Science - USA (2018-12-21)

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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