Supplementary Figure 1 a

Size: px

Start display at page:

Download "Supplementary Figure 1 a"

Emil Brown
5 years ago
Views:

% DAPI + Are Supplementry Figure 1 MOI 1 MOI.5 MOI.25 5 4 3 MOPC MOPC, LCMV reltive VL4 expression 2 1 d1 d2 d3 MOI.125 MOI.

1 % DAPI + Are Supplementry Figure 1 MOI 1 MOI.5 MOI MOPC MOPC, LCMV reltive VL4 expression 2 1 d1 d2 d3 MOI.125 MOI.625 untreted Supplementry Figure 1: LCMV replictes in MOPC without ffecting cell prolifertion : Quntifiction of Immunofluorescence of MOPC cells untreted or infected with LCMV (MOI 1) 72 hours fter infection (n = 3/group) (relted to Fig. 1c)., non-significnt. : Non-impired clonogenic survivl of MOPC cells fter in vitro LCMV WE infection. Representtive photogrph of long-term colony formtion following incution of MOPC with LCMV WE of indicted MOI.

LCMV(log 1 PFU/ml) Tumor Norml tissue VSV (PFU/well) Supplementry Figure 2 RCC RCC HCC HCC CRC CRC HNSCC HNSCC SW48 HeL HepG2 FADU c 6 4 2 Sw48 hifn- mifn- mifn- SKCM SKCM control 2 4 6 81 1 2 3

2 LCMV(log 1 PFU/ml) Tumor Norml tissue VSV (PFU/well) Supplementry Figure 2 RCC RCC HCC HCC CRC CRC HNSCC HNSCC SW48 HeL HepG2 FADU c Sw48 hifn- mifn- mifn- SKCM SKCM control IFNAR1 (reltive expression) IFNAR2 (reltive expression) IFNAR1 (reltive expression) IFNAR2 (reltive expression) IFN-I (U/ml) IFN-I (U/ml) d MOPC MC38 B16F1 LoxP-TAg MT/ret Control Ifnr1 (reltive expression) e DAPI p-my f WT, orophrynx MOPC WT, colon MC38 WT, liver LoxP-Tg WT, liver LoxP-TAg, liver tumor WT, colon MC38 tumor WT, orophrynx MOPC tumor * * M y l2 mrna (Fold c hnge) g Signl trduction Uncoting Vesiculr trport Tipin Atp6p2 Atp6v Pkmyt1 Arcn1 * * MOPC tumor Phrynx h 6 5 Control sirna B-My sirna 1 B-My sirna 2 * * Trltion regultion GPCR signling Cop1 Cop Mt2 Eif3g Eif3 Arhgp mrna (reltive expression, log scle) * * * * Supplementry Figure 2: B-My expression, nd not IFN-I unrespoiveness, is one fctor promoting renvirus repliction : mrna expression levels of Ifnr1 nd Ifnr2 for the cncer types renl cell cncer (RCC; n=534), heptocellulr cncer (HCC; n=373), colorectl cncer (CRC; n=382), hed nd neck squmous cell cncer (HNSCC; n=522) nd skin cutneous melnoms (SKCM; n=471). Downloded from the TCGA dtse y use of cbioportl ( Dt re shown s men nd minimum-mximum whiskers. : qrt-pcr of humn Ifnr1 nd Ifnr2 mrna expression in Sw48, HeL, HepG2, FADU cells nd controls (murine mrna, n=5/group). c: Numer of virl plques in Sw48 cell lyer (n=6/group, left pnel) nd HeL cell lyer (n=5/group, right pnel), which were treted with different concentrtio of humn IFN 4, murine IFN 4 or murine IFN 2 nd infected with VSV (MOI.1) 24 hours erlier. d: qrt-pcr of murine Ifnr1 mrna in MOPC cells, MC38 cells, B16F1 cells, LoxP-Tg tumors, MT/ret cells nd control (humn mrna, n=5/group). e&f: Immunofluorescence of phospho-b-my (e, n=3/group) nd qrt-pcr of B-My (=Myl2) mrna (f, n = 9-1/group) in MOPC, MC38 nd LoxP-Tg tumors compred to the correlting helthy tissue (orophryngel epitheli, colon nd liver). Scle r, 2µm. g: qrt-pcr of virl host fctors in MOPC tumors (n=5) nd phrynx tissue (n=5). h: Infectious LCMV prticles in superntnt of MCF7 cells, which were treted with control sirna or two seprte B-My sirnas nd then 24 hours lter infected with LCMV (MOI 1) nlyzed 24 hours fter infection (n = 6/group). Dt re shown s men ± SEM nd nlyzed y unpired Student`s t-test., non significnt; *p <.5; p <.1; *p < <3

ALT (U/ml) Flnk tumor Shoulder tumor Supplementry Figure 3 LCMV (

) untreted LCMV-NP 15 1 * 4/4 1/5 /3 5 LoxP-TAg control LoxP-TAg

to virl repliction in metstsis : Immunohistochemistry for LCMV-NP in

simultneously sucutneously 5 x 1 5 MOPC cells in the flnk nd

the flnk or intrvenously on dy (n=4-5/group). Scle r, 2µm.

induced tumor ering LoxP-TAg mice (11 month old) which were left

3 ALT (U/ml) Flnk tumor Shoulder tumor Supplementry Figure 3 LCMV ( into Flnk LCMV (i.v.) untreted LCMV-NP 15 1 * 4/4 1/5 /3 5 LoxP-TAg control LoxP-TAg LCMV C57BL/6 LCMV Supplementry Figure 3: Intrvenous infection leds to virl repliction in metstsis : Immunohistochemistry for LCMV-NP in flnk nd shoulder tumor (dy 15) from C57BL/6 mice receiving simultneously sucutneously 5 x 1 5 MOPC cells in the flnk nd shoulder (dy -3), treted with or without 2x1 4 PFU LCMV given into the flnk or intrvenously on dy (n=4-5/group). Scle r, 2µm. : Serum lnin-minotrferse (ALT) levels of C57BL/6 (n=3) nd denovirlly induced tumor ering LoxP-TAg mice (11 month old) which were left untreted (n=4) or which were dditionlly treted with 2 x 1 6 PFU of LCMV-WE (n=5) mesured 2 dys fter infection. Vlues ove line indicte pthologicl liver disese.

4 Supplementry Figure 4 Supplementry Figure 4: Immune cell infiltrtes re required for tumor regression Tumor dimeter of MOPC-tumor ering WT nd Mp3k14 ly/ly mice (dy -3) treted with (n=8 WT; n=6 Mp3k14 ly/ly ) or without (n=7 WT; n=6 Mp3k14 ly/ly ) 2x1 4 PFU LCMV peritumorlly on dy. Dt re shown s men ± SEM nd nlyzed y unpired Student`s t-test., non significnt; *p <.1.

T u m o r d i m e t e r ( c m ) SSC count WT, αly6g (clone 1A8) WT, undepleted WT, αly6c+g (clone RB6-8C5) SSC count WT, undepleted Supplementry Figure 5 ng ng MC ng MC MC ng MC CD115 Ly6G Ly6C ng MC

5 T u m o r d i m e t e r ( c m ) SSC count WT, αly6g (clone 1A8) WT, undepleted WT, αly6c+g (clone RB6-8C5) SSC count WT, undepleted Supplementry Figure 5 ng ng MC ng MC MC ng MC CD115 Ly6G Ly6C ng MC ng MC ng MC ng MC CD115 Ly6G Ly6C c 1.2 W T W T, L y 6 G WT, LCMV WT, Ly6G,LCMV < T im e ( d ) Supplementry Figure 5: Tumor infiltrting Ly6C+ cells re indispele for ntitumorl LCMV therpy MOPC tumor ering C57BL/6J mice were treted with 2x1 4 PFU LCMV peritumorlly on dy. Depletion ws performed y i.p. injection of either () αly6c+g (clone RB6-8C5) 2µg or ( nd c) αly6g (clone 1A8) 5µg strting on d-2 nd repeted on d2 nd d7. &: Representtive dot lots of peripherl vein lood collected on d4 nd stined for monocyte nd grnulocyte cell mrkers fter preselection of CD11 positive cells. c: Tumor dimeter in C57BL/6 mice injected with or without αly6g (1A8) ntiody (n=3-4/group). Dt re shown s men ± SEM nd nlyzed y unpired Student`s t-test., non-significnt.

NK1.1 Tumor dimeter (cm) Tumor dimeter (cm) Tumor dimeter (cm) Supplementry Figure 6 1.2 WT Tcr / WT, LCMV Tcr /, LCMV 1..8.6.4.2 <.1. 5 1 15 2 25 3 1.4 1.2 1..8.6.4 WT Jh /.2 <.1. 5 1 15 2 25 3 5 1 15 2 25 3 WT, LCMV Jh /, LCMV 5 1 15 2 25 3 c 1.

6 NK1.1 Tumor dimeter (cm) Tumor dimeter (cm) Tumor dimeter (cm) Supplementry Figure WT Tcr / WT, LCMV Tcr /, LCMV < WT Jh /.2 < WT, LCMV Jh /, LCMV c 1. WT WT, NK1.1 W T, LC M V W T, NK 1.1, L C M V d < CD3 Supplementry Figure 6: Impct of dptive immune system nd NK cells on LCMV-induced nti-tumorl immunity. : Tumor dimeter of MOPC-tumor ering WT nd Tcr / mice (dy -3) treted with or without 2x1 4 PFU LCMV peritumorlly on dy (n=5/group). : Tumor dimeter of MOPC-tumor ering WT nd Jh / mice (dy -3) treted with or without 2x1 4 PFU LCMV peritumorlly on dy (n=5/group). c&d: Tumor dimeter (c) nd representtive dot lot from peripheril vein lood collected t dy 2 nd stined for NK cell mrkers (d) of WT mice which were NK depleted y i.p. injection of 4µl NK1.1 ntiody on dy -3 nd dy -1 (n = 4/group). Dt re shown s men ± SEM nd nlyzed y unpired Student`s t-test., non-significnt.

of 12 immunogenes mrnas of primry smples from 34 orophryngel cncer ptients. : Liner regression etween IRF7 nd the humn monocyte mrkers CD14 nd CD16.

7 IRF7 normlized expression IRF7 normlized expression Supplementry Figure 7 15 R squre.4812 P vlue <.1 15 R squre.5668 P vlue < CD14 normlized expression CD16 normlized expression c Supplementry Figure 7: Monocyte recruitment in humn cncers correltes with IFN-I induction : Hetmp of correltion nlyses of 12 immunogenes mrnas of primry smples from 34 orophryngel cncer ptients. : Liner regression etween IRF7 nd the humn monocyte mrkers CD14 nd CD16. c: Gene-set enrichment nlysis (GSEA) of humn IFN-I induced genes y usge of the Browne interferon-relted gene-set. IFN-I-induced expression profile nd genes rnked y extent of differentil expression in TCGA melnom, heptocellulr crcinom, renl cncer, hed nd neck cncer nd colorectl cncer dtset. NES, normlized enrichment score; FDR, flse discovery rte.

8 Tumor dimeter (cm) Survivl (%) Tumor dimeter (cm) Survivl (%) T u m o r d i m e t e r ( c m ) Supplementry Figure <. 1 W T, L C M V m e m o r y Jh /, L C M V T i m e ( d ) WT Rg1 / WT, LCMV Rg1 /, LCMV WT, LCMV Rg1 /, LCMV c Rg1 /, Isotype control, LCMV Rg1 /, IFNAR, LCMV * Rg1 /, Isotype control, LCMV Rg1 /, nti-ifnar, LCMV Supplementry Figure 8: Virus-specific memory CD8 + T cells prevent LCMV-induced tumor regression : Tumor dimeter of MOPC-tumor ering WT or memory Jh / mice (pretreted with 2 PFU LCMV i.v. 1 dys efore tumor inocultion) treted with LCMV 2x1 4 PFU intrvenously on dy (n = 5/group). : Tumor dimeter nd survivl from MOPC-tumor trplnted WT nd Rg1 / mice (dy - 1) treted with or without 2x1 4 PFU LCMV intrtumorlly on dy (n = 5/group) c: Tumor dimeter nd survivl from MOPC-tumor trplnted Rg1 / mice (dy -1) treted with nti-ifnar1 ntiody or isotype control (n = 5/group). Dt re shown s men ± SEM nd nlyzed y unpired Student`s t-test. Survivl is shown in Kpln-Meier method nd nlyzed y log-rnk test., non-significnt; *p <.5; p <.1; *p <.1.

9 Tumor dimeter (cm) Supplementry Figure Control LCMV C57BL/6J Ifnr / C57BL/6J, LCMV Ifnr /, LCMV < Supplementry Figure 9: Anti-tumorl effect of LCMV is independent of host IFNAR expression. Tumor dimeter of Ifnr / nd C57BL/6 control mice injected with 5x1 5 MOPC cells (dy -3) sucutneously nd either left untreted (left) or treted with 2x1 4 PFU LCMV peritumorlly (right, dy ) (n = 6-7/group). Mesurements were performed on the indicted dys., non-significnt.

Hypoxic re (%) DAPI Cleved Cspse 3 Pimonidzol DAPI Ki 67 M V D ( m m ³ )

Tumor Tumor, LCMV Tumor Tum or Tumor, LCMV Tumor, LCMV c 8 6 3 2 * 4 2 1

LCMV 6 4 2 Control LCMV Supplementry Figure 1: Reduced vsculristion

MOPC-tumor ering WT mice (dy -3) treted with or without 2x1 4 PFU LCMV

&c: Immunofluorescence (, n= 3/group, CD31, red; DAPI, lue) nd

3/group) from MOPC-tumor ering C57BL/6 mice (dy -3) which were treted

d: Immunohistochemistry (dy 6) nd quntifiction (dy 9) of hypoxic res from

2x1 4 PFU LCMV peritumorlly on dy (n= 3 mice/group).

treted with or without 2x1 4 PFU LCMV peritumorlly on dy (n= 3/group,

10 Hypoxic re (%) DAPI Cleved Cspse 3 Pimonidzol DAPI Ki 67 M V D ( m m ³ ) V e s s e l-ve s s e l d is tnc e (µ m ) DAPI CD31 Supplementry Figure 1 Tumor Tumor, LCMV Tumor Tum or Tumor, LCMV Tumor, LCMV c * Log Control LCMV Control LCMV d Tumor Tumor, LCMV e Tumor Tumor, LCMV Control LCMV Supplementry Figure 1: Reduced vsculristion correltes with renvirus medited tumor regression : qrt-pcr from tumors of MOPC-tumor ering WT mice (dy -3) treted with or without 2x1 4 PFU LCMV peritumorlly (dy ), nlyzed on dy 6 (n= 3/group). &c: Immunofluorescence (, n= 3/group, CD31, red; DAPI, lue) nd quntifiction of microvessel deity (MVD) nd vessel vessel distnces (c, n = 3/group) from MOPC-tumor ering C57BL/6 mice (dy -3) which were treted with or without 2x1 4 PFU LCMV peritumorlly (dy ) mesured on dy 6. d: Immunohistochemistry (dy 6) nd quntifiction (dy 9) of hypoxic res from tumors of MOPC-tumor ering C57BL/6 mice (dy -3) treted with or without 2x1 4 PFU LCMV peritumorlly on dy (n= 3 mice/group). e: Immunofluorescence of tumors from MOPC-tumor ering C57BL/6 (dy -3) treted with or without 2x1 4 PFU LCMV peritumorlly on dy (n= 3/group, Ki-67 or cleved cspse 3, red; DAPI, lue). Dt re shown s men ± SEM nd nlyzed y unpired Student`s t-test., non-significnt; *p <.5 ; p <.1; *p <.1; Scle r = 2µm.

11 Supplementry Tle 1 Antiody Mnufcturer Ctlogue numer Anti-B MyB (phospho T487) Origene TA31225 Anti-CD9.2 ebiosciences Anti-CD4 BD Phrmingen Anti-CD8 ebiosciences Anti-CD45R (B22) BD Phrmingen Anti-Ly6C ebiosciences Anti-Ly6G ebiosciences Anti-CD115 ebiosciences Anti-CD11 ebiosciences Anti-CD11c ebiosciences Anti-mPDCA Miltenyi Anti-CD31 ebiosciences Anti-Ki-67 Thermo Scientific RM-916-S Anti-Cleved Cspse-3 (Asp175) Cell signlling #9579 Anti-CD274 (PD-L1, B7-H1) ebioscience Anti-CD279 (PD-1) ebioscience Anti-CD127 (IL-7R) ebioscience Anti- CD25 (IL-2R) ebioscience Anti-NK1.1 ebioscience Anti-CD3 ebioscience Supplementry Tle 1: List of ntiodies used for immunofluorescence. All ntiodies listed were used in 1:1 dilutio to their originl concentrtion for flow-cytometry nd/or for immunohistochemistry.

12 Supplementry Tle 2 Primer numer Eukryotic 18S RNA #433376T GAPDH Tipin Atp6p2 Atp6v Pkmyt1 Arcn Cop1 Cop Mt2 Eif3g Eif3 Arhgp23 MYBL2 Ifnr1 Ifnr2 TGFß MMP14 MMP2 MMP9 PDGFß VEGFA VEGFB VEGFC VEGFD (Figf) FGFR1 FGFR2 FGFR3 EGF ANGPT1 SELL CCL5 CXCL3 Csf3 CXCL15 CXCL1 Mm332249_g1 Mm6456_m1 Mm51396_m1 Mm _g1 Mm139244_m1 Mm524375_m1 Mm44633_m1 Mm55231_m1 Mm728688_s1 Mm469383_m1 Mm468721_m1 Mm _m1 Mm48534_m1 Mm439544_m1 Mm494916_m1 Mm117882_m1 Mm48554_m1 Mm439498_m1 Mm442991_m1 Mm44677_m1 Mm43736_m1 Mm44212_m1 Mm43731_m1 Mm _m1 Mm43893_m1 Mm126993_m1 Mm433294_m1 Mm438696_m1 Mm45653_m1 Mm441291_m1 Mm132427_m1 Mm171838_m1 Mm438334_m1 Mm441263_m1 Mm42746_m1 Supplementry Tle 2: List of murine Primers for Tqmn qpcr List of commercilly ville TqMn Gene Expression Assys from Life technologies used for detection of murine gene expression vi qpcr.

13 Supplementry Tle 3 Primer Gpdh IFN 4 IFNß IRF7 OAS1 ISG15 Ly6C CCR2 CCL2 numer QT QT QT QT QT15648 QT QT24764 QT QT Supplementry Tle 3: List of murine Primers for SYBR Green-sed qpcr List of commercilly ville QuntiTect Primer Assys used for detection of murine gene expression vi qpcr.

14 Supplementry Tle 4 Primer numer Eukryotic 18S RNA #433376T IFNAR1 Hs166116_m1 IFNAR2 Hs12259_m1 CCR2 Hs7472_s1 ITGAM Hs16734_m1 SELP Hs9279_m1 CXCL9 Hs17165_m1 MMP9 Hs957562_m1 CD14 Hs _s1 CD16 Hs _m1 IRF7 Hs11489_g1 IFNB1 Hs177958_s1 OAS1 Hs973635_m1 USP18 Hs276441_m1 PRKRA Hs269379_m1 Supplementry Tle 4: List of humn Primers for Tqmn qpcr List of commercilly ville TqMn Gene Expression Assys from Life technologies used for detection of humn gene expression vi qpcr. Supplementry Tle 5 Primer LCMV WE NP Forwrd LCMV WE NP Reverse Sequence 5 - CAA AGT ATT CAC ACG GCA TGG A 5 - TGG GAG AGC ACC TAT AAC TGA TGA Supplementry Tle 5: LCMV-NP Primer Sequences

TNF-α (pg/ml) IL-6 (ng/ml)

Xio, et l., Supplementry Figure 1 IL-6 (ng/ml) TNF-α (pg/ml) 16 12 8 4 1,4 1,2 1, 8 6 4 2 med Cl / Pm3CSK4 zymosn curdln Poly (I:C) LPS flgelin MALP-2 imiquimod R848 CpG TNF-α (pg/ml) IL-6 (ng/ml) 2 1.6