Download Supplementary Figure S5

Supplementary Figure S1
SNParray
CNV
Flow cytometry +
cytokine analysis
Treatment
implanted in mice
Targeted sequencing
WES + CNV
Transcriptomic
analysis
Anti-tumor activity
Supplementary Figure S1: Summary of experiments
Supplementary Figure S2
Supplementary Figure S2: Gating strategy for
identification of 9 major cell types, granzyme B+ NK cells
and ‘reinvigorated’ CD8+ T cells
A: Identification of live CD45+ cells by exclusion of
doublets, debris, dead cells and CD45- cells. This was carried
out before any further gating for all samples. B: Identification
of macrophages as CD11b+, F4/80+, Ly6C-, Ly6G- cells. C:
Identification of gMDSCs as CD11b+, Ly6G+, Ly6Clow
cells and of mMDSCs as CD11b+, Ly6G-, Ly6Chigh cells.
D: Identification of B cells as CD19+ cells. E: Identification
of NK cells as CD3-, NKp46+ cells. Identification of
granzyme B+ NK cells and fluorescence minus one (FMO)
control for granzyme B staining. F: Identification of NKT
cells as CD3+, NKp46+ cells. G: Identification of CD8+ T
cells as CD3+, CD8+, CD4- cells, of CD4+ T cells as CD3+,
CD8-, CD4+, FoxP3- cells and of Tregs as CD3+, CD8-,
CD4+, FoxP3+ cells. H: Identification of ‘reinvigorated’
CD8+ T cells as CD3+, CD8+, CD4-, Eomes+, PD-1+,
granzyme B+, Ki67+ cells. I: Fluorescence minus one
controls for Eomes, PD-1, granzyme B and Ki67 staining.
Supplementary Figure S3
Supplementary Figure S3:
Profiling by array CGH, whole-exome and targeted sequencing across 11
cell lines showing the percentage of altered genes from 41 cancer
associated genes. Somatic mutations and copy number changes are marked
for each gene with the percentage of samples altered along the side of the
heatmap. Sideway bargraphs show the total number of genomic alterations
across samples, which orders genes in the heatmap from most to least
altered. Human homolog genes are stated in brackets beside the mouse
gene names where these differ.
Supplementary Figure S4
A
B
% frequency
Supplementary Figure S4: Comparison of mutational profiles of murine syngeneic tumor cell lines and TCGA patient tumors
A: Percent of all mutations in each murine syngeneic cell line also found in the TCGA (The Cancer Genome Atlas) patient cohort of the
corresponding cancer type (lung sq: squamous cell lung cancer). B: Population frequencies of the top 10 recurrent mutations in each TCGA cancer
type is plotted beside the mutation profile of their corresponding murine syngeneic cell line.
Supplementary Figure S5
A
LYMPH NODE
Canonical Pathway
4T1
CT26
Renca
EIF2 Signaling
0.00
LXR/RXR Activation
0.00
-1.63
-1.73
iCOS-iCOSL Signaling in T
Helper Cells
0.00
-2.83
-3.64
Role of BRCA1 in DNA Damage
Response
2.24
3.00
Cell Cycle: G2/M DNA Damage
Checkpoint Regulation
-1.51
-0.53
MC38
B16F10_NCI
B16F10_ATCC
B16F0_ATCC
2.45
PPARα/RXRα Activation
0.00
-1.63
-2.31
ILK Signaling
0.00
0.82
2.98
Role of NFAT in Regulation of
the Immune Response
0.00
-3.00
CD28 Signaling in T Helper
Cells
0.00
Fc Epsilon RI Signaling
B16F0_ECACC TRAMPC1 TRAMPC2
3.46
-2.00
-2.00
2.65
-1.00
0.00
0.00
TC1
2.65
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
-2.98
0.00
0.00
-2.24
-3.46
0.00
0.00
-2.00
-3.16
0.00
0.00
0.00
0.00
0.00
0.00
EL4
P815
3.61
0.00
-0.82
0.00
0.00
LL2
0.00
2.24
0.00
0.00
PAN02
2.00
-2.00
-1.89
-1.00
0.38
0.38
-1.63
0.00
0.00
-2.45
0.00
0.00
0.00
SPLEEN
B
Canonical Pathway
4T1
Cyclins and Cell Cycle
Regulation
Cell Cycle: G2/M DNA
Damage Checkpoint
Regulation
MC38
B16F10_NCI
0.00
2.24
0.00
0.00
-2.65
0.00
1.63
Mitotic Roles of Polo-Like
Kinase
CT26
Renca
0.00
Estrogen-mediated S-phase
Entry
0.00
0.00
Role of BRCA1 in DNA
Damage Response
0.00
0.00
Protein Kinase A Signaling
0.00
cAMP-mediated signaling
1.00
0.00
0.00
0.00
p38 MAPK Signaling
B16F10_ATCC
B16F0_ATCC
B16F0_ECACC
TRAMPC1
TRAMPC2
PAN02
TC1
LL2
EL4
P815
0.00
0.00
0.00
0.00
2.24
2.45
2.45
2.83
0.00
0.00
0.00
0.00
0.00
-2.12
-1.51
-1.51
-1.39
0.00
0.00
0.00
0.00
0.00
0.00
2.12
2.53
2.12
2.71
0.00
0.00
0.00
0.00
0.00
0.00
2.00
2.00
2.24
0.00
0.00
0.00
0.00
0.00
0.00
1.63
1.63
1.90
0.00
0.00
0.00
0.00
0.00
0.00
-0.82
-1.63
-1.63
-0.71
0.00
0.00
0.00
0.00
0.00
2.00
1.63
0.00
-2.24
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
2.00
2.24
0.00
-2.00
-2.00
0.00
2.00
0.00
LXR/RXR Activation
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Actin Cytoskeleton
Signaling
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
2.00
Supplementary Figure S5:
Differentially-expressed
gene-sets per syngeneic
model (FC +/- 2.0, FDR
≤0.05) after IPA core
analysis where the top five
highest and lowest pathway
Z scores per line were
selected, sorted for nondisease canonical pathways
and ranked by absolute
activation score (summed
absolute Z score) (IPA
comparison tool) for (A)
lymph node and (B) spleen.
Supplementary Table S1
Inoculum
Endpoint
Day
In vivo passaged
during original
derivation?
1e5
14
No
2.5e6
11 (12)
Yes
5e5
14 (12)
Yes
Cell line
Supplier
Strain
Sex
Tissue of
origin
4T1
ATCC
Balb/c
Female
Breast
Spontaneous
Renca
ATCC
Balb/c
Female
Kidney
Spontaneous
CT26
ATCC
Balb/c
Female
Colon
NMU
RPMI 1640 (ATCC
modification), 10% FBS
RPMI 1640 (ATCC
modification), 10% FBS, 1%
NEAA, 1% sodium pyruvate,
1% Glutamax-I
RPMI 1640, 10% FBS
MC38
NCI
C57BL/6
Female
Colorectal
Dimethylhydrazine
DMEM, 10% FBS
5e5
18 (11)
Yes
B16F10 AP-3
AZ
C57BL/6
Female
Melanoma
Spontaneous
MEM, 10% FBS, 1% NEAA,
1% sodium pyruvate
5e3 in 50%
matrigel
(12)
Yes (then 3 extra
passages at AZ)
5e4
11
Yes
Method of generation
Culture conditions
B16F10
NCI
C57BL/6
Female
Melanoma
Spontaneous
MEM, 10% FBS, 1% NEAA,
1% sodium pyruvate, 1.5%
MEM Vitamins, 1% GlutamaxI, 3% sodium bicarbonate
B16F10
ATCC
C57BL/6
Female
Melanoma
Spontaneous
DMEM, 10% FBS
5e4
13
Yes
DMEM, 10% FBS
4
17 or 20
Yes
4
15
Yes
6
34 or 42
No
5e
6
42
No
Orthotopic
-
Yes
29-43
Yes
B16F0
ATCC
C57BL/6
Female
Melanoma
Spontaneous
5e
B16F0
ECACC
C57BL/6
Female
Melanoma
Spontaneous
TRAMPC1
ATCC
C57BL/6
Male
Prostate
Probasin-SV40
TRAMPC2
ATCC
C57BL/6
Male
Prostate
Probasin-SV40
Hepa1-6
ATCC
C57BL/6
Female
Liver
Spontaneous
DMEM, 10% FBS
DMEM, 5% FBS, 5% NuSerum IV, 5.5µg/ml bovine
insulin, 3ng/ml transdehydroandrosterone
DMEM, 5% FBS, 5% NuSerum IV, 5.5µg/ml bovine
insulin, 3ng/ml transdehydroandrosterone
DMEM, 10% FBS
PAN02
NCI
C57BL/6
Female
Pancreas
MCA
RPMI 1640, 10% FBS
LL2
ECACC
Uni Kansas
Medical Centre
ATCC
C57BL/6
Female
Lung
Spontaneous
DMEM, 10% FBS
5e
13 (11)
Yes
C57BL/6
Female
Ovary
Spontaneous in vitro
DMEM, 4% FBS, 0.2% ITS
-
-
No
C57BL/6
Female
Blood
DMBA
Human c-Ha-ras, HPV16
E6 and E7 in vitro in lung
epithelium
MCA
DMEM, 10% horse serum
RPMI 1640 (ATCC
modification), 10% FBS, 1%
NEAA
DMEM, 10% FBS
1e5
12
Yes
5e
4
15
No
1e5
15 or 26
Yes
ID8
EL4
TC-1
Johns Hopkins
Uni
C57BL/6
Female
Lung
P815
ATCC
DBA/2
Female
Mast cells
5e
5e
5e5
5
Supplementary Table S1: Cell line details
The cell line, supplier, genetic strain and sex of the mouse from which the cell line was derived, tissue of origin of the
tumor from which the cell line was originally derived, culture media for the cell line, number of cells injected
subcutaneously in 100µl PBS (unless otherwise stated) to form tumors and the day post implantation that samples were
collected for transcriptomic analysis or immune cell profiling by flow cytometry (values in brackets) and whether cells
were in vivo passaged during original derivation. NMU: N-nitroso-N-methylurethane, DMBA: 9,10-dimethyl-1,2benzanthracene, MCA: 3-methylcholanthrene
Supplementary Table S2
Cell line
B16F10 NCI
CT26
Hepa1-6
ID8
Renca
4T1
B16F0 ATCC
PAN02
EL4
P815
B16F0
ECACC
TRAMPC1
LL2
TRAMPC2
MC38
B16F10
ATCC
Homozygous
deletion = 0
copies
Heterozygous
deletion = 1 copy
Diploid
Gain= 3 copies
Amplification = 4
copies
-3.32
-3.32
-3.32
-3.32
-3.32
-3.32
-3.32
-3.32
-3.32
-3.32
-0.86
-0.86
-0.86
-0.86
-0.86
-0.86
-0.86
-0.86
-0.86
-0.86
0
0
0
0
0
0
0
0
0
0
0.6556
0.6337
0.585
0.6296
0.585
0.6034
0.7483
0.6748
0.585
0.7262
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
-3.32
-0.86
0
0.5901
0.93
-3.32
-3.32
-3.32
-3.32
-0.86
-0.86
-0.86
-0.86
0
0
0
0
0.6278
0.5987
0.6738
0.6439
0.93
0.93
0.93
0.93
-3.32
-0.86
0
0.6559
0.93
Supplementary Table S2: Copy Number Variation
Probe log2 ratios were converted to copy number variation (CNV) values by determining the median log2 ratio for duplicate
probes, mapping gene identities to the probes (mapping from Agilent_244A_014695_D_GeneList_20070207 using R),
calculating a median log2 ratio per gene and converting to a gene copy number value using cut offs determined by adjusting
absolute values for 10% heterogeneity and technical variance (-4.322 for homozygous deletion, -1 for heterozygous deletion,
0 for diploid and 1 for homozygous amplification). For gains, the minimum cut off value was set to 0.585 (3 copies absolute
value) and adjusted per cell line where 90% quantile log2 ratio values were higher. Copy number calls greater than 3 were
calculated from the log2 ratio. Genes with non diploid CNV calls and <5 probes were compared to their concomitant
segmentation calls (minimal log2ratio difference = 0.15, minimum marker count =4, Heuristic search p value cut off = 0.05
and significant segment p value cut off = 1e-5 (Omicsoft ArrayStudio)) and confirmed if amplification log2ratios were
matched to a gained segment, as were deletions with a loss segment. Where these did not match but there were 3 or 4 probes
the result was flagged for checking against chromosomal probe plots (Omicsoft ArrayStudio) while genes with <3 probes
were reset to diploid.
Supplementary Table S3
Abl1
Akt1
Alk
Apc
Atm
Braf
Brca1
Brca2
Cdh1
Cdkn2a
Cebpa
Csf1r
Ctnnb1
Egfr
Erbb2
Erbb4
Esr1
Ezh2
Fbxw7
Fgf2
Fgfr1
Fgfr2
Fgfr3
Flt3
Gata1
Gata2
Gna11
Gnaq
Hnf1a
Hras1
Idh1
Idh2
Igf1r
Igf2
Jak2
Jak3
Kdr
Kit
Kras
Map2k1
Mek1
Met
Mlh1
Mpl
Nf2
Notch1
Npm1
Nras
Pdgfra
Pik3ca
Ptch1
Pten
Ptpn11
Rb1
Ret
Runx1
Smad4
Smarcb1
Smo
Src
Stk11
Trp53
Vhl
Wt1
Supplementary Table S3: List of 64 genes investigated by targeted sequencing selected among commonly
mutated genes in cancer patients (based on proprietary datasets and the COSMIC database)
Supplementary Table S4
Antibody
Supplier
F4/80 Alexa Fluor 647
AbD Serotec
CD45 BV786
BD Biosciences
CD11b BUV395
BD Biosciences
CD8 BUV737
BD Biosciences
CD4 BUV395
BD Biosciences
CD3 BV650
Biolegend
Ly6C BV711
Biolegend
Ly6G Alexa Fluor 700
Biolegend
Granzyme B Alexa Fluor 647
Biolegend
NKp46 BV605
Biolegend
CD19 BV421
Biolegend
PD-1 PE-Alexa Fluor 610
eBioscience
Ki67 eFluor 450
eBioscience
Eomes PE-Cy7
eBioscience
FoxP3 PE
eBioscience
Supplementary Table S4: Fluorescent antibodies used
Target, fluorophore and supplier of the fluorescent antibodies used for immunoprofiling.
Supplementary Methods
Linear mixed-effect model
The effectiveness of the therapy (Tr) with respect to the baseline treatment performance is assessed with a linear mixed-effect model (1, 2). The Yij,
representing log10-transformed ith tumor volume observed at jth assessment point (T), follows the linear growth model:
Yij = a0i + a1i * Tj + eij,
where a0i and a1i denote individual intercept and slope parameters, respectively, and eij ~ N(0,σ) represents model error. Both intercept and slope are
assumed to express random effects:
a0i = g00 + g01*Tri + u0i,
a1i = g10 + g11*Tri + u1i.
with u0i ~ N(0, σ0) and u1i ~ N(0, σ1). The parameters g00, g10, and g01, g11 represent the parameter’s fixed-effects; σ, and σ0, σ1 correspond to intraand inter-tumor variance, respectively.
References
1.
Demidenko E. Statistical comparison of color cancer cell images. Oncol Rep. 2006;15 Spec no.:1077-9.
2.
Heitjan DF. Biology, models, and the analysis of tumor xenograft experiments. Clin Cancer Res. 2011 Mar 1;17(5):949-51.