Nature - 15.08.2019

Letter reSeArCH

To investigate whether the protection against phagocytosis con-

ferred by CD24 could be recapitulated in vivo, GFP-luciferase+

MCF-7 wild-type or MCF-7(ΔCD24) cells were engrafted into NOD.

Cg-PrkdcSCIDIl2rgtm1Wjl/SzJ (NSG) mice^22. Three weeks after engraft-

ment, we found that CD24-deficient tumours exhibited augmented lev-

els of in vivo phagocytosis by infiltrating TAMs as compared to wild-type

tumours, and TAMs that infiltrated the CD24-deficient tumours were

also of a more inflammatory phenotype (Extended Data Figs. 7, 8a, b).

Over weeks, we observed a robust reduction in the growth of ΔCD24

tumours as compared to wild-type tumours (Fig. 4a, b). Notably, the

sublines assessed had no measurable cell-autonomous differences in

proliferation in vitro (Extended Data Fig. 8c). After 35 days of growth,

the polyclonal ΔCD24 tumours had become largely CD24+, which

is consistent with the selection against CD24– cells by TAMs and the

0

103

104

0 103104

IgG control

Phagocytosis

1.36 %^0

103

104

0 103 104

Anti-CD24

Phagocytosis

18.6 %

0

103

104

0 103 104

Anti-CD47

Phagocytosis

6.52 %^0

103

104

0 103 104

Anti-CD24, anti-CD47

Phagocytosis

30.8 %

MCF-7 (FITC) MCF-7 (FITC)

MCF-7 (FITC) MCF-7 (FITC)

CD11b (A647)

CD11b (A647)

CD11b (A647)

CD11b (A647)

a b CD24+ cancer cell lines CD24–

0

0.5

1.0

Panc1 U-87 MG

0

0.5

1.0

APL1

IgG control

Anti-CD24

Anti-CD47

Anti-CD24,

anti-CD47

NS****

NS

****

************

****

**** ****

NS

0

0.5

1.0

Normalized phagocytosis 0

0.5

1.0

MCF-7

****

*********

c

**

Siglec-10 KO

0

2

4

6

Anti-CD24 response

(fold change)

Cas9 control

0 500 1,000 1,500 2,000

0

2

4

6

8

8

14

APL1

Panc1

U-87 MG

MCF7

H82

R^2 = 0.297

Pearson’s r = 0.840 (*)

CD24 expression (MFI)

Anti-CD24 response

(fold change)

d Ovarian

cancer

ascites

Donor-derived

macrophages

Anti-CD47

PhagocytosisFACS

Primary ovarian cancer

0

0.5

1.0

Normalized phagocytosis

****

****

***

**

IgG control

Anti-CD24

Anti-CD47

Anti-CD24,

Anti-CD47

e f

Fig. 3 | Treatment with anti-CD24 mAb promotes phagocytic clearance

of human cancer cells. a, Representative flow-cytometry plots depicting

the phagocytosis of MCF-7 cells treated with anti-CD24 mAb, CD47 mAb

or dual treatment, compared with the IgG control. Plots are representative

of five donors. FITC, fluorescein isothiocyanate. b, Phagocytosis of MCF-7

(n = 5 donors), APL1 (n = 8 donors) and Panc1 (n = 8 donors) cell lines

(left) and of the U-87 GM cell line (n = 3 donors; solid bars) (right) in the

presence of anti-CD24 mAb, anti-CD47 mAb or dual treatment, compared

with IgG control (one-way ANOVA with multiple comparisons correction;

MCF-7 F(3,16) = 145.6, APL1 F(3,28) = 144.7, Panc1 F(3,28) = 220.7, U-87

MG F(3,8) = 200.4; NS, not significant; **P = 0.0092, ***P = 0.0001,

****P < 0.0001). c, Response to anti-CD24 mAb treatment by Siglec-10

knockout compared with Cas9 control macrophages (n = 4 donors,

connecting lines indicate matched donor. Paired, one-tailed Student’s

t-test, **P = 0.0089). d, Pearson correlation between CD24 expression

(x axis) and mean anti-CD24 mAb response (y axi s) (n values are the same

as those listed in b and Extended Data Fig. 5c. Linear regression is shown.

Data are mean ± s.e.m. *P = 0.0375). MFI, median fluorescence intensity.

e, Workflow to measure the phagocytosis of primary ovarian cancer.

f, Phagocytosis of primary ovarian cancer cells treated with anti-CD24

mAb, anti-CD47 mAb or dual treatment, compared with IgG control

(n = 10 macrophage donors, n = 1 primary ovarian cancer ascites donor)

(one-way ANOVA with multiple comparisons correction, F(2.110, 18.99)

= 121.5, **P = 0.0078, ***P = 0.0006, ****P < 0.0001). Data are

mean ± s.e.m.

b

0

0.5

1.0

1.5

2.0

5.0

Day 7142821 714 2821 7142821 7142821

To tal ux (×10

11 photons s

–1)

WT ΔCD24

NS *** * WT, vehicle

ΔCD24, vehicle

ΔCD24, TAM depletion

WT, TAM depletion

Radiance

(photons s

–1 cm

–2) ×10

9

5

4

3

2

1

IgG controlAnti-CD24

2.5

2.0

1.5

1.0

0.5

Radiance

(photons s

–1 cm

–2) ×10

9

WT ΔCD24

Total ux

(×10

10 photons s

–1)

****

IgG control Anti-CD24

57912141628579 12 14 16 28

0

5

10

15

20

P = 0.0197

ΔCD24

WT

020406080

0

50

100

Days

Survival (%)

a

c d e

Day

Fig. 4 | CD24 protects cancer cells from macrophage attack in vivo.
a, Representative bioluminescence images of day-21 tumours in mice
engrafted with MCF-7 wild-type and MCF-7(ΔCD24) tumours. Image
representative of two independent experimental cohorts. b, Burden of
MCF-7 wild-type compared with MCF-7(ΔCD24) tumours in mice with
TAMs (vehicle) or in TAM-depleted mice (treated with anti-CSF1R) as
measured by bioluminescence (WT vehicle, n = 14; WT TAM depletion,
n = 5; ΔCD24 vehicle, n = 13; ΔCD24 TAM depletion, n = 5. Two-
way ANOVA with multiple comparisons correction, tumour genotype
F(3,33) = 11.75, *P = 0.0296, ***P = 0.0009). c, Survival analysis of

vehicle-treated mice in b; P value computed by a log-rank (Mantel–Cox)

test (WT, n = 5; ΔCD24, n = 5). d, Representative bioluminescence image

of day-33 tumours in mice with MCF-7 tumours treated with either IgG

control or anti-CD24 mAb (image representative of two experimental

cohorts). Data are mean ± s.e.m. e, Burden of MCF-7 wild-type tumours

treated with IgG control (blue) and anti-CD24 mAb (red) as measured by

bioluminescence (IgG, n = 10; anti-CD24 mAb, n = 10. The days on which

the treatments were administered are indicated by arrows. Data from

two experimental cohorts. Two-way ANOVA with multiple-comparisons

correction, tumour treatment F(1,126) = 5.679, ****P < 0.0001).

15 AUGUSt 2019 | VOL 572 | NAtUre | 395