Administration of the hypoxia markers EF5 (60mg/kg; provided by Dr. Cameron Koch, University of. Pennsylvania, PA) or pimonidazole (60 mg/kg; Hypoxyprobe, ...
Cell Reports, Volume 23
Supplemental Information
Intravital Imaging to Monitor Therapeutic Response in Moving Hypoxic Regions Resistant to PI3K Pathway Targeting in Pancreatic Cancer James R.W. Conway, Sean C. Warren, David Herrmann, Kendelle J. Murphy, Aurélie S. Cazet, Claire Vennin, Robert F. Shearer, Monica J. Killen, Astrid Magenau, Pauline Mélénec, Mark Pinese, Max Nobis, Anaiis Zaratzian, Alice Boulghourjian, Andrew M. Da Silva, Gonzalo del Monte-Nieto, Arne S.A. Adam, Richard P. Harvey, Jody J. Haigh, Yingxiao Wang, David R. Croucher, Owen J. Sansom, Marina Pajic, C. Elizabeth Caldon, Jennifer P. Morton, and Paul Timpson
A
Ki67
DAPI
B
Pimonidazole
KPflC
Ki67 positive cells (%)
30
KPflC
20
ns
10
N
KPC
Ki67 positive cells (%)
30
a ox i H yp
or m
ox ia
0
KPC ns
20
10
2
KPC
1
2
0. 1%
2
O
2
2
O
2
O
2
O
0. 1%
30 20 10 2
2
O
0.
1%
1%
2
O
ia
O 5%
N
or
m
ox
2
2
O
ox ia m N or
O
0 %
2
O
2
O
*
**
40
N
1%
* *
*
*
50
0. 1%
1%
O 0. 2 1% O or m 2 ox ia 5% O
2
O 5%
N
or m
ox
ia
0
KPC
*
*
60
0. 1
*
**
2
O
N 1%
*
Pimonidazole
KPflC
70
2
*
3
N
O 2
O
1%
1%
0.
2
O
5%
O 2
m ox
N or
O
1%
0.
1%
ox
2
O
m or N
H Pimonidazole
1%
**
**
ns
O
KPflC
4
ns
0
5%
ns
5
KPC
ia
Densitometry (HIF1α/Actin)
5
ns
ia
KPflC 5%
F
HIF1α
0.
or m N
G
ns
ns
10
2
3
Day
15
2
0
O
2
100
ia
1
200
ox
0
300
KPC
20
2
0
400
25
O
500
500
1%
1000
Annexin V positive cells (%)
Growth rate (Cells/day)
Cells/FOV (#)
0.1% O2
1500
ns
600
Normoxia
2000
1%
or m N
KPC
2500
0
ox ia
3
5%
2
Day
ox ia
1
5
5%
0
0
10
or m
0
100
ns
15
Densitometry (Pimonidazole/Actin)
Actin
200
KPflC ns
20
or m
500
300
ns
25
2
1000
400
O
0.1% O2
500
0. 1%
Normoxia
1500
E
ns
600
ox ia
2000
Cells/FOV (#)
KP
KP
p53
KPflC
2500
Growth rate (cells/day)
D
C
Cfl
C
Annexin V positive cells (%)
N
H
or
yp
m ox
ox
ia
ia
0
Actin
*** *** 2
O
1%
2
2
O
O
1%
0.
ia ox
m
N or
5%
2
O
2
2
O
1%
1%
0.
O
0.0
2
O
**
***
0.2
1%
2
0.
2
O
O
1%
2
ia ox
5%
m
O
N or
1%
2
O
0.
O
2
1%
m or
5%
ia ox
2
O
1%
2
2
O
1%
0.
O 0. 2 1% O N or m 2 ox ia 5% O
2
1%
ia
O
ox
5%
m
0.4
5%
1 0
or N
ns
*
0.6
ia
0
*
*
ns
2
ns
0.8
ox
ns
1
3
*
1.0
m
*
KP C fl
p70-S6K(Thr389) ns ns ns KPflC KPC
or
* ns
4
N
*
3 2
Akt(Thr308) ns ns ns KPC *
5
N
Densitometry (Akt(Ser473)/Akt)
KP C fl
K Densitometry (p70-S6K(Thr389)/p70-S6K)
Akt(Ser473) ns ns ns * KPC
5 4
J Densitometry (Akt(Thr308)/Akt)
I
Figure S1, related to Figure 1. The presence of hypoxia and the associated growth and molecular effects in the KPflC and KPC mouse models of PDAC. (A) Immunofluorescence staining of the GEM KPflC and KPC PDAC mouse models for Ki67 (red), DAPI (cyan) and pimonidazole (green). Scale bars: 50 µm. (B) Quantification of Ki67 positive nuclei as a fraction of total nuclei, in pimonidazole negative (normoxic) and positive (hypoxic) regions (n=5 tumours/mouse model). Mean ± SEM. A onesample t-test was performed on normalized data. (C) Representative western blot of p53 loss or gainof-function mutant expression in the KPflC and KPC primary PDAC cell lines respectively, after 48 hours of culture in normoxia (n=5). P values are from a Student’s two-tailed parametric t-test. (D)
Assessment of growth rate in normoxic and hypoxic (0.1% oxygen) conditions in the KPflC and KPC cell lines, over a 3 day time course (n=5). Mean ± SEM. (E) Quantification of Annexin V positive KPflC and KPC cells by FACS analysis, after incubation for 48 hours in normoxia or hypoxia (5%, 1% and 0.1% oxygen). Mean ± SEM. P values are from a Student’s two-tailed parametric t-test. (F) Densitometry of HIF1α western blot in Figure 1D (n=5). Mean ± SEM. A one-sample t-test was performed on normalized data. (G) Representative western blot of pimonidazole adducts in the KPflC and KPC primary PDAC cell lines after 48 hours of culture in normoxia and 2 hours treatment with pimonidazole (236 µM, n=5). (H) Densitometry of pimonidazole western blot (n=5). Mean ± SEM. (IK) Densitometry of Akt(Ser473), Akt(Thr308) and p70-S6K(Thr389) western blots in Figure 1D (n=5). Mean ± SEM. A one-sample t-test was performed on normalized data. *p?,.">#'N&++@ DEFG/C",H
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Figure S2, related to Figure 2. Assessment of hypoxia within organotypic matrices, as well as drug
!"#$%&'()*'%&+,-&.'-/'!"#$%&')0 penetrance and response. Representative images of (A) laurdan (green; 2.5 µM) and (B) doxorubicin
(red; 1 µM) treated organotypic matrices with invading KPC cells (14 day invasion), after 2 hours with the compound (n=3). SHG of the organotypic collagen matrix is shown as grey on the image. Scale bars: 50 µm. (C,D) Representative immunohistochemistry (IHC) images of lactate dehydrogenase and GLUT1 stained organotypic matrices with invading KPC cells (14 day invasion; n=4). Scale bars: 50 µm, scale bars (inset): 10 µm. (E) Representative western blot of the KPC primary PDAC cell line, incubated for 48 hours in normoxia or hypoxia (5%, 1% or 0.1% oxygen; n=5). (F) Densitometry of lactate dehydrogenase, GLUT1 and NDRG1(Thr346) western blots (n=5). Mean ± SEM. (G) IC50 curve of KPC cells treated with the HAP TH-302 in both normoxic (black lines) and hypoxic (0.1%
oxygen, red lines) conditions (n=3). An extra sum-of-squares F test was performed between the best-fit parameters of each curve. (H,I) Representative images of invading KPC cells stained with NDRG1(Thr346) or γH2AX, with quantification of the percentage of positively stained KPC cells on the surface (“Normoxia”) or invading into (“Hypoxia”) the organotypic matrices (n=4). Scale bars: 50 µm, scale bars (insets): 10 µm. Mean ± SEM. A one-sample t-test was performed on all normalized data. *p
