Se-ries1

0.0E+00

9.0E+03

1.8E+04

2.7E+04

3.6E+04

4.5E+04 No TumourK1492K1492+PBS

Nu

mb

er o

f ce

lls in

tu

mou

r-b

ear-

ing

hem

isp

her

e

CD45high

“Infiltrating Leukocytes”

CD45lowCD11b+

“Microglia”CD45highCD11b+

“GIMs”

CD45highCD11b-

“Lymphocytes”

**

**

**

**

CD45high

“Infiltrating Leukocytes”

CD45lowCD11b+

“Microglia”CD45highCD11b+

“GIMs”CD45highCD11b-

“Lymphocytes”

Supplementary Figures

Suppl. Figure S1: Immunophenotyping of the K1492 glioma microenvironment in response to sterile injury. C57Bl/6 mice implanted with K1492 cells and analyzed by flow cytometry at 15 days-post implantation, 3 days post-treatment with intracranial PBS (PBS, n=4) or untreated (NoTx, n=4). Non-tumour bearing mice were used as a control (NT, n=4). Quantified numbers of each cell type isolated from the tumour-bearing hemisphere (top). Error bars represent standard error, and asterisks indicate statistical difference (p<0.05). Scatter graph of isolated immunocytes as a percent of the total CD45 population present in the tumour-bearing hemisphere compared to PBS treatment (bottom).

AK

1492

14d

ays

post

-im

plan

tati

on, I

ba1

stai

n

H&

E

Iba1

K14

92 +

1dp

Tx

MY

XV

K14

92 +

3dp

Tx

MY

XV

K14

92 +

7dp

Tx

MY

XV

M-T

7

Su

pp

l. F

igu

re S

2: I

mm

unop

heno

typi

ng o

f th

e K

1492

gli

oma

mic

roen

viro

nmen

t af

ter

Myx

oma

viru

s tr

eatm

ent.

A –

14

day

K14

92 t

umou

r in

w

ildt

ype

mic

e w

ith

Iba1

sta

inin

g de

mon

stra

ting

the

‘gr

adie

nt o

f ac

tiva

tion

.’ S

erie

s of

thr

ee p

hoto

s st

itch

ed t

oget

her

from

10X

obj

ecti

ve. W

hite

li

ne d

enot

es a

ppro

xim

ate

tum

our

bord

er.

B -

C57

Bl/

6 m

ice

wer

e im

plan

ted

wit

h K

1492

cel

ls a

nd t

reat

ed w

ith

MY

XV

14

days

pos

t-im

plan

tati

on.

For

mal

in-f

ixed

par

affi

n se

ctio

ns w

ere

stai

ned

wit

h H

&E

, M

yxom

a vi

rus

prot

ein

M-T

7 or

for

the

mic

rogl

ial/

mac

roph

age

mar

ker

Iba1

(F

irst

row

25X

; S

econ

d ro

w 2

00X

). K

1492

NoT

x Ib

a1 2

00x

pane

ls r

epre

sent

wit

hin

tum

our

(lef

t) a

nd a

djac

ent

to t

umou

r (r

ight

) w

itho

ut

MY

XV

tre

atm

ent.

Rep

rese

ntat

ive

pict

ures

of

2-3

anim

als/

grou

p. W

hite

arr

ows

show

are

as o

f po

lym

orph

onuc

lear

cel

l in

filt

rati

on w

hile

bla

ck

arro

w s

how

are

as o

f fo

cal n

ecro

sis.

B

H&

EM

-T7

1dpTx

1dpTx H&EMagnification

Suppl. Figure S3: Virus infected areas of K1492 tumours are accompanied by polymorphonuclear cell infiltration. Formalin-fixed paraffin sections were stained using Hematoxylin and Eosin (H&E) or immunohistochemical for MYXV early protein M-T7. White arrows show areas of intense infiltration of polymorphonuclear cells. Far right picture is 400X displaying polymorphonuclear phenotype.

H&E IbaI

Hig

h G

rad

eM

id-H

igh

Gra

de

B

A K1861

Suppl. Figure S4: K1861 and Spontaneous NPcis gliomas in C57Bl/6 mice have significant microglia and/or GIM recruitment. A – NPcis cell line K1861 was orthotpically grafted into C57Bl/6 mice and tumours were stained for Iba1 14 days post-implantation. B - C57Bl/6 mice heterozygous for Nf1 and Tp53 inactivating mutations spontaneously develop astrocytomas. Two such spontaneous astrocytomas, a high grade (top) and mid-high grade (bottom) were stained with haematoxylin and eosin (H&E) or with the microglia/macrophage marker Iba1.

1dpTx 2dpTx 3dpTx 5dpTx 7dpTx1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07WTCCR2

Tot

al F

LU

XA

B

Suppl. Figure S5: Non-tumour bearing CCR2-deficient animals are only slightly impaired in clearing MYXV. Naïve CCR2-null mice (n=5) or WT (n=5) were infected with 5x106 FFUs of vMyx-FLuc and followed for bioluminescence (A) and survival (B). Experiment was terminated after 100 days.

**

**

NK T0.0E+00

2.5E+03

5.0E+03

7.5E+03

1.0E+04

WT-K1492

WT-K1492 + MYXV

CCR2-K1492

CCR2-K1492 + MYXV

Nu

mb

er o

f ce

lls in

tu

mou

r-b

eari

ng

hem

isp

her

e

NK1.1-CD3+

“T cells”NK1.1+CD3-

“NK cells”

*

+*

K1492-WT K1492-CCR20.0E+00

1.0E+03

2.0E+03

3.0E+03

4.0E+03

5.0E+03 NoTxMYXV

NK1.1+DX5+ Cells

Nu

mb

er o

f C

ells

in

Tu

mou

r-b

eari

ng

Hem

isp

her

e

WT- K1492

CD

45

WT-K1492 + MYXV CCR2- K1492 CCR2-K1492 + MYXV

A

B

C

Suppl. Figure S6: CCR2-deficient mice have an exaggerated recruitment of NK and T cells to the K1492 glioma in response to Myxoma treatment. Wildtype (WT) or CCR2-deficient (CCR2) C57Bl/6 mice implanted with K1492 cells and analyzed by flow cytometry at 15 days-post implantation, 3 days post-treatment with Myxoma virus (MYXV) or untreated (NoTx). Initial live gating was around small lymphocyte population followed by interrogation of the CD45high population within this gate. Quantified numbers of each cell type isolated from the tumour-bearing hemisphere demonstrating: A - NK cells as measured by NK1.1+CD3- and T cells as NK1.1-CD3+; B – Representative scatter plots from the small gated lymphocytes on the CD45/CD11b scatter plot. NK1.1 staining is grey population. C - Subsequent quantification of flow cytometry experiment using only the NK1.1 and DX5 antibodies to look at NK cell populations recruited to K1492 gliomas 3 dpTx. Error bars represent standard error and asterisks represent significant differences within mouse strain but between treatment groups. Plus signs represent significant differences between mouse strains within treatment groups. (n=3, p<0.05).

CD11b

B

A

1dpTx 2dpTx 3dpTx 5pdTx 7pdTx1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06 MYXV (n=5)MYXV + Mino (n=7)

Tot

al F

LU

X*

Suppl Figure S7: Minocycline administration mimics CCR2-null viral clearance kinetics but does not result in a survival advantage. Minocycline hydrochloride (Sigma) was prepared fresh in DMSO and heated to 37oC immediately before every treatment. Wildtype C57Bl/6 mice implanted with K1492 cells were treated with Minocycline (Mino) or 50 mg/Kg twice a day starting at 10-11dpi, 50 mg/Kg once a day from 12-16dpi, and then 25 mg/Kg from 17-20dpi or with DMSO (Vehicle). Viral administration was given at 14dpi. A - Real-time monitoring of viral infection with bioluminescence using vMyx-FLuc. Error bars represent standard error and asterisks represent significant differences (Mann-Whitney, p<0.05; MYXV n=5, MYXV+Mino n=7). B – Survival of animals treaded with this regimen (Log-rank, Mantel-Cox, p=0.8630).

NK T0.0E+00

9.0E+02

1.8E+03

2.7E+03

3.6E+03

4.5E+03 WT-K1492

WT-K1492 + MYXV

IL2Rg-K1492

IL2Rg-K1492 + MYXVN

um

ber

of

cells

in t

um

our-

bea

rin

g h

emis

ph

ere

CD11b

CD

45

K1492-IL2Rγ K1492-IL2Rγ MYXV

++

NK1.1+CD3-

“NK cells”NK1.1-CD3+

“T cells”

++

K1492 K1492 + MYXV

A

B

* *

Suppl. Figure S8: K1492 tumours in IL2Rγ mice are significantly depleted of NK and T cell populations.Wildtype (WT) or IL2Rγ-deficient (IL2Rγ) C57Bl/6 mice implanted with K1492 cells and analyzed by flow cytometry at 15 days-post implantation, 3 days post-treatment with Myxoma virus (5x106 FFUs vMyx-GFP; MYXV) or untreated. Initial live gating was around small lymphocyte population followed by interrogation of the CD45high population within this gate. Quantified numbers of each cell type isolated from the tumour-bearing hemisphere demonstrating: A - NK cells as measured by NK1.1+CD3- and T cells as NK1.1-CD3+; Error bars represent standard error and asterisks represent significant differences within mouse strain but between treatment groups. Plus signs represent significant differences between mouse strains within treatment groups (n=3, p<0.05). B – Representative scatter plots from the small gated lymphocytes on the CD45/CD11b scatter plot. NK1.1 staining is grey population.

1dpTx 2dpTx 3dpTx 5dpTx 7dpTx1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

WTILR2γ

Tot

al F

LU

X

A

* *

B

Suppl. Figure S9: Non-tumour bearing IL2Rγ-deficient animals are impaired in clearing MYXV. Naïve IL2Rγ-null mice (n=5) or WT (n=5) were infected with 5x106 FFUs of vMyx-FLuc and followed for bioluminescence (A) and survival (B). Experiment was terminated after 100 days.

Suppl Figure S10: Single low-dose cyclophosphamide combined with Myxoma treatment did result in improved treatment efficacy (A) or viral infection (B). However, repeated low-dose treatments resulted in severe lymphoablation of tumour-resident as well as treatment recruited leukocytes (C).

CD11b

CD

45

1dpTx 2dpTx 3dpTx 5dpTx 7dpTx1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06 MYXV

MYXV+CPA

Day Post MYXV Tx

Tot

al F

LU

X

A

C

NoTx vs. CPA, p=0.0771CPA vs. MYXV + CPA, p=0.1191