Supplementary Materials for · Fig. S10. Flow cytometry on CD44 and CD69 T cell markers on CD4 T...
16
stm.sciencemag.org/cgi/content/full/12/532/eaay8707/DC1 Supplementary Materials for High-dose vitamin C enhances cancer immunotherapy Alessandro Magrì, Giovanni Germano, Annalisa Lorenzato, Simona Lamba, Rosaria Chilà, Monica Montone, Vito Amodio, Tommaso Ceruti, Francesco Sassi, Sabrina Arena, Sergio Abrignani, Maurizio D’Incalci, Massimo Zucchetti, Federica Di Nicolantonio*, Alberto Bardelli* *Corresponding author. Email: [email protected] (A.B.); [email protected] (F.D.N.) Published 26 February 2020, Sci. Transl. Med. 12, eaay8707 (2020) DOI: 10.1126/scitranslmed.aay8707 The PDF file includes: Fig. S1. VitC quantification in plasma. Fig. S2. High doses of VitC are required for maximal antiproliferative effects in murine breast tumors. Fig. S3. Administration of antioxidant NAC does not impair VitC anticancer effect. Fig. S4. Flow cytometry for IFN-γ in spleen-derived T lymphocytes. Fig. S5. Isolation of T cells for adoptive T cell transfer. Fig. S6. Depletion of CD4 T cells for adoptive cell transfer experiments. Fig. S7. Tumor growth of individual mice treated as shown in Fig. 3 (A and B). Fig. S8. Tumor growth of individual mice treated as shown in Fig. 3 (C to E). Fig. S9. Modulation of tumor immune infiltration induced by VitC. Fig. S10. Flow cytometry on CD44 and CD69 T cell markers on CD4 T lymphocytes. Fig. S11. Representative CD44- and CD69-positive events on CD8 T lymphocytes by flow cytometry. Fig. S12. VitC effect on growth of MMR-deficient tumors in NOD-SCID mice. Fig. S13. Tumor growth of individual mice bearing MLH1-KO tumors and treated with ICT and VitC as shown in Fig. 5D. Fig. S14. Tumor volume variations since treatment start of TS/A MLH1-KO and CT26 MLH1- KO tumors treated with ICT and VitC. Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/532/eaay8707/DC1) Data file S1 (Microsoft Excel format). Tumor measurements of experiments in Fig. 1. Data file S2 (Microsoft Excel format). Tumor measurements of experiments in Fig. 2. Data file S3 (Microsoft Excel format). Tumor measurements of experiments in Fig. 3. Data file S4 (Microsoft Excel format). Immuno-phenotiping of CD4 and CD8 T cells.
Transcript of Supplementary Materials for · Fig. S10. Flow cytometry on CD44 and CD69 T cell markers on CD4 T...
stm.sciencemag.org/cgi/content/full/12/532/eaay8707/DC1
Supplementary Materials for
High-dose vitamin C enhances cancer immunotherapy
Alessandro Magrì, Giovanni Germano, Annalisa Lorenzato, Simona Lamba, Rosaria Chilà, Monica Montone, Vito Amodio,
Tommaso Ceruti, Francesco Sassi, Sabrina Arena, Sergio Abrignani, Maurizio D’Incalci, Massimo Zucchetti, Federica Di Nicolantonio*, Alberto Bardelli*
*Corresponding author. Email: [email protected] (A.B.); [email protected] (F.D.N.)
Published 26 February 2020, Sci. Transl. Med. 12, eaay8707 (2020)
DOI: 10.1126/scitranslmed.aay8707
The PDF file includes:
Fig. S1. VitC quantification in plasma. Fig. S2. High doses of VitC are required for maximal antiproliferative effects in murine breast tumors. Fig. S3. Administration of antioxidant NAC does not impair VitC anticancer effect. Fig. S4. Flow cytometry for IFN-γ in spleen-derived T lymphocytes. Fig. S5. Isolation of T cells for adoptive T cell transfer. Fig. S6. Depletion of CD4 T cells for adoptive cell transfer experiments. Fig. S7. Tumor growth of individual mice treated as shown in Fig. 3 (A and B). Fig. S8. Tumor growth of individual mice treated as shown in Fig. 3 (C to E). Fig. S9. Modulation of tumor immune infiltration induced by VitC. Fig. S10. Flow cytometry on CD44 and CD69 T cell markers on CD4 T lymphocytes. Fig. S11. Representative CD44- and CD69-positive events on CD8 T lymphocytes by flow cytometry. Fig. S12. VitC effect on growth of MMR-deficient tumors in NOD-SCID mice. Fig. S13. Tumor growth of individual mice bearing MLH1-KO tumors and treated with ICT and VitC as shown in Fig. 5D. Fig. S14. Tumor volume variations since treatment start of TS/A MLH1-KO and CT26 MLH1-KO tumors treated with ICT and VitC.
Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/532/eaay8707/DC1)
Data file S1 (Microsoft Excel format). Tumor measurements of experiments in Fig. 1. Data file S2 (Microsoft Excel format). Tumor measurements of experiments in Fig. 2. Data file S3 (Microsoft Excel format). Tumor measurements of experiments in Fig. 3. Data file S4 (Microsoft Excel format). Immuno-phenotiping of CD4 and CD8 T cells.
Data file S5 (Microsoft Excel format). Tumor measurements of experiments in Fig. 5. Data file S6 (Microsoft Excel format). Plasma VitC analysis. Data file S7 (Microsoft Excel format). Tumor measurements of experiments in figs. S2 and S3. Data file S8 (Microsoft Excel format). Tumor measurements of experiments in fig. S6. Data file S9 (Microsoft Excel format). Tumor measurements of experiments in fig. S8. Data file S10 (Microsoft Excel format). Tumor measurements of experiments in fig. S12.
Supplementary figures:
Fig. S1. VitC quantification in plasma. Plasma was collected from Balb/c mice
without tumor and Balb/c mice orthotopically injected with TS/A tumor cells
(100,000 cells, 50% Matrigel), one hour after VitC i.p. administration. Heparinized
plasma samples were supplemented with 10% metaphosphoric acid and analyzed
by a LC-MS/MS based method. Individual values are shown. Mean and +/- SEM are
also shown. P values were calculated by student’s t test. NS, not significantly
different. Ctrl, control. VitC, vitamin C.
Balb/c mice without tumors
Balb/c mice with TS/A breast tumors
Plasma
P<0.0001 P<0.0001
Fig. S2. High doses of VitC are required for maximal antiproliferative effects
in murine breast tumors. TS/A cancer cells were orthotopically injected into
immunocompetent syngeneic mice (100,000 cells, 50% matrigel). Treatment with
the indicated VitC doses was initiated when tumors reached approximately 100
mm3 in volume, as indicated by the black arrow. Each experimental group was
composed of at least 5 mice. Data and error bars indicate mean +/- SEM. P values
were calculated by one-way ANOVA. NS, not significantly different. Ctrl, control.
VitC, vitamin C.
0 5 10 15 20 25
500
1000
1500
2000
Tu
mo
r V
olu
me (
mm
3)
Days
CTRL
VITC 4 g/kg
VITC 2 g/kg
VITC 1 g/kg
VITC 1.5 g/kg
VITC 0.5 g/kg
P<0.0001
n.s.
n.s.
TS/A - breast
Fig. S3. Administration of antioxidant NAC does not impair VitC anticancer
effect. A, TS/A cells were orthotopically injected into immunocompetent mice
(100,000 cells, 50% matrigel), and N-acetyl cysteine (1.2 g/kg) was administered
by daily gavage. The arrow indicates the timepoint at which treatment was started.
B, Tumors were explanted at the end of the experiment, and FFPE sections were
stained for 8-oxo guanine as a marker for ROS-induced DNA damage. Scale bar is
representative of 100 μm. Each experimental group was composed of at least 6
mice. Data and error bars indicate mean +/- SEM. P value was calculated using
one-way ANOVA at the indicated timepoint. NS, not significantly different. Ctrl,
control. VitC, vitamin C. NAC, N-acetyl cysteine.
0 5 10 15 20 25
250
500
750
1000
1250
1500
1750
Tu
mo
r V
olu
me (
mm
3)
Days
CTRL
VITC
NAC + VITC
NACP<0.0001
n.s.
TS/A - breast
CTRL NAC VITC NAC + VITC
#1
#2
#3
8-oxo-guanine
A
B
Fig. S4. Flow cytometry for IFN-γ in spleen-derived T lymphocytes. A-B,
Representative IFNγ positivity on CD4 T cells (A) and CD8 T cells (B) shown in Fig.
2 A and B. The fraction of positive cells was calculated on CD4 and CD8 positive
live events, respectively (500,000 events were acquired for each sample).
IFNγ+
CD
4+
4.2% 2% 4.8%
Control
Mouse 1 Mouse 2 Mouse 4
Mouse 1 Mouse 2 Mouse 5
Vitamin C
IFNγ+
CD
8+
5% 14.9% 10%
2% 2.4% 4.2%
Mouse 2 Mouse 3 Mouse 4
Mouse 1 Mouse 3 Mouse 4
Control
Vitamin C
A B
2% 1.9% 3.2%
Fig. S5. Isolation of T cells for adoptive T cell transfer. A-B, CD4 T cells (A) and
CD8 T cells (B) were isolated from the spleens of syngeneic mice (by negative
selection) and analyzed for purity by flow cytometry. The fraction of positive cells
was calculated on total live events (500,000 events were acquired for each
sample).
CD
4+
CD45+
CD
8+
1%
96%
Isolation of CD8 T cells
Isolation of CD4 T cells
90%
2%
CD
4+
CD45+
CD
8+
A B
Fig. S6. Depletion of CD4 T cells for adoptive cell transfer experiments. A,
TS/A cells (100,000) were orthotopically injected into immunocompetent
syngeneic mice. Isotype and CD4-depleting (indicated as antiCD4) antibodies were
administered according to the same experimental setting indicated in Figure 2. The
arrow indicates the timepoint at which VitC treatment was started. B, Blood was
collected and analyzed by flow cytometry to confirm effective CD4 T cell depletion.
A representative flow cytometry plot from a single animal is shown. C, The
percentages of CD4+ and CD8+ T cells were quantified by flow cytometry in blood
from animals (n= 4 per group) that received the indicated treatments. After 30
days, spleens were harvested and CD8 T cells isolated and adoptively transferred
into immunocompromised mice shown in Fig. 2H. Data and error bars indicate
mean +/- SEM. P values were calculated using one-way ANOVA. αCD4, anti-CD4.
αCD8, anti-CD8. Ctrl, control. VitC, vitamin C.
A B
C
0 5 10 15 20 25 300
500
1000
1500
2000
Tu
mo
r V
olu
me (
mm
3)
Days
CTRL
antiCD4
VITC
antiCD4 + VITC P<0.0001n.s.
TS/A – breast
Isoty
pe
Isoty
peVitC
VitC
anti-
CD4
anti-
CD4
anti-
CD4
+ VitC
anti-
CD4
+ VitC
0
5
10
15
20
25
30
35
40
45
50
% p
osit
ive c
ells
CD4+
CD8+
Isoty
pe
Isoty
peVitC
VitC
anti-
CD4
anti-
CD4
anti-
CD4
+ VitC
anti-
CD4
+ VitC
0
5
10
15
20
25
30
35
40
45
50
% p
osit
ive c
ells
CD4+
CD8+
Isotype
VitC
αCD4
CD
4+
CD45+
CD
8+
αCD4 + VitC
45% 19%
42% 19%
0.1% 29%
0.1% 23%
Fig. S7. Tumor growth of individual mice treated as shown in Fig. 3 (A and B).
A, PDAC pancreatic cancer cells were injected subcutaneously (500,000 cells) into
syngeneic mice that were treated as indicated. B, 4T1 breast cancer cells were
injected orthotopically (100,000 cells) into syngeneic mice that were treated as
indicated. VitC (4 g/kg) was administered i.p. 5 days/week starting when tumors
reached a volume around 100 mm3. Anti-CTLA-4 (200 μg/mouse) and anti-PD1
(250 μg/mouse) were given at the timepoints indicated by the dashed vertical
lines in the graphs. In combinatorial treatments, VitC was administered starting
with the first cycle of immunotherapy. The arrows indicate the timepoint at which
VitC treatment was started. Individual values are shown. Ctrl, control. VitC, vitamin
C.
0 10 20 30 40
500
1000
1500T
um
or
Vo
lum
e (
mm
3)
Days
4T1 - breast
PDAC - pancreatic
0 10 20 30 40
500
1000
1500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
2000
Tu
mo
r V
olu
me (
mm
3)
Days0 10 20 30 40
500
1000
1500
2000
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
2000
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
Tu
mo
r V
olu
me (
mm
3)
Days
CTRL
VITC
αCTLA4 + αPD1
αCTLA4 + αPD1 + VITC0 10 20 30 40
500
1000
1500
Tu
mo
r V
olu
me (
mm
3)
Days
CTRL
VITC
αCTLA4 + αPD1
αCTLA4 + αPD1 + VITC
A
B
0 10 20 30 40
500
1000
1500
Tu
mo
r V
olu
me (
mm
3)
Days
CTRL
VITC
αCTLA4 + αPD1
αCTLA4 + αPD1 + VITC
0 10 20 30 40
500
1000
1500
2000
Tu
mo
r V
olu
me (
mm
3)
Days
Fig. S8. Tumor growth of individual mice treated as shown in Fig. 3 (C to E). A,
TS/A breast cancer cells were injected orthotopically (100,000 cells) into
syngeneic mice that were treated as indicated in Fig. 3C. B, CT26 colorectal cancer
cells were injected subcutaneously (500,000 cells) into syngeneic mice that were
treated as indicated in Fig. 3E. VitC (4 g/kg) was administered i.p. 5 days/week
starting when TS/A and CT26 tumors reached a volume of around 100 mm3 or
800-1000 mm3, respectively. Anti-CTLA-4 (200 μg/mouse) and anti-PD1 (250
μg/mouse) were given at the timepoints indicated by the dashed vertical lines in
the graphs. In combinatorial treatments, VitC was administered starting with the
first cycle of immunotherapy. The arrows indicate the timepoint at which
treatment was started. Individual values are shown. Ctrl, control. VitC, Vitamin C.
0 10 20 30 40 50
500
1000
1500
2000
2500
3000
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
2000
2500
3000
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40 50
500
1000
1500
2000
2500
3000
Tu
mo
r V
olu
me (
mm
3)
Days
TS/A - breast
0 10 20 30 40
500
1000
1500
2000
Tu
mo
r V
olu
me (
mm
3)
Days0 10 20 30 40
500
1000
1500
2000
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
Tu
mo
r V
olu
me (
mm
3)
Days
CTRL
VITC
αCTLA4 + αPD1
αCTLA4 + αPD1 + VITC
0 10 20 30 40 50
500
1000
1500
2000
2500
3000
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40 50
500
1000
1500
2000
2500
3000
Tu
mo
r V
olu
me (
mm
3)
Days0 10 20 30 40 50
500
1000
1500
2000
2500
3000
Tu
mo
r V
olu
me (
mm
3)
Days
CT26 - colorectal
0 10 20 30 40
500
1000
1500
Tu
mo
r V
olu
me (
mm
3)
Days
CTRL
VITC
αCTLA4 + αPD1
αCTLA4 + αPD1 + VITC
0 10 20 30 40
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days0 10 20 30 40
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
A
B
0 10 20 30 40
500
1000
1500
2000
Tu
mo
r V
olu
me (
mm
3)
Days0 10 20 30 40
500
1000
1500
2000
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40 50
500
1000
1500
2000
2500
3000
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40 50
500
1000
1500
2000
2500
3000
Tu
mo
r V
olu
me (
mm
3)
Days
Experiment 1
Experiment 2
Experiment 1
Experiment 2
Fig. S9. Modulation of tumor immune infiltration induced by VitC. TS/A
orthotopic tumors were explanted, single cell suspended, and analyzed by flow
cytometry. Indicated cell percentages were gated according to the following gating
strategy: live CD45+, CD4+, CD127-, CD25+, FoxP3+ are indicated as Treg cells; live
CD45+, CD11b+, F4/80+ as macrophages; live CD45+, CD3-, CD49b as natural killer
cells; live CD45+, F4/80-, CD11c+ as dendritic cells. The fraction of positive cells
was calculated on CD45+ live events (500,000 events were acquired for each
sample). Individual values are shown. Mean and +/- SEM are also shown.
CTR
L
VIT
C
CO
MBO
TRIP
LA
0
10
20
30
40
50
% M
ac
rop
ha
ge
s
(of
live c
ells)
CTR
L
VIT
C
CO
MBO
TRIP
LA
0
20
40
60
80%
CD
45+
(of
live c
ells)
CTR
L
VIT
C
CO
MBO
TRIP
LA
0
5
10
15
20
25
% D
C
(of
live c
ells)
CTR
L
VIT
C
CO
MBO
TRIP
LA
0
5
10
15
20
% N
K
(of
live c
ells)
CTR
L
VIT
C
CO
MBO
TRIP
LA
0
1
2
3
4
% T
reg
(of
live c
ells)
Isoty
pe-C
trl
VitC
aPD1
+ aC
TLA4
aPD1
+ aC
TLA4
+ VitC
0
20
40
60
80
CD
8+
T c
ell
s /
HP
F
Isotype
VitC
αCTLA-4 + αPD-1
αCTLA-4 + αPD-1 + VITC
CD45+
Macrophages Dendritic Cells Natural Killers
Treg
P=0.0282
Fig. S10. Flow cytometry on CD44 and CD69 T cell markers on CD4 T
lymphocytes. The fraction of positive cells was calculated on CD45+ CD4+ live
events (500,000 events were acquired for each sample). VitC, Vitamin C.
CD45+
CD
4+
VitC αCTLA4 + αPD1 αCTLA4 + αPD1 + VitC
CD44+
CD
4+
CD69+
CD
4+
Mouse 1 Mouse 3 Mouse 4 Mouse 2 Mouse 1 Mouse 3 Mouse 4 Mouse 6
4,6% 13% 20.6% 16.3% 17% 15.3% 48% 24%
9,7% 8,7% 20.1% 16.8% 7,4% 7% 16% 13%
Isotype
5.3% 14.1% 26% 17% 21.6% 8.1% 25.9% 22%
Fig. S11. Representative CD44- and CD69-positive events on CD8 T
lymphocytes by flow cytometry. The fraction of positive cells was calculated on
CD45+ CD8+ live events (500,000 events were acquired for each sample). VitC,
Vitamin C.
Isotype
CD45+
CD
8+
VitC αCTLA4 + αPD1 αCTLA4 + αPD1 + VitC
CD44+
CD
8+
CD69+
CD
8+
Mouse 2 Mouse 4 Mouse 2 Mouse 1 Mouse 2 Mouse 4 Mouse 5 Mouse 3
15% 22% 28% 28% 22% 32% 27% 45%
10% 25% 27% 35% 17% 27% 12% 36%
7.2% 10% 21% 21% 11% 25% 20% 20%
Fig. S12. VitC effect on growth of MMR-deficient tumors in NOD-SCID mice.
Effect of VitC on MMR-proficient and MMR-deficient tumors in
immunocompromised mice that were treated with Vitamin C when tumors
reached approximately 100 mm3 in volume. The arrows indicate the timepoint at
which VitC treatment was started. Data and error bars indicate mean +/- SEM. P
values were calculated by one-way ANOVA. NS, not significantly different. WT,
wild-type. KO, knock-out. VitC, Vitamin C.
0 5 10 15 20 25 30
500
1000
1500
2000
Tu
mo
r V
olu
me (
mm
3)
Days
MLH1 WT
MLH1 WT + VITC
MLH1 KO
MLH1 KO + VITC
0 5 10 15 20 25 30
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
MLH1 WT
MLH1 WT + VITC
MLH1 KO
MLH1 KO + VITC
TS/A - breast CT26 - colorectal
n.s. n.s.
Fig. S13. Tumor growth of individual mice bearing MLH1-KO tumors and
treated with ICT and VitC as shown in Fig. 5D. A, Breast TS/A and B, colorectal
CT26 tumor-bearing mice. MLH1-KO tumors were transplanted into
immunocompetent syngeneic mice and treated with ICT and VitC (4 g/kg) starting
at a tumor volume of 800-1000 mm3. Anti-CTLA-4 (200 μg/mouse) and anti-PD1
(250 μg/mouse) were given at the timepoints indicated by the dashed vertical
lines in the graphs. Individual values are shown. Ctrl, control. VitC, Vitamin C.
A B TS/A – MLH1-KO
0 10 20 30 40
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days0 10 20 30 40
500
1000
1500
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2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
CTRL
VITC
αPD1
αCTLA-4
αPD1 + VITC
αCTLA-4 + VITC
αCTLA4 + αPD1
0 10 20 30 40 50
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40 50
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
CTRL
VITC
αPD1
αCTLA-4
αPD1 + VITC
αCTLA-4 + VITC
αCTLA4 + αPD1
CT26 – MLH1-KO
0 10 20 30 40
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
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2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40 50
500
1000
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2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40 50
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1500
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Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40 50
500
1000
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2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days0 10 20 30 40 50
500
1000
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2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
0 10 20 30 40 50
500
1000
1500
2000
2500
Tu
mo
r V
olu
me (
mm
3)
Days
Fig. S14. Tumor volume variations since treatment start of TS/A MLH1-KO
and CT26 MLH1-KO tumors treated with ICT and VitC. Tumor volume
variations at the indicated timepoints since tumor transplantation of individual
mice shown in Fig. 5D. Individual values are shown. Mean and +/- SEM are also
shown. Ctrl, control. VitC, vitamin C.
TS/A - MLH1-KO day 25
CTR
L
VIT
C
aPD1
aCTLA
-4
aPD1
+ VIT
C
aCTLA
-4 +
VIT
C
aCTLA
4 + a
PD1
-100
-50
0
50
100
150
200
250
300
350
400
% T
um
or
vo
lum
e v
ari
ati
on
CTRL
VITC
aPD1
aCTLA-4
aPD1 + VITC
aCTLA-4 + VITC
aCTLA4 + aPD1
CTR
L
VIT
C
aPD1
aCTLA
-4
aPD1
+ VIT
C
aCTLA
-4 +
VIT
C
aCTLA
4 + a
PD1
-100
-50
0
50
100
150
200
250
300
350
400
% T
um
or
vo
lum
e v
ari
ati
on
CTRL
VITC
aPD1
aCTLA-4
aPD1 + VITC
aCTLA-4 + VITC
aCTLA4 + aPD1
CTR
L
VIT
C
aPD1
aCTLA
-4
VIT
C +
aPD1
VIT
C +
aCTLA
-4
aCTLA
4 + a
PD1
-100
-50
0
50
100
150
200
250
300
350
400
% T
um
or
vo
lum
e v
ari
ati
on
CT26 - MLH1-KO day 24
CTR
L
VIT
C
aPD1
aCTLA
-4
aPD1
+ VIT
C
aCTLA
-4 +
VIT
C
aCTLA
4 + a
PD1
-100
-50
0
50
100
150
200
250
300
350
400
% T
um
or
vo
lum
e v
ari
ati
on
CTRL
VITC
aPD1
aCTLA-4
aPD1 + VITC
aCTLA-4 + VITC
aCTLA4 + aPD1
0 10 20 30 40 50
500
1000
1500
2000
2500
Tum
or
Vo
lum
e (
mm
3)
Days
CTRL
αPD1
VITC
αCTLA4
αPD1 + VITC
αCTLA4 + VITC
αPD1 + αCTLA4
0 10 20 30 40 50
500
1000
1500
2000
2500
Tum
or
Vo
lum
e (
mm
3)
Days
CTRL
αPD1
VITC
αCTLA4
αPD1 + VITC
αCTLA4 + VITC
αPD1 + αCTLA4
