Investigating the differential response to immunotherapy...
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Investigating the differential Investigating the differential response response to immunotherapy of to immunotherapy of orthotopic tumors compared orthotopic tumors compared to subcutaneous tumors to subcutaneous tumors Michael Kershaw Michael Kershaw Peter MacCallum Cancer Center, Melbourne, Peter MacCallum Cancer Center, Melbourne, Australia Australia
Transcript of Investigating the differential response to immunotherapy...
Investigating the differential Investigating the differential responseresponse to immunotherapy of to immunotherapy of orthotopic tumors comparedorthotopic tumors comparedto subcutaneous tumorsto subcutaneous tumors
Michael KershawMichael KershawPeter MacCallum Cancer Center, Melbourne, Peter MacCallum Cancer Center, Melbourne,
AustraliaAustralia
Subcutaneous Renca tumors respond well to Trimab when compared to kidney tumors
0
20
40
60
80
100
0 20 40 60 80 100
Days since tumor injected
perc
ent s
urvi
val
S.c control S.c TrimAb
I.K control I.K TrimAb
SC: 86%
IK:14%
◊ Trimab
1 experiment representative of 3
N=7
Monitor
Renca (Ch+/Luc+)
S.C
I.K
◊ TrimAb
D0 D11 - 20 ....D100
Days since tumor injection
Per
cent
sur
viva
l
TrimabDR5CD40
CD137
No differences in tumor growth rate between SC and IK
1 experiment representative of 3
TrimAbControl
11 13 15 18 20 25 27 29 31 34 36 390
50
100
150
200
250
tum
or s
ize
(mm
2)SC tumors
11 17 27 38 441000
10000
100000
days since tumor injected
biol
umin
esce
nt e
mis
sion
(cou
nts)
TrimAb Control
D11
D17
D27
IK tumors
AIM: Determine the reasons behind the differential responses to immunotherapy
of tumors in different locationsImmune related differences:cells and molecules of the tumor microenvironmentbefore treatment
Differences in intrinsic tumor qualities:resistance to apoptosis, MHC expression, morphological/structural differences
Cytometry gating for immune cells in tumors
Before treatment (D12 after tumor cell injection)
Gated on viability
CD11b
F4/8
0
100
101
102
103
10410
0
101
102
103
104
CD11b
Ly6G
100
101
102
103
10410
0
101
102
103
104
neutrophilsmacrophages
TCR B10
010
110
210
310
4100
101
102
103
104
TCRb NegTCRb Neg
CHERRY100 101 102 103 104
100
101
102
103
104
CherryNegCherryNeg
TCR B10
010
110
210
310
4100
101
102
103
104
TCRb pos
CD11c10
010
110
210
310
4100
101
102
103
104
DCs B cells_
CD1910
010
110
210
310
4100
101
102
103
104
_
CD8
CD
25
100
101
102
103
10410
0
101
102
103
104
CD25+/CD8-CD25+/CD8-
_
FR4100 101 102 103 104
100
101
102
103
104
TregCD8
No differences in frequency of immune cells in kidney tumors compared to subcutaneous tumors
4 independent experiments pooled
Neutrophils
SC IK0
5
10
15
% of total cells
TCRβ‐/Ly6G+/CD11b+B cells
SC IK0
2
4
6
8
10
% o
f tot
al c
ells
TCRβ‐/CD19+
DCs
SC IK0
10
20
30
40
50
% o
f tot
al c
ells
TCRβ‐/CD11c+Macrophages
SC IK0
10
20
30
40
50
% o
f tot
al c
ells
TCRβ‐/F4/80+/CD11b+
CD4 Tcells
SC IK0
10
20
30
40
50
% o
f tot
al c
ells
TCRβ+/CD4+CD8 T cells
SC IK0
10
20
30
40
50
% o
f tot
al c
ells
TCRβ+/CD8+
TReg
SC IK0
2
4
6
8
10
% o
f tot
al c
ells
TCRβ+/CD8‐/CD25+/FR4high
Macrophages
SC IK0
5
10
15
% o
f tot
al c
ells
Differences in macrophage profile between SC/IK tumors
3 independent experiments pooled
SC IK
CD11bhi F4/80low
CD11bint F4/80hi
CD11b
F4/8
010
010
110
210
310
4100
101
102
103
104
CD11b
F4/8
0
100 101 102 103 104100
101
102
103
104
i i t t
0
2
4
6
8
10
% o
f tot
al c
ells *
*
SC SC IKIKCD11bint F4/80hi CD11bhi F4/80low
F4/80hiCD11bint macrophages express FoxP3 and the mannose receptor (CD206)
CD11bhiF4/80low
0
20
40
60
80
100
% o
f mac
roph
ages
SC SC IKIK
CD11bint F4/80hi
FoxP3
Foxp300 101 102 103 104
0
2
3
4
2.43%2.43% 97.57%
Foxp3100 101 102 103 1040
2
3
4
89.07% 10.93%89.07%
CD11bhi F4/80lowCD11bint F4/80hi
IK
CD11b
F4/8
0
100 101 102 103 104100
101
102
103
104
SC IK0
20
40
60
80
100
% o
f mac
roph
ages
SC IK0
20
40
60
80
100
% o
f mac
roph
ages
CD206CD206
100 101 102 103 104100
101
102
103
104
18.75%18.75% 81.25%
CD20610
010
110
210
310
4100
101
102
103
104
96.19% 3.81%96.19%
% o
f M
% o
f M
M1 M2
Mantovani A et al, 2002
TraffickingCCL2, CCL1 CX3CL1, CCL6
Growth factorsGM‐CSFM‐CSF
↑ IK tumors
Factors for the M2 switch LIF
ActivityArginase IL‐10
↑ SC tumors
M2 macrophage markers predominate in kidney tumors
Scavenger receptor AScavenger receptor BCD163Mannose receptor
Membrane receptors
CD14
FcRII
IL-1ra
IL-10
Decoy IL-1R type II
CCL17, CCL22CCL24
CCL18CCL16
CCR2CXCR1, CXCR2
arginase
TLR2, TLR4
TNF-
CD80, CD86
IL-1
IL-6IL-12
Type I IFN
IL-1 R type 1
CXCL9, CXCL10, CXCL11
CCL2, CCL3, CCL4CCL5, CXCL8
CCR7
iNOS
ROI
Effector molecules
Chemokine receptors
Chemokines
Cytokine receptors
Cytokines
CD16, CD32, CD64
CD4+ T cell depletion triggers regression of SC tumors but not IK tumors
S.C
I.K
Renca injected
D 0
Depleting anti‐CD4 antibody
Tumor monitoring
Survival
D 11‐12‐13 ‐‐‐D100
0
20
40
60
80
100
0 20 40 60 80 100
Days since tumor injected
perc
ent s
urvi
val
S.c controlS.c Treg deplik controlik Treg depl
SC 84% (n=13)
IK 0% (n=10)
T reg depletion (D11,12,13)
1 experiment representative of 3
N=11
TReg
SC IK0
2
4
6
8
10
% o
f tot
al c
ells
Immune response after CD4+ depletion may be systemic
S.CI.K
I.K+S.C
S.C+S.C
D 0
Depleting anti‐CD4 antibody
Tumor monitoring
Survival
D 11‐12‐13 ‐‐‐D100
0 20 40 60 80 1000
20
40
60
80
100SC+IK CtleSC+IK TRDIK CtleIK TRDSC CtleSC TRDSC+SC CtleSC+SC TRD
days
Perc
ent s
urviv
al
***
***
***
**
3 independent experiments pooled
N=14‐20
T reg depletion (D11,12,13)
SC -CD4
SC+SC -CD4
SC+IK -CD4
Kidney tumor inhibits rejection of subcutaneous tumor
1 experiment representative of 3
9 14 18 23 28 32 37 42 46 510
50
100
150
200
250
SC T Reg DSC Control
N=6
Tumor size (m
m2 )
SC alone
Anti‐CD4 days 11, 12 and 13
1/66/6
9 14 18 23 28 32 37 42 46 510
50
100
150
200
T Reg D rightT Reg leftControl rightControl left
SC + SCN=6
Days since tumor injected
6/6
2/6
9 14 18 23 28 32 37 42 460
100
200
300
400
SC+IK TReg DSC+IK Control
N=8
SC + IK
7/88/8
Kidney tumors do not respond as well as subcutaneous tumors when pieces are transplanted under the skin
0
20
40
60
80
100
0 20 40 60 80 100
Days after tumor transplant
Perc
ent s
urvi
val
sc - sc untreatedSsc - sc a-CD4ik - sc untreatedik - sc a-CD4
one experiment (n=5)
S.C
I.K
RencaD 0
Tumors harvested
D 15Tumor pieces reinjected SC
anti‐CD4 Ab11 days later
Monitor survival
‐‐‐D100
ik sc -CD4
sc sc -CD4
Days after tumor transplant
Per
cent
sur
viva
l
AIM: Determine the reasons for the differential responses to immunotherapy
of tumors in different locationsImmune related differences:cells and molecules of the tumor microenvironment
Differences in intrinsic tumor qualities:resistance to apoptosis, MHC expression, morphological/structural differences
‐What do the tumors look like before treatment?
SC and IK tumors are same size / weight before treatment
19.70 21.16
05
1015202530
Volume
mm
3
SC
IK
14.60 12.55
0
5
10
15
20
Weight
mg
SC
IK
1 experiment representative of 3
IK tumors
SC tumors
D10 before treatmentV
olum
e (m
m2 )
Wei
ght (
mg)
sc ik sc ik
Phenotype of tumors: Higher level of MHC I in SC tumor and higher level of DR5 and expression of Fas L in IK tumors
2 exp pooled
SC IK 0
2000
4000
6000
MFI
MHC I00 101 102 103 104
0.12% 99.88%
_
MHC I0
010
110
210
310
4
0.26% 99.74%
SC IK
***
MHC I DR5
SC IK 0
20
40
60
80
100
% o
f tu
mo
ur c
ells
SC IK 0
50
100
150
200
MFI
***
DR50
010
110
210
310
4
9.38% 90.62%9.38%
SC IK_
DR50
010
110
210
310
4
97.43%2.57%
Fas L0
010
110
210
310
4
99.40% 0.60%99.40%
FasL
p
SC IK 0
5
10
15
20
% o
f tum
our c
ells ***
Fas L0
101
102
103
104
93.12% 6.88%93.12%
SC IK PD L1 SC IK
SC IK0
20
40
60
80
100
% o
f tum
our c
ells
PD-L1100 101 102 103 1040
2
3
4
19.10% 80.90%19.10%
PD-L110
010
110
210
310
40
1
2
3
4
15.13%15.13% 84.87%
MFI
sc ik
% tu
mor
cel
ls
sc ikM
FI
% tu
mor
cel
ls
sc ik sc ik
sc ik% tu
mor
cel
ls
020000400006000080000100000120000140000160000
1
Positiv
e pixels fo
r CD31
IK SC
IK SC
CD31
isotype
T
K
TS
X20
X20
X20(5 tumors, 10 fields/tumor)
Kidney tumors are more highly vascularized
P<0.00116
12
8
4
0
Pos
itive
pix
els
(x 1
0-4)
ik scCD
31
No difference in tumor vessel permeability
Elution in formamide
Renca Ch+ luc+ cells
D 0
S.C
I.K
Evans blue IV injections
D 14
tumors taken
26 min
DO reading at 620nm
24 h
N = 5 tumors, representative of 3 experiments
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Ctle IK IK Ctle SC SC
Ev B
l (ng
/mg
tissu
e)
Kidney Subcutaneous
no dye no dye+ dye + dye
Evans b
lue (ng/mg tissue)
Summary
• Subcutaneous tumors eradicated by Trimab or Treg depletion but kidney tumors are not
• M2 macrophage microenvironment in kidney tumors
• Higher frequency of F4/80hiCD11bintFoxP3+macrophages in kidney tumors
• Immunosuppression may be systemic• More blood vessels and higher MHCI in subcutaneous tumors
Thanks
Immunology program
Christel DevaudJenny WestwoodPhil DarcyMark SmythMichele TengLiza John Jacqueline FlynnConnie DuongCarmen Yong
FACS facility
Peter Mac animal facility
Angiogenesis labSophie Paquet-FifieldMarc Achen
Peter Mac molecular genomics facility
Histology lab
Cancer Council of Victoria
National Health and Medical Research Council
Peter MacCallum Cancer Center
Funding:
