Supplementary Figure 1: LCMV replicates in MOPC without affecting cell proliferation a: Quantification of Immunofluorescence of MOPC cells untreated or infected with LCMV (MOI 1) 72 hours after infection (n = 3/group) (related to Fig. 1c). ns, non-significant. b: Non-impaired clonogenic survival of MOPC cells after in vitro LCMV WE infection. Representative photograph of long-term colony formation following incubation of MOPC with LCMV WE of indicated MOI.

Supplementary Figure 1

d1 d2 d30

10

20

30

40

50

% D

AP

I+ A

rea relative VL4 expression

MOPC, LCMV

MOPC

MOI 1 MOI 0.5 MOI 0.25

MOI 0.125 MOI 0.0625 untreated

b a

ns

ns

ns

Supplementary Figure 2: B-Myb expression, and not IFN-I unresponsiveness, is one factor promoting arenavirus replication a: mRNA expression levels of Ifnar1 and Ifnar2 for the cancer types renal cell cancer (RCC; n=534), hepatocellular cancer (HCC; n=373), colorectal cancer (CRC; n=382), head and neck squamous cell cancer (HNSCC; n=522) and skin cutaneous melanomas (SKCM; n=471). Downloaded from the TCGA database by use of cBioPortal (www.cbioportal.org). Data are shown as mean and minimum-maximum whiskers. b: qRT-PCR of human Ifnar1 and Ifnar2 mRNA expression in Sw480, HeLa, HepG2, FADU cells and controls (murine mRNA, n=5/group). c: Number of viral plaques in Sw480 cell layer (n=6/group, left panel) and HeLa cell layer (n=5/group, right panel), which were

treated with different concentrations of human IFN4, murine IFN4 or murine IFN2 and infected with VSV (MOI 0.01) 24 hours earlier. d: qRT-PCR of murine Ifnar1 mRNA in MOPC cells, MC38 cells, B16F10 cells, LoxP-Tag tumors, MT/ret cells and control (human mRNA, n=5/group). e&f: Immunofluorescence of phospho-B-Myb (e, n=3/group) and qRT-PCR of B-Myb (=Mybl2) mRNA (f, n = 9-10/group) in MOPC, MC38 and LoxP-Tag tumors compared to the correlating healthy tissue (oropharyngeal epithelia, colon and liver). Scale bar, 200µm. g: qRT-PCR of viral host factors in MOPC tumors (n=5) and pharynx tissue (n=5). h: Infectious LCMV particles in supernatant of MCF7 cells, which were treated with control siRNA or two separate B-Myb siRNAs and then 24 hours later infected with LCMV (MOI 1) analyzed 24 hours after infection (n = 6/group). Data are shown as mean ± SEM and analyzed by unpaired Student`s t-test. ns, non significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Supplementary Figure 2

0 2000 4000 6000 800010000

SKCM

HNSCC

CRC

HCC

RCC

IFNAR1 (relative expression)

0 1000 2000 3000

SKCM

HNSCC

CRC

HCC

RCC

IFNAR2 (relative expression)

a b

0.01 0.1 1 10

control

FADU

HepG2

HeLa

SW480

IFNAR1(relative expression)

0.01 0.1 1 10

IFNAR2(relative expression)

10 100 1000100000

hIFN-

mIFN-

mIFN-

IFN-I (U/ml)

10 100 100010000

0

20

40

60

0

IFN-I (U/ml)

VS

V (

PF

U/w

ell)

Sw480 c

d

0.1 1 10 100 1000

Control

MT/ret

LoxP-TAg

B16F10

MC38

MOPC

Ifnar1(relative expression)

WT, oropharynx

MOPC

WT, colon

MC38

WT, liver

LoxP-Tag

Norm

al tissue

T

um

or

e DAPI p-bmyb f

0.01 0.1 1 10 100

MOPC tumor

WT, oropharynx

MC38 tumor

WT, colon

LoxP-TAg, liver tumor

WT, liver

M y bl2 m RNA (F o ld c hange)

**

***

*

32 641 2 4 8 16

mRNA (relative expression, log scale)

TipinMOPC tumor

Pharynx*

**

**

***

ns

*

*

*

****

**

*Arhgap23

Atp6ap2

Atp6v0b

Pkmyt1

Arcn1

Copb1

Copa

Mat2a

Eif3g

Eif3a

Signal transduction

Uncoating

Translation regulation

Vesicular transport

GPCR signaling

g

3

4

5

6

Control siRNAB-Myb siRNA 1B-Myb siRNA 2

* *

<3

LC

MV

(log

10P

FU

/ml)

h

Supplementary Figure 3: Intravenous infection leads to viral replication in metastasis a: Immunohistochemistry for LCMV-NP in flank and shoulder tumor (day 15) from C57BL/6 mice receiving simultaneously subcutaneously 5 x 10

5 MOPC cells in the flank and shoulder (day -3),

treated with or without 2x104 PFU LCMV given into the flank or intravenously on day 0 (n=4-5/group).

Scale bar, 200µm. b: Serum alanin-aminotransferase (ALT) levels of C57BL/6 (n=3) and adenovirally induced tumor bearing LoxP-TAg mice (11 month old) which were left untreated (n=4) or which were additionally treated with 2 x 10

6 PFU of LCMV-WE (n=5) measured 20 days after infection. Values

above line indicate pathological liver disease.

LCMV ( into Flank LCMV (i.v.) untreated

Should

er

tum

or

Fla

nk tu

mor

LC

MV

-NP

Supplementary Figure 3

a

b

0

500

1000

1500

LoxP-TAgcontrol

LoxP-TAgLCMV

C57BL/6LCMV

4/4 1/5 0/3

*

ALT

(U

/ml)

Supplementary Figure 4: Immune cell infiltrates are required for tumor regression Tumor diameter of MOPC-tumor bearing WT and Map3k14

aly/aly mice (day -3) treated with (n=8 WT;

n=6 Map3k14aly/aly

) or without (n=7 WT; n=6 Map3k14aly/aly

) 2x104 PFU LCMV peritumorally on day 0.

Data are shown as mean ± SEM and analyzed by unpaired Student`s t-test. ns, non significant; ***p < 0.001.

Supplementary Figure 4

Supplementary Figure 5: Tumor infiltrating Ly6C+ cells are indispensable for antitumoral LCMV therapy MOPC tumor bearing C57BL/6J mice were treated with 2x10

4 PFU LCMV peritumorally on day 0.

Depletion was performed by i.p. injection of either (a) αLy6C+G (clone RB6-8C5) 200µg or (b and c) αly6G (clone 1A8) 500µg starting on d-2 and repeated on d2 and d7. a&b: Representative dot blots of peripheral vein blood collected on d4 and stained for monocyte and granulocyte cell markers after preselection of CD11b positive cells. c: Tumor diameter in C57BL/6 mice injected with or without αLy6G (1A8) antibody (n=3-4/group). Data are shown as mean ± SEM and analyzed by unpaired Student`s t-test. ns, non-significant.

SSC

CD115

WT

, un

dep

lete

d

WT

, αly

6G

(c

lon

e 1

A8

)

MC

nG

nG

MC

nG MC nG MC

Ly6G

cou

nt

Ly6C

SSC

CD115

WT

, un

dep

lete

d

WT

, αL

y6

C+

G

(clo

ne

RB

6-8

C5

)

MC

nG

nG

MC

nG MC nG MC

Ly6G

cou

nt

Ly6C

0 5 1 0 1 5

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

< 0 .1

W T , L y 6 G

W Tn s

T im e ( d )

Tu

mo

r d

ia

me

te

r (c

m)

0 5 10 15

WT, Ly6G,LCMV

WT, LCMV ns

Time (d)

c

a

b

Supplementary Figure 5

Supplementary Figure 6: Impact of adaptive immune system and NK cells on LCMV-induced anti-tumoral immunity. a: Tumor diameter of MOPC-tumor bearing WT and Tcrab

–/– mice (day -3) treated with or without

2x104 PFU LCMV peritumorally on day 0 (n=5/group). b: Tumor diameter of MOPC-tumor bearing WT

and Jh–/–

mice (day -3) treated with or without 2x104 PFU LCMV peritumorally on day 0 (n=5/group).

c&d: Tumor diameter (c) and representative dot blot from peripherial vein blood collected at day 2 and stained for NK cell markers (d) of WT mice which were NK depleted by i.p. injection of 400µl NK1.1 antibody on day -3 and day -1 (n = 4/group). Data are shown as mean ± SEM and analyzed by unpaired Student`s t-test. ns, non-significant.

Supplementary Figure 6

a

0 5 10 15 20 25 30

WT, LCMVTcrb–/–, LCMV

Time (d)

0 5 10 15 20 25 300.0

0.2

0.4

0.6

0.8

1.0

1.2

<0.1

WTTcrb–/–

Time (d)

Tu

mor

dia

mete

r (c

m)

ns ns

b

0 5 10 15 20 25 30

Jh–/–, LCMVWT, LCMV

Time (d)

0 5 10 15 20 25 300.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

<0.1

WTJh–/–

Time (d)

Tu

mor

dia

mete

r (c

m)

ns ns

c

0 5 10 15 20

0.2

0.4

0.6

0.8

1.0

<0.1

WT, NK1.1

WT

Time (d)

Tum

or

dia

me

ter

(cm

)

0 5 1 0 1 5 2 0

W T, NK 1 .1 , L C M V

W T , LC M V

Time (d)

ns ns

NK

1.1

CD3

d

Supplementary Figure 7: Monocyte recruitment in human cancers correlates with IFN-I induction a: Heatmap of correlation analyses of 12 immunogenes mRNAs of primary samples from 34 oropharyngeal cancer patients. b: Linear regression between IRF7 and the human monocyte markers CD14 and CD16. c: Gene-set enrichment analysis (GSEA) of human IFN-I induced genes by usage of the Browne interferon-related gene-set. IFN-I-induced expression profile and genes ranked by extent of differential expression in TCGA melanoma, hepatocellular carcinoma, renal cancer, head and neck cancer and colorectal cancer dataset. NES, normalized enrichment score; FDR, false discovery rate.

Supplementary Figure 7

0 2 4 6 80

5

10

15

CD16 normalized expression

IRF

7 n

orm

alized e

xpre

ssio

n R square 0.5668P value < 0.0001

0 2 4 6 80

5

10

15

CD14 normalized expression

IRF

7 n

orm

alized e

xpre

ssio

n R square 0.4812

P value < 0.0001

c

a b

Supplementary Figure 8: Virus-specific memory CD8

+ T cells prevent LCMV-induced tumor

regression a: Tumor diameter of MOPC-tumor bearing WT or memory Jh

–/– mice (pretreated with 200 PFU LCMV

i.v. 100 days before tumor inoculation) treated with LCMV 2x104 PFU intravenously on day 0 (n =

5/group). b: Tumor diameter and survival from MOPC-tumor transplanted WT and Rag1–/–

mice (day -10) treated with or without 2x10

4 PFU LCMV intratumorally on day 0 (n = 5/group) c: Tumor diameter

and survival from MOPC-tumor transplanted Rag1–/–

mice (day -10) treated with anti-IFNAR1 antibody or isotype control (n = 5/group). Data are shown as mean ± SEM and analyzed by unpaired Student`s t-test. Survival is shown in Kaplan-Meier method and analyzed by log-rank test. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Supplementary Figure 8

a

- 1 0 - 5 0 5 1 0 1 5 2 0

0 . 3

0 . 6

0 . 9

1 . 2

1 . 5

< 0 . 1

W T , L C M V

m e m o r y J h – / – , L C M V

T i m e ( d )

Tu

mo

r d

ia

me

te

r (

cm

)

**

b

0 10 20 30 40 50 60 700.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Rag1–/–WT

Time (d)

Tum

or

dia

me

ter

(cm

)

0 20 40 60 800

20

40

60

80

100

Rag1–/–

, LCMV

WT, LCMV

Time (d)

Surv

ival (%

)

0 10 20 30 40 50 60 70

WT, LCMV

Rag1–/–, LCMV

Time (d)

** ** ns

c

0 5 10 15 20 25 30 350.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Rag1–/–, aIFNAR, LCMV

Rag1–/–, Isotype control, LCMV

Time (d)

Tum

or

dia

mete

r (c

m)

0 20 40 60 800

20

40

60

80

100

Rag1–/–, Isotype control, LCMV

Rag1–/–, anti-IFNAR, LCMV

Time (d)

Surv

ival (

%)

*** **

Supplementary Figure 9: Anti-tumoral effect of LCMV is independent of host IFNAR expression. Tumor diameter of Ifnar

–/– and C57BL/6 control mice injected with 5x10

5 MOPC cells (day -3)

subcutaneously and either left untreated (left) or treated with 2x104 PFU LCMV peritumorally (right,

day 0) (n = 6-7/group). Measurements were performed on the indicated days. ns, non-significant.

Supplementary Figure 9

Control LCMV

0 5 10 15 20 25

0.2

0.4

0.6

0.8

1.0

1.2

<0.1

Time (d)

Tum

or

dia

mete

r (c

m)

0 5 10 15 20 25

Time (d)

C57BL/6J

Ifnar–/–

C57BL/6J, LCMV

Ifnar–/–

, LCMV

ns

ns

Supplementary Figure 10: Reduced vascularisation correlates with arenavirus mediated tumor regression a: qRT-PCR from tumors of MOPC-tumor bearing WT mice (day -3) treated with or without 2x10

4 PFU

LCMV peritumorally (day 0), analyzed on day 6 (n= 3/group). b&c: Immunofluorescence (b, n= 3/group, CD31, red; DAPI, blue) and quantification of microvessel density (MVD) and vessel–vessel distances (c, n = 3/group) from MOPC-tumor bearing C57BL/6 mice (day -3) which were treated with or without 2x10

4 PFU LCMV peritumorally (day 0) measured on day 6. d: Immunohistochemistry (day

6) and quantification (day 9) of hypoxic areas from tumors of MOPC-tumor bearing C57BL/6 mice (day -3) treated with or without 2x10

4 PFU LCMV peritumorally on day 0 (n= 3 mice/group). e:

Immunofluorescence of tumors from MOPC-tumor bearing C57BL/6 (day -3) treated with or without 2x10

4 PFU LCMV peritumorally on day 0 (n= 3/group, Ki-67 or cleaved caspase 3, red; DAPI, blue).

Data are shown as mean ± SEM and analyzed by unpaired Student`s t-test. ns, non-significant; *p < 0.05 ; **p < 0.01; ***p < 0.001; Scale bar = 200µm.

a Tumor, LCMV Tumor

Log2

-10 0 10

Supplementary Figure 10

DA

PI C

D31

Tumor

Tumor, LCMV

b Tumor Tumor, LCMV

c

Control LCMV0

1 0 0

2 0 0

3 0 0

Ve

ss

el-

ve

ss

el

dis

tan

ce

(µ

m)

***

Control LCMV0

2 0 0

4 0 0

6 0 0

8 0 0

MV

D (

mm

³)

* *

d

Pim

onid

azo

l

Tumor Tumor, LCMV

Control LCMV0

20

40

60

Hyp

oxi

c a

rea (

%)

**

Tumor Tumor, LCMV

DA

PI K

i 6

7

DA

PI

Cle

ave

d C

asp

ase 3

e

Supplementary Table 1: List of antibodies used for immunofluorescence. All antibodies listed were used in 1:100 dilutions to their original concentration for flow-cytometry and/or for immunohistochemistry.

Antibody Manufacturer Catalogue number

Anti-B MyB (phospho T487) Origene TA301225

Anti-CD90.2 eBiosciences 11-0903

Anti-CD4 BD Pharmingen 17-0042

Anti-CD8 eBiosciences 553035

Anti-CD45R (B220) BD Pharmingen 17-0452

Anti-Ly6C eBiosciences 17-5932

Anti-Ly6G eBiosciences 11-5931

Anti-CD115 eBiosciences 12-1152

Anti-CD11b eBiosciences 47-0112

Anti-CD11c eBiosciences 11-0114

Anti-mPDCA Miltenyi 130-091-964

Anti-CD31 eBiosciences 11-0311-81

Anti-Ki-67 Thermo Scientific RM-9106-S

Anti-Cleaved Caspase-3 (Asp175)

Cell signalling #9579

Anti-CD274 (PD-L1, B7-H1) eBioscience 12-5982

Anti-CD279 (PD-1) eBioscience 11-9981

Anti-CD127 (IL-7Ra) eBioscience 17-1271

Anti- CD25 (IL-2Ra) eBioscience 12-0251

Anti-NK1.1 eBioscience 12-5941

Anti-CD3 eBioscience 11-0031

Supplementary Table 1

Supplementary Table 2: List of murine Primers for Taqman qPCR List of commercially available TaqMan® Gene Expression Assays from Life technologies used for detection of murine gene expression via qPCR.

Primer number

Eukaryotic 18S RNA #4333760T

GAPDH Mm03302249_g1

Tipin Mm00600456_m1

Atp6ap2 Mm00510396_m1

Atp6v0b Mm01193846_g1

Pkmyt1 Mm01309244_m1

Arcn Mm00524375_m1

Copb1 Mm00446330_m1

Copa Mm00550231_m1

Mat2a Mm00728688_s1

Eif3g Mm00469383_m1

Eif3a Mm00468721_m1

Arhgap23 Mm01722379_m1

MYBL2 Mm00485340_m1

Ifnar1 Mm00439544_m1

Ifnar2 Mm00494916_m1

TGFß Mm01178820_m1

MMP14 Mm00485054_m1

MMP2 Mm00439498_m1

MMP9 Mm00442991_m1

PDGFß Mm00440677_m1

VEGFA Mm00437306_m1

VEGFB Mm00442102_m1

VEGFC Mm00437310_m1

VEGFD (Figf) Mm01131929_m1

FGFR1 Mm00438930_m1

FGFR2 Mm01269930_m1

FGFR3 Mm00433294_m1

EGF Mm00438696_m1

ANGPT1 Mm00456503_m1

SELL Mm00441291_m1

CCL5 Mm01302427_m1

CXCL3 Mm01701838_m1

Csf3 Mm00438334_m1

CXCL15 Mm00441263_m1

CXCL1 Mm04207460_m1

Supplementary Table 2

Supplementary Table 3: List of murine Primers for SYBR Green-based qPCR List of commercially available QuantiTect Primer Assays used for detection of murine gene expression via qPCR.

Primer number

Gapdh QT01658692

IFN4 QT01774353

IFNß QT00249662

IRF7 QT00245266

OAS1 QT01056048

ISG15 QT02274335

Ly6C QT00247604

CCR2 QT02276813

CCL2 QT00167832

Supplementary Table 3

Supplementary Table 4: List of human Primers for Taqman qPCR List of commercially available TaqMan® Gene Expression Assays from Life technologies used for detection of human gene expression via qPCR.

Supplementary Table 5: LCMV-NP Primer Sequences

Primer number

Eukaryotic 18S RNA #4333760T

IFNAR1 Hs01066116_m1

IFNAR2 Hs01022059_m1

CCR2 Hs00704702_s1

ITGAM Hs00167304_m1

SELP Hs00927900_m1

CXCL9 Hs00171065_m1

MMP9 Hs00957562_m1

CD14 Hs02621496_s1

CD16 Hs04334165_m1

IRF7 Hs01014809_g1

IFNB1 Hs01077958_s1

OAS1 Hs00973635_m1

USP18 Hs00276441_m1

PRKRA Hs00269379_m1

Primer Sequence

LCMV WE NP Forward 5’- CAA AGT ATT CAC ACG GCA TGG A

LCMV WE NP Reverse 5’- TGG GAG AGC ACC TAT AAC TGA TGA

Supplementary Table 4

Supplementary Table 5