AU87non-stem U87stem-like

U251non-stem U251stem-like

0

200

400

600

800

MF

I/FO

V:T

LR9

**

0200400

1,000

1,400

MF

I/FO

V:p

ST

AT

3

600800

1,200

***

0

200

400

800

1,000

600

*** **

0

500

1,000

2,000

2,500

MF

I/FO

V:T

LR9

1,500

pSTAT3TLR9

Hoechst

non-stemstem-like

non-stemstem-like

B C

non-

stem

stem

-like

25

0

20

15

10

5

Tlr9

mR

NA

P = 0.06

GL261 DBT

- TLR9 (180 kDa)

- -Actin - TLR9 (115 kDa)

Mr [kDa]

100 -

150 -250 -

D

- pStat3 (91 kDa)

- Stat3 (91 kDa)

- MSI-1 (39 kDa)

- SOX2 (40 kDa)

- Actin (42 kDa)

GL261 DBT

- Nestin (177 kDa)

non-

stem

stem

-like

non-

stem

stem

-like

non-

stem

stem

-like

non-

stem

stem

-like

Figure S1, Herrmann, A., Cherryholmes, G. et al.

Figure S1. Increased TLR9 expression in induced glioma spheres.

(A) TLR9 and activated STAT3 are significantly increased in glioma stem cell enriched culture using sphere formation assay. TLR9 and pSTAT3 expression in cells grown in non-cancer stem cell conditions, compared to sphere forming culture condition (left panels). Scale bar 20 μm. Quantification of mean fluorescent intensity (MFI) of TLR9 and pSTAT3, respectively (right panels). (B) Quantitative RT-PCR to analyze expression of Tlr9 mRNA in murine GL261 glioma cells, in sphere stem-cell culture condition compared to adherent non-stem cell culture condition. (C) Expression of TLR9 protein and (D) CSC-associated factors by Western blotting in murine glioma cells cultured in sphere stem-cell culture condition, compared to adherent non-stem cell culture condition.

MF

I/FO

V:p

ST

AT

3

F

- TLR9

- TLR9

GL261

DBT

Input

IgG Sta

t3Ig

G Sta

t3

stem-like non-stem

ChIP:

TLR

9 m

RN

A

ST

AT

3 m

RN

A

scr-shRNASTAT3-shRNA

0

0.2

0.4

0.6

0.8

1.0

1.2

0

0.2

0.4

0.6

0.8

1.0

1.2

GP = 0.01 P = 0.03

A

**

**

none, n = 4 CpG-ODN1668, n = 4

0 2 4 6

B

050

100150200250

No.

of s

pher

es 300350400

80

5

10

15

20

25

No.

of s

pher

es, Ø

1-2

mm

30

35

none CpG

P = 0.007

0

2

3

4

Sox

2 m

RN

A

5

1

0

4

6

8

Nes

tin m

RN

A

10

2

none CpG

P = 0.03 P = 0.03

Days

0

50

100

150

200

250

No.

of s

pher

es

0 2 4 6

Days

300

350

DBTnt-shRNA, n = 9

******

******

DBTTlr9-shRNA (1), n = 9DBTTlr9-shRNA (2), n = 9

DC

Tlr9-shRNA nt-shRNA

0

2.0

1.0

0.5

Mus

ashi

-1 m

RN

A

1.5

P = 0.051

0

2.0

1.0

0.5Sox

2 m

RN

A 1.5

P = 0.013

0

2.5

1.0

0.5Nes

tin m

RN

A

1.5

P = 0.013

2.0

nt-shRNA, n = 24

TLR9shRNA1, n = 24

TLR9shRNA2, n = 24

– 4

– 3

– 2

– 1

0 U251

non-

resp

ondi

ng [l

og]

number of cells

5004003002001000

E

Figure S2, Herrmann, A., Cherryholmes, G. et al.

BLANK2nd ab ctrlDBT-nt-RNADBT-Tlr9shRNA#1

TLR9

DBT-Tlr9shRNA#2

% o

f Max

.

Figure S2. TLR9 and STAT3 form a feed-forward loop in GSCs.

(A) Sphere formation assay to phenocopy cancer stem cell-like cells upon TLR9 signaling activation in DBT glioma spheres triggered with CpG-ODN. SD and significance are shown: *P ≤ 0.05; **P ≤ 0.01. Quantification of sphere diameter at day 8 of sphere formation is shown. SD shown and significance are indicated. (B) Quantitative RT-PCR of Sox2 and Nestin mRNA levels in DBT spheres with or without CpG-ODN treatments. SD and significance are shown. (C) Effect of TLR9 signaling on human GSC-like cells using limiting dilution assays. U251 glioma spheres stably transduced with indicated TLR9shRNAs were grown in stem cell-enriching (sphere-forming) culture conditions, followed by a limiting dilution assay to assess the frequency of GSCs. (D) Tlr9shRNA was introduced into DBT glioma cells and TLR9 protein knockdown efficacy was determined by flow cytometry (left panel). Sphere formation assay of murine DBT glioma cells expressing shRNA against Tlr9 in sphere stem-cell culture condition. SD and significance shown: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 (right panel). (E) Analysis of mRNA expression levels of stem cell-associated factors in glioma such as Msi-1, Sox2, and Nestin by RT-PCR upon introduction of Tlr9 shRNA; SD and significance are shown. (F) Chromatin immunoprecipitation (ChIP) of Stat3 binding to the Tlr9 promoter in GL261 and DBT murine glioma cells cultured in sphere stem-cell culture condition, compared to adherent non-stem cell culture condition. (G) Expression of STAT3 mRNA and TLR9 mRNA levels upon STAT3 knockdown in human glioma cells grown in sphere culture condition analyzed by quantitative RT-PCR. SD and significance are shown.

Figure S2 cont’d, Herrmann, A., Cherryholmes, G. et al.

A

Days

DBT

0

250

1,000

1,500

Tum

or v

olum

e [m

m3]

500

750

1,250

8 10 12 14*

* *

vehicle, n = 4

CpG-Stat3siRNA , n = 4CpG-lucsiRNA, n = 4

NeovasculatureECMAnnexin V

none CpG-lucsiRNA CpG-Stat3siRNAC

Figure S3, Herrmann, A., Cherryholmes, G. et al.

none CpG-Stat3siRNAFITC

1.17 86.30

SS

C-H

FL-1A:: FITC

none CpG-Stat3siRNAFITC

B

DDBT GL261

none noneCpG-lucsiRNA CpG-lucsiRNACpG-Stat3siRNA CpG-Stat3siRNA

HoechstpStat3

Hoechstcl.Casp.3

CD31Hoechst

E

0

25Per

cent

Sur

viva

l

20 40 80

Days

0

50

75

100

125

Vehicle, n = 8CpG, n = 8CpG-Stat3siRNA, n = 8

60 100

CpG-Stat3siRNAFITC

Hoechst33342

Figure S3. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits tumor growth.

(A) Internalization of CpG-Stat3siRNA-FITC into glioma tumors engrafted subcutaneously visualized by in vivo multi-photon imaging (upper panel, scale bar 50 m) and quantified by flow cytometry (lower panel). (B) Tumor growth of DBT murine glioma in immunocompromised mice treated locally as indicated. SD and significance shown (*P ≤ 0.05). (C) Tumor vasculature and induction of cell apoptosis in DBT murine glioma treated with CpG-Stat3siRNA, CpG-lucsiRNA, or left untreated was analyzed by in vivo multi-photon imaging. Scale bar 100 m. (D) Antitumor effects on glioma treated with CpG-Stat3siRNA, CpG-lucsiRNA, or vehicle control was analyzed in two rodent glioma models DBT and GL261, respectively, showing decreased Stat3 activation (upper panel), diminished tumor CD31+ vasculature (middle panel) and cleaved caspase 3+ induced tumor cell apoptosis (lower panel). Scale bar 100 m. (E) Improved survival rate as a result of locally administered CpG-Stat3siRNA was acquired in a syngenic model by treating mice bearing brain tumors twice a week for two weeks. CpG1668 and vehicle treatment were included as controls. Significance shown (**P ≤ 0.01).

Figure S3 cont’d, Herrmann, A., Cherryholmes, G. et al.

AS

SC

-H

0.0 91.7 93.7 95.6

0’ 30’ 60’ 120’

DBTsphere

CpG-Stat3siRNA-FITC0

200

400

600

800

1,000

No.

of s

pher

es

none, n = 6

0 2Days

CpG-lucsiRNA, n = 6CpG-Stat3siRNA, n = 6

DBT

64

B

**

Tlr9

mR

NA

Sox

2 m

RN

A

SS

EA

1 m

RN

A

none, n = 3CpG-lucsiRNA, n = 3CpG-Stat3siRNA, n = 3

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0

1.0

1.5

2.0

2.5

0.5

3.0

00.20.40.60.81.01.2

1.61.4

Sta

t3 m

RN

A

0

1.0

2.0

3.0

4.0

C

none CpG-lucsiRNA CpG-Stat3siRNA

pStat3NestinHoechst

NestinTLR9

Hoechst

D

Figure S4, Herrmann, A., Cherryholmes, G. et al.

P=0.024 P=0.008 P=0.008 P=0.001

Figure S4. Stat3 silencing by local CpG-Stat3siRNA delivery inhibits GSCs.

(A) Internalization of CpG-Stat3siRNA-FITC into DBT glioma sphere culture determined by flow cytometry. (B) Sphere formation capacity of DBT murine glioma cells upon CpG-Stat3siRNA treatment compared to controls as indicated. SD and significance shown (*P ≤ 0.05; **P ≤ 0.01). (C) Quantitative RT-PCR analyses showing mRNA levels of stem cell associated factors in glioma tumors grown subcutaneously and treated as indicated. SD and P values shown. (D) Confocal microscopic analyses showing protein levels of pStat3 and TLR9 in Nestin+ tumor areas from tumors treated with indicated CpG-siRNAs. Scale bar 100 m.

Figure S4 cont’d, Herrmann, A., Cherryholmes, G. et al.

none CpGlucsiRNACpGscrRNA

BLU

106

Figure S5, Herrmann, A., Cherryholmes, G. et al.

2

4

6

8

0

3

5

7

1

none

CpGlucsiRNA

CpGscrRNA

P = 0.07

P = 0.25

Figure S5. Targeting Brain tumors systemically with CpG-siRNA reaches the tumor site.

Xenogen imaging of GL261luc+ brain tumors orthotopically engrafted in C57BL/6 mice showing decreasing bioluminescent signals upon treatment with CpG-lucsiRNA as well as indicated controls (left panel). Luciferase signals upon indicated treatments was acquired by non-invasive xenogen imgaging and quantified. P values shown (right panel).