Salmonella typhimurium suppresses tumor growth via the pro-inflammatory cytokine interleukin-1β

Jung-Eun Kim3,4*, Thuy Xuan Phan1*, Vu Hong Nguyen1*, Hong-Van Dinh-Vu1, Jin Hai Zheng1, Misun Yun1, Sung-Gyoo Park3, Yeongjin Hong2, Hyon E. Choy2, Michael Szardenings5, Won Hwang3,4, Jin-A Park4, SunHee Park4, Sin-Hyeog Im4,6#, and Jung-Joon Min1,2#

SUPPLEMENTARY DATA

Supplement 1.

A

B

SLΔppGpp

Day 0

± anti IL-1β

Day -1

Injection of anti-IL-1β

Blocking of IL-1β

SLΔppGpp

Day 0

Injection of rIL-1β every 2 days

Addition of recombinant IL-1β

± rIL-1β

Day 5

SL∆ppGpp SL∆ppGpp + IL-1β Ab SL∆ppGpp + rIL-1β

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

3 7 11

Bac

teria

(C

FU/m

g of

tiss

ue)

Day post infection

C

ND

ns

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

Sup

pres

sion

Re-

grow

th

Sup

pres

sion

Re-

grow

th

Sup

pres

sion

Re-

grow

th

Control SLΔppGpp MG1655

Bact

eria

l CFU

(C

FU/m

g tis

sue)

*

Figure S1. Number of bacteria in tumor tissue. (A) E. coli (MG1655; 5 × 107 CFU) or S. typhimurium (SLΔppGpp; 4.5 × 107 CFU) were injected into tumor-bearing mice, and the number of bacteria in the tumor tissues was counted during the suppression stage (3 dpi) and recurrence stage (when tumor sizes reach around 1200mm3). (B) Protocols for co-treatment with an IL-1β blocking antibody (left) or recombinant IL-1β (right), plus PBS or SLΔppGpp. (C) CT26 cells were transplanted into mice. Mice then received an intravenous (i.v.) SLΔppGpp (white bars). IL-1β depletion: mice received an i.v. injection of anti-IL-1β-specific antibody (IL-1β Ab) 1 day before SLΔppGpp treatment (Day -1). The antibody was then injected twice a week for 2 weeks (grey bars). Treatment with recombinant IL-1β (rIL-1β): mice received an intratumoral (i.t.) injection of rIL-1β on 5 dpi (black bars), followed by another i.t injection every other day up until 11 dpi. The number of bacteria in the tumor tissues was counted at 3, 7 and 11 dpi. Data represent the mean ± SD of three independent experiments. *P < 0.001.

Supplement 2.

A B Control SL∆ppGpp MG1655

FSC

CD

45 0.69 0.88 0.43

F4/80

CD

11b 0.20 0.11 0.097

Ly6C

CD68

MHCII C

D11

c 0.058 0.76 0.051

Ly6C

Ly6G

1.89 │ 35.2 1.21 │ 22.7 2.94 │ 14.7

3.85│ 3.85 3.70 │ 0 14.3 │ 4.76

Annexin V

7AA

D

SL∆ppGpp MG1655 Control

Imm

une cells Tum

or cells

23.3

35.6

0.48

1.84

32.4

17.6

36.4

13.7

24.2

13.7

30.9

15.0

C

Figure S2. Immune cell population in tumor at the re-growing stage. (A) Apoptosis was examined in tumor-resident cells at re-growing stage using Annexin V and 7AAD. Cells isolated from tumor (tumor volume > 1200 mm3 or at 15 dpi) were separated into tumor cells and infiltrating immune cells using magnetic bead method. The upper and lower panels show infiltrating immune cells and tumor cells, respectively. (B) The immune cell population at the re-growing stage was examined using multi-color flow cytometry. The indicated percentages represented hematopoietic cells (CD45+), neutrophils (CD11b+Gr1+), macrophages (CD11b+F4/80+), and dendritic cells (CD11c+MHCII+), respectively. (C) The population of M1/M2 macrophages was examined by measuring CD68 and Ly6C expression in CD11b+F4/80+ cells (M1: CD68int Ly6Cint, M2: CD68hi Ly6Chi). MDSCs within CD11b+ cell population were categorized as M-MDSC (monocytic MDSC, CD11b+Ly6ChiLy6G-) or PMN-MDSC (polymorphonuclear MDSC, CD11b+ Ly6Clow Ly6G+). Data are representative of two individual experiments, each with similar results.

Supplement 3.

Ly6C

CD68

Ly6C

Ly6G

13.2 │ 0 28.8 │ 0.11 24.2 │ 0.55

48.7 │ 7.69 41.8 │ 27.8 34.1 │ 27.5

Ly6C

CD68

Ly6C

Ly6G

15.6│ 3.15 23.1 │ 1.41 20.1 │ 1.36

70.3 │ 9.80 66.0 │ 8.93 76.2 │ 6.70

D E Control SL∆ppGpp MG1655 Control SL∆ppGpp MG1655

Control SL∆ppGpp MG1655

Gr1

CD

11b 0.49 1.90 1.09

CD

11c 1.50 2.03 2.44

MHCII

F4/80

CD

11b 0.38 0.73 0.69

0.39 0.73 0.48

Control SL∆ppGpp MG1655

Gr1

CD

11b 7.81 6.62 5.19

CD

11c 4.25 4.39 3.98

MHCII

F4/80

CD

11b 1.24 6.88 4.18

2.39 5.71 3.57

B C

Control SL∆ppGpp MG1655

×40

A

Figure S3. Colonization by SLΔppGpp leads to increased tumor infiltration by immune cells. (A) Neutrophils infiltrating into the tumor were examined by immunohistochemistry as described in the legend to Figure 2A. Brown signals indicate Ly-6G/Ly-6C+ cells. Sections were counterstained with hematoxylin. In addition to the experiments depicted in Figure 2B, the proportion of the immune cells in tumor-draining lymph nodes (B) and spleens (C) were analyzed by flow cytometry at 2dpi. The indicated percentages represent neutrophils (CD11b+Gr1+), macrophages (CD11b+F4/80+), and dendritic cells (CD11c+MHCII+), respectively. The population of M1/M2 macrophages was examined by measuring CD68 and Ly6C expression in CD11b+F4/80+ cells (M1: CD68int Ly6Cint, M2: CD68hi Ly6Chi). MDSCs within CD11b+ cell population were categorized as M-MDSC (monocytic MDSC, CD11b+ Ly6Chi Ly6G-) and PMN-MDSC (polymorphonuclear MDSC, CD11b+ Ly6Clow Ly6G+). M1/M2 and MDSC population in cells isolated from tumor-draining lymph nodes (D) and spleen (E) were analyzed. Data are representative of two individual experiments,each with similar results.

36.5

Supplement 4.

A Re-growth

Control SL∆ppGpp MG1655

40.5

8.76

55.1

11.6

24.4

20.2

12.1

36.4

31.8

33.6

11.5

31.7

10.1

60.3

2.41

30.4

9.27

7.31

32.5 57.8

15.9

23.6

10.5

34.1

2.92

69.0

2.01

28.0

3.00

21.8

Suppression

Control SL∆ppGpp MG1655

18.2

30.0

0.75

13.3

27.2

49.2

23.4

20.5

4.08

8.91

10.7

60.1

27.9

0.37

11.7

47.5

29.4

28.1

30.3 1.15

13.1

21.6

47.8

46.4

13.8

0.48

11.7

54.3

16.9

Hel

ios

CD

25

CTL

A4

ICO

S

CD

103

Hel

ios

CD

25

CTL

A4

ICO

S

CD

103

Foxp3

Foxp3

Foxp3

Foxp3

Foxp3

Foxp3

Foxp3

Foxp3

Foxp3

Foxp3

B

Figure S4. SLΔppGpp reduced the numbers and altered characteristics of Treg. Cells were stained with CD4 and Foxp3 to compare the proportion of Tregs among control, MG1655-, or SLΔppGpp-treated tumors. CD4+-gated cells are shown in each plot. The CD4+Foxp3+ population was examined by co-staining with Treg-related markers, including CD25, Helios, CTLA4, ICOS, and CD103. The numbers are indicative of each population in tumor tissue at the suppression (2 dpi) (A) and re-growing (at 15 dpi or when tumor volume exceeded 1200 mm3) (B) stages. Data are representative of two individual experiments, each with similar results.

020406080

100120140

IL-1

β/H

PRT

**

00.10.20.30.40.50.60.7

IL-6

/HPR

T

00.0050.01

0.0150.02

0.0250.03

0.0350.04

0.045

IFN

-γ/H

PRT

00.10.20.30.40.50.60.70.80.9

1

TGF-

β/H

PRT

* **

02468

1012

TNF-

α/H

PRT

* *

Pro-IL-1β

β-actin

Caspase 1

Re Sup Re Sup Re Sup

IL-1β

Control SL∆ppGpp MG1655

Supplement 5.

A

B

Figure S5. Cytokine profile in SLΔppGpp- or MG1655-colonized tumors during the suppression and re-growing stages. (A) Cytokine expression patterns at the suppression (2dpi) and re-growing (tumor volume > 1200 mm3 or at 15 dpi) stages were examined by RT-PCR after intravenous injection of PBS (control) or bacteria into tumor-bearing mice. Data are expressed as the mean ± SD of three separate experiments. (B) Cells isolated from the tumors at both stages were lysed, and the levels of caspase-1 and IL-1β were examined by western blotting. Results are representative of at least three individual experiments. *P < 0.05, **P < 0.005, and *** P < 0.001.

A

0

500

1000

1500

2000

2500

0 3 6 9 12 15 18

Tum

or v

olum

e (m

m3 )

Day post injection

Control

SLΔppGpp

SLΔppGpp + TNF-α Ab

SLΔppGpp + rTNF-α

SLΔppGpp

Day 0

± anti TNF-α

Day -1

Injection of anti-TNF-α

Blocking of IL-1β or TNF-α

SLΔppGpp

Day 0

Injection of rTNF-α every 2 days

Addition of recombinant IL-1β or TNF-α

± rTNF-α

Day 5

Supplementary 6

C B

Con

trol

SLΔ

ppG

pp

SLΔ

ppG

pp

+ TN

F-α

Ab

Day 0 Day 5 Day 16

SLΔ

ppG

pp

+ rT

NF-

α

*

*

Figure S6. Role of TNF-α in SLΔppGpp-mediated cancer therapy. (A) Protocols for co-treatment with a TNF-α blocking antibody (left) or TNF-α (right) plus PBS or SLΔppGpp. (B) CT26 cells were transplanted into mice. Mice then received an intravenous (i.v.) injection of PBS (black squares) or SLΔppGpp (black circles). To deplete TNF-α, mice were treated with an anti-TNF-α antibody (TNF-α Ab) 1 day before SLΔppGpp treatment (Day -1). The antibody was then injected twice a week for 2 weeks (open squares). For treatment with recombinant TNF-α (rTNF-α), mice received rTNF-α via an intra-tumoral (i.t.) injection on Day 5 post-injection (pi) of SLΔppGpp (open circles), followed by another i.t. injection every other day up until 11 dpi. (E) Photographs of representative animals in each group were taken before (0 dpi) and after treatment (5 and 16 dpi). Data represent the mean ± SD. Results from at least three individual experiments are shown. *P < 0.05.