude mice (with defective T cell immunity). Tumor
volumes were analysed on day 12 comparing control untreated with TDG treated mice. The
reduction in tumor growth was ~47% in nude mice versus 67% in wild type immunocompetent
Balb/c mice. Three independent experiments were performed (total n=9-10). The data represent
the mean ± S.E. The statistical significance of the difference between samples compared was
determined by students-t test, *p<0.05
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Galectin, anti-oxidant, angiogenesis, TIL and cancer.
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Supplemental Figure 1. (S-1)
18 kDa
1
2
3
4
Wash
5
6 7 8
Elution
20 kDa
18 kDa
14.5 kDa
WT
P79R
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Galectin, anti-oxidant, angiogenesis, TIL and cancer.
Ito et al., Angiogenesis
Supplemental Figure 2. (S-2)
Trypan blue 4T1 cell counting
before injection
Trypan blue B16F10 cell counting
before injection
100
Live 4T1 cells (%)
75
50
25
75
50
25
200
x 103 cells
150
100
50
0
12
G
G
TD
4T1 WT
l
g/
m
Su
c2
0m
TD
G
TD
G
20
m
5m
g/
m
g/
m
l
l
on
tr
ol
C
Su
c
TD
Day 0
Day 1
Day 2
l
20
m
m
20
G
G
TD
225
200
175
150
125
100
75
50
25
0
g/
m
g/
m
5m
on
t
C
g/
m
l
l
ro
l
0
4T1 G1KD
225
200
175
150
125
100
75
50
25
0
250
x 103 cells
200
150
100
50
l
g/
m
20
m
Su
c
m
20
G
TD
TD
B16F10
4T1
140
Gal-3 MFI (% of WT)
140
Gal-3 MFI (% of WT)
g/
m
l
l
g/
m
G
C
Su
c
TD
5m
on
t
g/
m
20
m
m
20
G
G
TD
l
g/
m
l
l
g/
m
5m
on
t
C
C
ro
l
0
ro
l
x 103 cells
TD
C
B16F10 G1KD
B16F10 WT
x 103 cells
40
ro
l
G
on
t
12
0
80
TD
TD
G
TD
G
40
ro
l
on
t
C
B
80
0
0
G
Live B16F10 cells (%)
100
TD
A
120
100
80
60
40
20
120
100
80
60
40
20
0
0
WT
G1KD
WT
G1KD
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Galectin, anti-oxidant, angiogenesis, TIL and cancer.
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2.25
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
Tumour volume (x1000mm3)
Tumour volume (x1000mm3)
Supplemental Figure 3. (S-3)
Control WT
Sucrose WT
0
5
10
15
20
25
2.5
Control WT
shRNA control
G1KD pool
G1KD-2
2.0
1.5
1.0
0.5
0.0
0
3
6
9
12 15 18 21 24 27 30
Day
Day
Supplemental Figure 4. (S-4)
Isotype IgG/DAPI
CD31/CD8/DAPI
Supplemental Figure 5. (S-5)
Tumor volume suppression
by TDG (% of control)
52.63
60
50
40
Balb/c
Nude
p=0.0273
33.05
*
30
20
10
0
Balb/c
Nude
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Supplemental References
1. Scott SA, Scott K, Blanchard H (2007) Crystallization and preliminary crystallographic analysis of
recombinant human galectin-1. Acta Crystallogr Sect F Struct Biol Cryst Commun 63 (Pt 11):967-971.
doi:S1744309107050142 [pii]
10.1107/S1744309107050142
2. Scott SA, Bugarcic A, Blanchard H (2009) Characterisation of oxidized recombinant human galectin-1.
Protein Pept Lett 16 (10):1249-1255
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