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