Science - USA (2021-10-29)

(fig. S8A). In a second migration assay, mac-
rophage numbers selectively increased in the
peritoneal lavage 4 days after intraperitoneal
injection of CM-NS, but not after injection of
CM-NS fromp21-orRb-depleted IR MEFs (fig.

S8, B to F). The PASP also stimulated cell move-

ment in a standard scratch assay on cultured

MEFs(fig.S8,GandH),indicatingthatits

promigratory properties extend beyond macro-

phages. Nuclear factorkB (NF-kB) p65 (RELA)

appeared to have no role in establishing the

PASP or its macrophage-attractant properties

(fig. S9 and table S7).

To determine whether the PASP requires an

actual senescence-inducing stressor or merely

elevated p21 levels, we transduced MEFs with

a lentivirus harboringp21-Myc-Flag(fig. S10A).

p21-overexpressing (p21-OE) MEFs were sub-

ject to growth arrest, initially without elevated

p16and SA–b-galactosidase (b-Gal) activity (d4),

and later with these senescence markers (d10)

(fig.S10,BtoD).Atd4,p21-OEMEFsup-

regulated 295 SFs, 227 of which were also up-

regulated in d4 IR MEFs, indicating that p21

induction is sufficient to yield a PASP (Fig. 2F

and table S8). SMAD2, SMAD3, STAT1, and

STAT6 coimmunoprecipitated Rb from the

chromatin fraction of d2 p21-OE MEFs (fig.

S10E), further supporting the contention that

p21-induced, hypophosphorylated Rb interacts

with STAT and SMAD TFs at select gene pro-

moters to establish the PASP. PASP factors of

d4 p21-OE MEFs were largely preserved in d10

p21-OE MEFs (Fig. 2F and table S8). Thus, the

PASP becomes an integral part of the SASP as

cells senesce.

p21 enforces immunosurveillance through the

chemokine CXCL14

Functional annotation analysis on the PASP of

d4 p21-OE MEFs suggested that it has biologi-

cal properties similar to those of the PASP of

d4 IR-MEFs (fig. S10F). Indeed, CM from d4

p21-OE MEFs stimulated fibroblast migration

in our scratch assay and macrophage migra-

tion in our transwell assay and increased local

macrophage numbers when intraperitoneally

injected in mice (fig. S10, G to N). MEF-derived

PASPs consistently includedCxcl14(Fig. 1B and

table S8), a member of the CXC chemokine

family that acts as a chemoattractant for var-

ious immune cell types ( 26 – 28 ). The addition

of CXCL14-neutralizing antibodies to CM har-

vested from d4 p21-OE MEFs ablated stimula-

tion of macrophage migration in our transwell

assay, whereas control immunoglobulin G (IgG)

did not (Fig. 3A and fig. S11A). Moreover, CM

fromCxcl14-depleted d4 p21-OE MEFs failed

to evoke macrophage migration (Fig. 3B and

fig. S11, B and C), further demonstrating that

CXCL14 is the key macrophage attractant

of the PASP. Complementary experiments in

human dermal fibroblasts (HDFs) and human

umbilical vein endothelial cells (HUVECs) sug-

gested that the PASP is a common feature of

p21 induction and that CXCL14 is a signature

PASP component (fig. S12).

To study the PASP phenomenon at the

organismal level, we established a transgenic

approach in mice that allows for Cre-inducible

coexpression of Myc-Flag–tagged p21 and the

fluorescent reporter protein tdTomato (Tom)

(Fig. 3C). We induced Myc-Flag–tagged p21

and Tom in ~10% of hepatocytes by tail vein

Sturmlechneret al.,Science 374 , eabb3420 (2021) 29 October 2021 3 of 15

shp21 vs

shScr

(235 )

shRbvs

shScr

(171 )

92 143 28

shp21 vs

shScr

(188 )

shRbvs

shScr

(161 )

54 134 27

84 common p21- and Rb -controlled PASP factors

d4

d0 d2

IR

shScr,

shp21 or shRb

d4 d10 d13

RNA-seq RNA-seq

d13

shScr,

d0 d2 d4 d10 shp21 or shRb

SFs

351

IR SFs

450 503

241

(^2999)
40
d10 vs d0 SASP
(503 )
d4 vs d0
SFs (450 )
d2 vs d0
SFs (351 )
41 70
134
Immune system
–Log 10 (FDR)
Functional clusters:
ECM
Proteolysis
Migration/adhesion
GF/hormone
Cell proliferation
Angiogenesis
Stress response
Cell death/survival
27
14
26
11
14
34
15
11
12
0 5 10
A B
C D
d0 d2 d4 d6 d10 d13
CM-NS CM-S
d15
Migrating macrophages (fold)
n=5n=
5
n=
5
n=3n=
7
n=5
n=5

ns
ns
ns
ns
CM
Peritoneal
immune cells
Chemo-
taxis
Count adherent
lymphocytesCount macrophages
shScrorshp21
or shRb
shScrorshp21
or shRb
IR
CM cycling cells
CM-NS +shScr
CM-NS +shp21
CM-NS +shRb
CM-S +shScr
CM-S +shp21
CM-S +shRb
CM cycling
cells
CM-NS
+shp21
CM-NS
+shScr
Adherent macrophages
SFs
pTSIN-p21-Myc-Flag(p21-OE)
or
pTSIN-EV (EV)
68 227 223
d4 p21-OE
vs d4 EV
(450 SFs)
(295 SFs)
(295 SFs)
d0 d4 d10
RNA-seq RNA-seq
58 237 80
d4 p21-OE
vs d4 EV
(317 SFs)
E F
d4 IR
vs d0
d10 p21-OE
vs d10 EV
0
1
2
3
4
5
Fig. 2. The p21-activated secretory phenotype places cells under immunosurveillance as they arrest.
(A) Timeline and Venn diagrams based on RNA-seq depicting significantly up-regulated SFs. (B) Timeline and Venn
diagrams comparing significantly down-regulated SFs uponp21orRbdepletion. (C) Functional annotation analyses
of 84 PASP factors indicated in (B) displaying overrepresented functional clusters. ECM, extracellular matrix;
GF, growth factor. (D) Schematic of CM production and transwell migration assay of peritoneal immune cells in the
presence of CM. (E) Representative images and quantitation of adherent macrophages in the bottom transwell
chamber. Scale bar: 100mm. (F) Venn diagrams depicting significantly up-regulated PASP factors. Data represent
means ± SEM. Experiments in the above panels were performed once except for (E), where data from three
experiments were pooled. ns, not significant. **P< 0.01. One-way ANOVA with SidakÕs correction (E).
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