Ruscettiet al.,Science 362 , 1416–1422 (2018) 21 December 2018 5of7
D
B
C
E
Vehicle (Ren) Combo (Ren) Vehicle (p65) Combo (p65)
Vehicle lle Combo Vehicle Combo
Ren p65
***
n.s.
Change in tumor cells (%)
0
-10
-20
-30
-40
-50
10
e e
0
0
n.s. (^0) ***
NK1.1 (%)
- 0
5
10
15
0
5
10
15
20
Vehicle eeeCombo o Vehicle ee leCombo
Ren p65
Vehicle eCombo o Vehicle leCombo
Ren p65
Survival (%)
0
50
100
10 15 20 25 30
Time (d)
Isotype
Combo + Isotype
Combo + NK1.1
Combo + TNF-
P = 0.0057
P = 0.0173
Relative Expression ( AU)
**
0
1
2
3
4
5
0
5
10
15
20
25
0
1
2
3
4
0
1
2
3
4
0
5
10
15
20
0
5
10
15
20
25 * **
0
2
4
6
0
1
2
3 * **
Combo p65
Vehicle Ren
Vehicle
Combo
A
CD107a
- cells) (%)
(of NK1.1
F
Change in tumor cells (%) ***
Vehicle Combo Combo
Scramble Tnfa
n.s.
-30
-20
-10
0
10
Vehicle eCombo o Vehicle leCombo o^ Vehicle C
Isotype
5
0
10
15
NK1.1 - (%)
TNF-
n.s.
Vehicle eCombo o Vehicle leCombo
Isotype TNF-
0
5
10
15
25
CD107a
- cells) (%)
(of NK1.1
20
p65 Il15 Ccl2 Tnfa
Cxcl1 Icam1 Il6 Ccl5
αα
α
Fig. 3. NF-kB-mediated SASP and TNF-asecretion is required for
treatment-induced NK cell activity.(A) Quantitative reverse tran-
scription polymerase chain reaction analysis of SASP gene expression in
KP tumor cells transduced with indicated shRNAs after treatment
with vehicle or trametinib (25 nM) and palbociclib (500 nM) for 8 days.
Mean of two biological replicates associated with three technical replicates
is plotted. AU, arbitrary units. (B) Representative images of KP tumor
cells with indicated shRNAs (green) pretreated as in (A) and cocultured
with primary murine splenic NK cells (red) at a 20:1 E:T ratio for 20 hours
in the presence or absence of indicated drugs (scale bar, 25mm).
Quantification of NK cell cytotoxicity (by live cell imaging) is shown on
the right. Change in tumor cells is normalized to control wells lacking
NK cells. Mean of three biological replicates is plotted. (C) Flow
cytometry analysis of total and CD107a+degranulating NK cells within
the CD45+population in the lungs after 1-week treatment of mice
transplanted with KP tumor cells containing controlRenilla(Ren)orp65
shRNAs with vehicle or combined trametinib (1 mg/kg body weight)
and palbociclib (100 mg/kg body weight) (n≥3 mice per group).
(D) Kaplan-Meier survival curve of KP transplant mice treated with
vehicle or combined trametinib (1 mg/kg body weight) and palbociclib
(100 mg/kg body weight) and either an isotype control (HRPN), NK1.1
(PK136), or TNF-a(XT3.11) targeting antibody (n≥6 per group) (log-rank
test). (E) Flow cytometry analysis as in (C) after 1-week treatment of
KP transplant mice with vehicle or combined trametinib (1 mg/kg body
weight) and palbociclib (100 mg/kg body weight) and either an isotype
control (HRPN) or TNF-a(XT3.11) targeting antibody (n= 5 per group).
(F) Quantification of NK cell cytotoxicity (by live cell imaging) toward
KP tumor cells with indicated shRNAs pretreated as in (B) and cocultured
with primary murine splenic NK cells at a 20:1 E:T ratio for 20hours
in the presence or absence of indicated drugs. (A to C and E and F)
One-way ANOVA. Error bars, mean ± SEM. P< 0.01, *P< 0.001,
**P< 0.0001.
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