Science - USA (2021-10-29)

revealed that coordinated induction of growth

arrest and immunosurveillance is a distinctive

feature of p21 (fig. S17 and table S8).

Oncogene-induced p21 triggers

immunosurveillance

We sought to critically test the physiological

relevance of p21-dependent immunosurveil-

lance in a cancer-related context. To this end,

we adapted our transgenic approach for co-

induction of Tom and p21 in hepatocytes by

replacingp21withKRASG12V(Fig. 5A), an

oncoprotein that can induce p21 via mitogenic

stress ( 4 ). Indeed, ~25% of d4 Tom+KRASG12V

hepatocytes had elevated levels of p21 (Fig. 5B

and fig. S18A). These hepatocytes attracted

macrophages, whereas those that failed to

induce p21 did not (Fig. 5C). Use of a newly

generatedp21conditional knockout strain con-

clusively demonstrated that d4 Tom+KRASG12V

hepatocytes recruit macrophages in a p21-

dependent manner (Fig. 5, A to C, and fig.

S18B). Furthermore, d4 Tom+KRASG12Vhepa-

tocytes in whichRbwas conditionally knocked

out retained p21 induction but nevertheless

failed to attract macrophages, validating cell

culture experiments indicating that p21 places

cells under immunosurveillance in an Rb-

dependent fashion (Fig. 5, A to C, and fig. S18B).

Hepatocytes from d4 Tom+KRASG12Vmice

had a PASP, which they lost with condition-

al inactivation ofp21(Fig.6A).ThePASP

includedCxcl14, explaining why d4 Tom+

KRASG12Vhepatocytes attract macrophages

and their counterparts lacking p21 do not.

Tom+hepatocyte numbers remained largely

unchanged at d4, d12, and d28 after induc-

tion when KRASG12Vwas absent (Fig. 6B), but

progressively declined because of cell death

when KRASG12Vwas coexpressed (Fig. 6, B and

C). However, no such decline occurred when

p21was inactivated upon KRASG12Vinduction.

This was not due to compensatory cell prolif-

eration becausep21inactivation had no im-

pact on the mitotic index of Tom+KRASG12V

hepatocytes (fig. S18, C and D). Consistent

with p21-dependent cell elimination, Tom+

KRASG12Vhepatocytes with high p21 levels

gradually decreased from d4 to d28 (Fig. 5B).

At d12, Tom+KRASG12Vhepatocytes were

surrounded by M1 macrophages and T lym-

phocytes, whereas their d4 counterparts were

not (Fig. 6, D and E). Thus, p21-mediated mac-

rophage recruitment represents an essential

first step toward cell elimination upon KRASG12V

induction, which then appears to be executed

by subsequently recruited T lymphocytes. This

conclusion is supported by the earlier observa-

tion that clearance of hepatocytes expressing

oncogenic NRAS depends on monocytes and

macrophages and on CD4 T cells ( 4 ).

Regardless of whetherp21was intact or in-

activated, Tom+KRASG12Vhepatocytes hard-

ly proliferated and showed signs of cellular

Sturmlechneret al.,Science 374 , eabb3420 (2021) 29 October 2021 5 of 15

***

Tom

+

hepatocytes (%)

ns

ns

A

iNOS

Tom

DNA

Tom

+

hepatocytes joined

by

1 B220

+

cells (%)

***

**

ns

Tom

+

hepatocytes joined

by

1 CD3

+

cells (%)

CD3

Tom

DNA

ns ns

***

B

n=5n=5n=5

n=5

n=4n=4

n=4

n=4n=4

n=4

n=4n=4

n=5n=5n=5

n=5

n=4

n=4

***

ns

n=5

n=5

n=5

n=4

Tom

+

hepatocytes joined

by

1 iNOS

+

cells (%)

Dying Tom

+

p21-OE hepatocytes

joined by

1 iNOS

+

cells (%)

n=4

Dying Tom

+

hepatocytes (%)

D ***

ns

ns

Tom

DNA n=4

n=4

n=5n=5

n=5

n=5

C

E F

B220

Tom

DNA

Ai14

Ai14;L-p21

d2d4 d8

0

10

20

30

50

40

60

0

5

10

15

0

10

20

30

0

10

20

40

30

0

10

20

50

30

40

0

25

50

75

100

Fig. 4. Hepatocytes under surveillance die upon macrophage differentiation and lymphocyte
recruitment.(A) Representative image and quantification of Tom+hepatocytes associated with≥1 B220+
cells (arrowhead). (B) Representative image and quantification of Tom+hepatocytes associated
with≥1 CD3e+cells (arrowhead). (C) Proportion of Tom+and healthy (not dying) hepatocytes.
(D) Representative image and quantification of dying Tom+hepatocytes. (E) Representative image
and quantification of Tom+hepatocytes associated with≥1 iNOS+cells (arrowhead). (F) Quantitation
of dying p21-OE Tom+hepatocytes associated with≥1 iNOS+cells. Scale bars: 10mm [(A), (B), (D),
and (E)]. Mice used were on a C57BL/6×129Sv mixed genetic background. Data represent means ± SEM.
Experiments in the above panels were performed once. ns, not significant. **P< 0.01; ***P< 0.001.
One-way ANOVA with SidakÕscorrection[(A)to(E)].

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