a lung met 1 lung met 2 lung met 3 - media.nature.com · parental line 24hrs metastatic line 24hrs...
TRANSCRIPT
-1
-0.5
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2Lung Met H3K4me1
Lung Met H3K27ac
Primary H3K4me1
Primary H3K27ac
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Lung Met H3K4me1
Lung Met H3K27ac
Primary H3K4me1
Primary H3K27ac
-0.8-0.6-0.4-0.2
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Lung Met H3K4me1
Lung Met H3K27ac
Primary H3K4me1
Primary H3K27ac
-0.4!
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1.2!MNNG H3K4me1!MNNG H3K27ac!MNNG DNase!HOS H3K4me1!HOS H3K27ac!HOS DNase!
-0.2!
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1.2!MNNG H3K4me1!MNNG H3K27ac!MNNG DNase!HOS H3K4me1!HOS H3K27ac!HOS DNase!
-0.4!-0.2!
0!0.2!0.4!0.6!0.8!
1!1.2!1.4!1.6!1.8!
143B H3K4me1!143B H3K27ac!143B DNase!HOS H3K4me1!HOS H3K27ac!HOS DNase!
-0.2!
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1.2!143B H3K4me1!143B H3K27ac!143B DNase!HOS H3K4me1!HOS H3K27ac!HOS DNase!
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1.6!LM7 H3K4me1!LM7 H3K27ac!LM7 DNase!SaOS H3K4me1!SaOS H3K27ac!SaOS DNase!
-0.4!
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0!
0.2!
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1.2!
1.4!LM7 H3K4me1!LM7 H3K27ac!LM7 DNase!SaOS H3K4me1!SaOS H3K27ac!SaOS DNase!
-0.5
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2M112 H3K4me1M112 H3K27acM112 DNaseHu09 H3K4me1Hu09 H3K27acHu09 DNase
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1.4M112 H3K4me1M112 H3K27acM112 DNaseHu09 H3K4me1Hu09 H3K27acHu09 DNase
Lost Met-VELs
Gained Met-VELs Lost Met-VELs
b-1
-0.50
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Lung Met H3K4me1
Lung Met H3K27ac
Primary H3K4me1
Primary H3K27ac
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Lung Met H3K27ac
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Primary H3K27ac
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Lung Met H3K4me1
Lung Met H3K27ac
Primary H3K4me1
Primary H3K27ac
-1
-0.5
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2Lung Met H3K4me1
Lung Met H3K27ac
Primary H3K4me1
Primary H3K27ac
Gai
ned
Met
-VEL
sLo
st M
et-V
ELs
Gai
ned
Met
-VEL
sLo
st M
et-V
ELs
Lung Met 1 Lung Met 2 Lung Met 3
Lung
Met
5
MNNG 143B LM7
M11
2
a
Gained Met-VELs
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 1: Met-VEL profiles of osteosarcoma patient lung metastases and human osteosarcoma cell lines. a, Aggregate plots showing H3K4me1 ChIP-seq and H3K27ac ChIP-seq signal +/- 3Kb from mid-points of gained and lost Met-VELs in paired patient lung metastases and primary tumors. b, Aggregate plots showing H3K4me1 ChIP-seq, H3K27ac ChIP-seq and DNase-seq signal +/- 3Kb from mid-points of gained and lost Met-VELs in metastatic/parental human osteosarcoma cell lines.
Nature Medicine: doi:10.1038/nm.4475
a
c d
H3K
4me1
100kb
GHR
MG63 (Parental) MG63.3 (Metastatic)
Gained Met-VEL Cluster
0.5
0
Gained Met-VEL
H3K
4me1
100kb
COL23A1
MG63 (Parental) MG63.3 (Metastatic)
Lost Met-VEL Cluster
1.2
0
Gained Met-VEL
b
Lung Met
1
Lung Met
2
Lung Met
3
Lung Met
4
Lung Met
5
MG63.3
LM7M11
2
MNNG14
3B0
50
100
outside clusterswithin clusters
% T
otal
Met
-VEL
s
Lung Met
1
Lung Met
2
Lung Met
3
Lung Met
4
Lung Met
5
MG63.3
LM7M11
2
MNNG14
3B0
50
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outside clusterswithin clusters
% T
otal
Met
-VEL
s
Gained Met-VELs Lost Met-VELs
Lung Met
1
Lung Met
2
Lung Met
3
Lung Met
4
Lung Met
5
MG63.3
LM7M11
2
MNNG14
3B0
200
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1000Gained Met-VELsLost Met-VELs
Num
ber o
f Clu
ster
s
Scaled Coordinates Chrom
osom
es
COL23A1
16
0Lost Met-VELs
in 200kb window
MG63.3 Lost Met-VEL Clusters
WASF3LHFP
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 2: Met-VEL clusters occur across metastatic cancers. a. UCSC browser view of H3K4me1 profiles in MG63.3 (metastatic) and MG63 (parental) cell lines illustrating an example of a gained (left) and lost (right) Met-VEL cluster. Met-VELs identified by black bars. 200kb Met-VEL clusters highlighted in gray. b. Genome-wide lost Met-VEL landscape for MG63.3 cell line. Rows represent scaled chromosomal coordinates. Peaks represent maximum gained Met-VEL counts in 200kb sliding windows. Predicted target genes for selected peaks are labeled. c. Gained and lost Met-VEL cluster counts in patient lung metastases/primary tumors and metastatic/parental cell line pairs. d. Percentage of total gained (top) and lost (bottom) Met-VELs within and outside of clusters in patient lung metastases/primary tumors and metastatic/parental cell line pairs.
Nature Medicine: doi:10.1038/nm.4475
a
RNA-seq
24hrs 24hrs 14 days
Parental Metastatic b
Parental line 24hrs Metastatic line 24hrs Metastatic line day 14
MNNG Pair
−2
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Gained Met-VEL Genes
Gained Met-VEL Cluster Genes
LostMet-VEL Genes
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log2
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M)
*** *** *** ***** ***
*
143B Pair
log2
(FPK
M)
−2
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Gained Met-VEL Genes
Gained Met-VEL Cluster Genes
LostMet-VEL Genes
Lost Met-VEL Cluster Genes
****** *** ***
*
*
<3-7
<7-10
<10-13
<13-15
>15
-10*log p-value
embryonic morphogenesisextracellular matrixproteinaceous extracellular matrixtranscription factor activitytube developmentECM-receptor interactioncell motionenzyme linked receptor protein signaling pathwayextracellular matrix part
MG
63.3
MN
NG
143B
phosphoproteinnucleusembryonic skeletal system developmentacetylation
Lost Met-VEL Genes
FOS
FOSL1
JUN
JUNDJUNB
MG63.3 MNNG 143B
Lost
Met
-VEL
Mot
ifs
MG
63.3
MN
NG
X143B
MAFFMAFAATF2TFCP2ZNF711MYCNFIBATF4TCF12SMAD4ZIC2E2F1E2F6TFAP2CZBTB7BTFAP4CTCFZIC1NFIXTFAP2ANFE2L2MAFBACH1JDP2JUNFOSUBP1JUNDJUNBFOSL1
UBP1
JDP2BACH1MAFNFE2L2TFAP2ANFIXZIC1CTCFTFAP4ZBTB7BTFAP2CE2F6E2F1ZIC2SMAD4TCF12ATF4NFIBMYCZNF711TFCP2ATF2MAFAMAFF
Color Key-10*log(p-value)
100 600
MN
NG
143B
Normalized Expression
-3 0 3
Met Day 1Met Day 14Parental Day 1
Met Day 1Met Day 14Parental Day 1
Early Constitutive Late
Early Constitutive Late
c
d
e
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 3: Assessment of Met-VEL associated gene expression during metastatic colonization of the lung. a. Schematic of experimental design for assessment of Met-VEL gene expression in parental and metastatic cell lines in ex vivo lung metastasis model. Image adapted from 29. b. Log2 quantile-normalized FPKM values for gained (left) and lost (right) Met-VEL and Met-VEL cluster genes in HOS/MNNG (top) and HOS/143B (bottom) cell line pairs. Asterisks indicate significant differences in FPKM distributions between parental and metastatic cell lines (* P<0.05; ** P<1E-3; *** P<1E-4). P-values calculated by Mann-Whitney Test. c. Heatmap of up-regulated gained Met-VEL genes in HOS/MNNG (top) and HOS/143B (bottom) cell line pairs. d. Expressed transcription factors with enriched motifs in lost Met-VELs in three metastatic/parental cell line pairs and corresponding motif enrichment p-values. e. Gene Ontology (GO) terms for lost Met-VEL genes in three metastatic/parental cell line pairs and corresponding p-values.
Nature Medicine: doi:10.1038/nm.4475
MG63.3
MNNG14
3B0
500
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Top up-regulated genesOverlapGained Met-VEL Genes
Num
ber o
f Gen
es
Gained Met-VELs
MG63.3
MNNG14
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Top up-regulated genesOverlapGained Met-VEL Genes
Num
ber o
f Gen
es
−4
−3
−2
−1
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p=1.74E-5
p=0.0017
−2
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MG63.3
log2
(fold
cha
nge
expr
essi
on)
p=0.076
p=8.5E-4
Met vs. Parental in vitro
Met 24hrs lung vs. Parental 24hrs lung
Met 14 days lung vs. Parental 24hrs lung
Met vs. Parental in vitro
Met 24hrs lung vs. Parental 24hrs lung
Met 14 days lung vs. Parental 24hrs lung
−2
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p=0.057
p<2.2E-16
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p<2.2E-16
p=0.15
−4
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p=1.12E-4
p=9.33E-5
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p=4.12E-12
a b
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Top down-regulated genesOverlapLost Met-VEL Genes
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f Gen
es
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MNNG14
3B0
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Top down-regulated genesOverlapLost Met-VEL Genes
Num
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f Gen
es
Lost Met-VELs
��
−6−4
−20
24
6
−6−4
−20
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6
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−20
24
6
cMG63.3 Pair MNNG Pair 143B Pair
log2
(fold
-cha
nge
expr
essi
on m
et. v
s. n
on-m
et)
All Expressed Genes Gained Met-VEL Genes Lost Met-VEL Genes
0
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MNNG 143B MG63.3 MNNG 143B
Gained Met-VEL Genes Lost Met-VEL Genes
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 4: Assessment of Met-VEL associated gene expression in vitro and in ex vivo lung culture. a. Log2 fold-change in quantile-normalized FPKM values of gained Met-VEL genes in metastatic cell lines versus parental cell lines in various conditions. P-values calculated by Mann-Whitney Test. b. Log2 fold-change in quantile-normalized FPKM values of lost Met-VEL genes in metastatic cell lines versus parental cell lines in various conditions. P-values calculated by Mann-Whitney Test. c. Violin plots of log2(fold-change quantile-normalized FPKM values) distributions for gene sets in three metastatic cell lines relative to parental non-metastatic lines. Fold-change values for all expressed genes and gained Met-VEL gene sets represent comparisons of expression in metastatic lines at day 14 in ex vivo lung culture to non-metastatic lines at 24hrs. Fold-change values for lost Met-VEL gene set represent comparison of expression in metastatic lines at 24hrs in ex vivo lung culture to non-metastatic lines at 24hrs. d. Bar charts indicating overlap between gained (left) and lost (right) Met-VEL gene sets and top 1000 up- and down-regulated genes in corresponding conditions, respectively.
Nature Medicine: doi:10.1038/nm.4475
Urothelial
Embyronic
EndothelialEpithelial
tube
Angiogenesis andEndothelial cell migrationRegulation of
cell migration
Neg. regulationof cell migration
Adherensjunction
Epithelial cellmigration Epithelial
cell proliferation
Growth
Pathways in cancer
Developmental Programs
Signaling Pathways
WNTHippo
Rap1
ERBB IL-6response
Ossification
Regulationof cell shape
Actin cytoskeletonorganization
Protein localizationto plasma membrane
Proteindephosphorylation
Negative regulationof protein phosphorylation
Node Color = FDR
0.05 <0.0050.01
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 5: Assessment of enriched functions of gained Met-VEL genes in osteosarcoma patient metastases. Enriched Map representation of all Gene Ontology (GO) terms for gained Met-VEL genes calculated by aggregating gene lists from two patient metastases (Lung Met 4 and Lung Met 5).
Nature Medicine: doi:10.1038/nm.4475
All Acti
vated
Genes
Lost Met-
VEL Genes
Activat
ed Gain
ed M
et-VEL Gen
es
Activat
ed Gain
ed M
et-VEL Gen
es no SEs
SE Genes
-15
-10
-5
0
5
10143B
% R
educ
tion
in E
xpre
ssio
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* *
All Acti
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Genes
Lost Met-
VEL Genes
Activat
ed Gain
ed M
et-VEL Gen
es
Activat
ed Gain
ed M
et-VEL Gen
es no SEs
SE Genes
-10
0
10
20
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40MNNG
% R
educ
tion
in E
xpre
ssio
n
**
*
143B
0 5 10 150
1
2
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4
250nM JQ1Vehicle
Time (days)
Nor
mal
ized
Met
asta
tic B
urde
nMNNG
0 5 10 150
1
2
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4
250nM JQ1Vehicle
Time (days)
Nor
mal
ized
Met
asta
tic B
urde
n
p=7.45E-8
p=1.73E-5
250nM JQ1Vehicle
MNNG
Vehicle 250nM JQ1
Mou
se 1
Mou
se 2
Vehicle 250nM JQ1
Mou
se 1
Mou
se 2
143B
ba
e
dMNNG Activated Gained Met-VEL Genes
Vehicle Treated JQ1 Treated
0
-0.6
NES = -1.83FDR < 0.005
ES
143B Activated Gained Met-VEL Genes
Vehicle Treated JQ1 Treated
0.2
-0.4
NES = -1.27FDR < 0.005
ES
0
−2
0
2
4
p<2.20E-16
MNNG Activated Met-VEL Gene Expression
Log2
(fold
-cha
nge
vs. p
aren
tal l
ine)
4
2
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-2 −2
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143B Activated Gained Met-VEL Genes
Log2
(fold
-cha
nge
vs. p
aren
tal l
ine)
4
2
0
-2
f
cDay 15 Vehicle
Day 15 250nM JQ1
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 6: Analysis of anti-metastatic and gene expression effects of BET inhibition with JQ1. a. Kinetics of metastatic outgrowth of MNNG (top) and 143B (bottom) metastatic cell lines in ex vivo lung culture with 250nM JQ1 or vehicle (DMSO) treatment. Metastatic burden measured as total GFP+ area per lung section normalized to GFP+ area on day 0. Values represent averages of 8 lung sections (4 sections per mouse x 2 mice) +/-SEM. P-value calculated by Mann-Whitney Test. Dashed line indicates time points chosen for RNA-seq studies. b. Representative 2.5x images of vehicle (left) and 250nM JQ1 treated (right) lung sections at day 15. Lung sections outlined with dashed white line. Scale bar = 500µm. c. 20X image of hematoxylin and eosin stained section of lung slice after 15 days in ex vivo culture treated with DMSO (top) and 250nM JQ1 (bottom) illustrating viable lung cells and architecture. Scale bar = 100µm. d. GSEA plots of 2-fold up-regulated gained Met-VEL gene sets in vehicle versus JQ1-treated MNNG (left) and 143B (right) cells isolated from ex vivo lung culture. Cells isolated at time points indicated by dashed lines in Supplemental Figure 2-5a. e. Log2 fold-change expression 2-fold up-regulated gained Met-VEL gene sets in vehicle versus JQ1-treated MNNG (left) and 143B (right) cells isolated from ex vivo lung culture at time points indicated by dashed lines in Extended Data Figure 7a relative to parental cell line. f. Percent reduction in gene expression with 250nM JQ1 treatment of all genes up-regulated 2-fold in metastatic cell lines relative to parental cell lines at sorting time points, lost Met-VEL genes, gained Met-VEL genes 2-fold up-regulated relative to parental cell lines at sorting time points, up-regulated gained Met-VEL genes without SE genes, and all SE genes in MNNG and 143B cells growing in ex vivo lung culture sorted at time points indicated in Extended Data Figure 7a.
Nature Medicine: doi:10.1038/nm.4475
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 7: Assessment of LT3REPIR shRNA construct induction and leakiness. a. Schematic of doxycycline-inducible LT3REPIR shRNA construct. Modified from (Fellmann et al., 2013). b. Cytometric analysis of GFP and DsRed expression in GFP+ MG63.3 cells transduced with LT3REPIR construct at baseline (left) and 40hrs after exposure to 5ug/ml doxycycline (right).
Nature Medicine: doi:10.1038/nm.4475
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 8: Assessment of Tissue Factor (F3) dysregulation in metastatic osteosarcoma. a. IGV browser view of H3K27ac, H3K4me1, and DNase profiles at F3 gained Met-VEL cluster locus in MG63.3 (metastatic) and MG63 (parental) cell lines. Top of figure shows local contact profile analysis of F3 locus in MG63.3. In the top panel (main trend), the contact intensity (black line) is calculated by using a running mean analysis of normalized read counts with a 1kb sliding window. The 20th and 80th percentile are visualized as a gray trend graph. In the bottom panel, contact intensities are computed using linearly increasing sliding windows (scaled 2-50 kb) and are displayed as a color-coded heatmap of positive 4C signals (maximum of interaction set to 1). Local color changes are log-scaled to indicate changes of statistical enrichment of captured sequences, corresponding to the enhancer-promoter interaction. Areas of significant contact highlighted. b. Fold-change quantile normalized F3 FPKM values in 3 metastatic cell lines at 24hrs and 14 days of metastatic outgrowth in ex vivo lung culture relative to parental line at 24hrs. c. Tissue Factor (F3) relative expression in human patient primary tumors and lung metastases normalized to expression in normal osteoblasts. d. IGV browser view of H3K27ac, H3K4me1, and DNase profiles at F3 gained Met-VEL cluster locus in MG63.3, MNNG, and 143B metastatic cell lines.
Nature Medicine: doi:10.1038/nm.4475
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 9: F3 expression in human osteosarcoma tumors. Immunohistochemical staining of F3 in human osteosarcoma lung metastases and primary tumors. Scale bars = 200µm.
Nature Medicine: doi:10.1038/nm.4475
F3
Pa
tie
nt
Lu
ng
Me
ts
MG63.3 DHS
MG63 H3K27ac
MG63.3 H3K27ac
ESC TADs
IMR90 TADs
−0.4 −0.2 0.0 0.2 0.4 0.6
0.0
0.5
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mean(met)−mean(non_met)
−log
10(w
ilcox
p_v
al)
SEs gained in MetsSEs lost in Mets
F3
a.
b.
711 kb
chr1
p36.32 p36.22 p36.13 p36.11 p35.1 p34.2 p33 p32.3 p32.1 p31.2 p31.1 p22.3 p22.1 p21.2 p13.3 p13.1 p11.1 q12 q21.1 q21.3 q23.1 q23.3 q24.2 q25.1 q25.3 q31.1 q31.3 q32.1 q32.2 q41 q42.11 q42.2 q43 q44
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 10: Assessment of Tissue Factor (F3) Met-VELs in patient lung metastases. a. IGV browser views of the F3 locus showing H3K27ac ChIP-seq tracks of lung metastases from 10 osteosarcoma patients. Blue bars below each track correspond to super enhancers defined using the ROSE script. Hi-C defined topologically associating domains (TADs) are displayed below. b. Volcano plot of 5571 total super enhancers detected in all osteosarcoma patient samples and cell lines used in this study. Points marked in red denote super enhancers meeting the threshold of significance (P < 0.05) for being gained or lost in metastatic samples. Points in grey denote super enhancers below the significance threshold. The F3 super enhancer is indicated by the arrow.
Nature Medicine: doi:10.1038/nm.4475
a b
Untrea
ted
RFP+ Frac
tion
Untrea
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RFP+ Frac
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Untrea
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RFP+ Frac
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shF3B
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Rel
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e G
APD
H E
xpre
ssio
n-D
ox v
s. +
Dox
-Dox +Dox -Dox +Dox
shF3-AshF3-B
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 11: Assessment of F3 knockdown by RT-qPCR. a, Amplification plots and standard curves of GAPDH RT-qPCR using 0.2ng, 2ng, 20ng, and 200ng of template cDNA demonstrating efficiency value of 100% and R2 value of 1.000. All Cq values used for quantification of GAPDH were confirmed to be within linear range of standards. b, Amplification plots and standard curves of F3 RT-qPCR using 0.2ng, 2ng, 20ng, and 200ng of template cDNA demonstrating efficiency value of 97.2% and R2 value of 0.999. All Cq values used for quantification of F3 were confirmed to be within linear range of standards. c, Relative GAPDH expression in DsRed+ (induced) cells transduced with F3 shRNA constructs after 40hrs treatment with 5ug/ml doxycycline relative to uninduced controls (average of 3 replicates +/-SEM). d, Relative F3 expression normalized to GAPDH in DsRed+ (induced) cells transduced with F3 shRNA constructs after 40hrs treatment with 5ug/ml doxycycline relative to uninduced controls (average of 3 replicates +/-SEM).
Nature Medicine: doi:10.1038/nm.4475
shF3A
no do
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shF3A
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l dox
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urde
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urde
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Nor
mal
ized
Met
asta
tic B
urde
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P=0.0003 P=0.0002 P=0.0013 P=0.0003
MG63.3 MNNG
-Dox +DoxInducible shF3A
-Dox +DoxInducible shF3B
-Dox +DoxInducible shF3A
-Dox +DoxInducible shF3B
b
c
Inducible shF3A Inducible shF3BMG63.3
-Dox
+Dox
(F3
kd)
MNNGInducible shF3A Inducible shF3B
Uninduced F3 kd26
27
28
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31
n=5 mice per condition
-Dox +Dox (F3 kd)
P<0.0001
log2
[tota
l are
a pe
r 2.5
x fie
ld(p
ixel
s)]
-Dox +Dox (F3 kd)
Mou
se 1
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se 2
Mou
se 3
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e
MG63.3 shF3A
0 50 1000
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MNNG shF3B untreatedMNNG shF3B 5ug/ml dox
a
Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 12: Assessment of F3 knockdown on metastatic osteosarcoma cell in vitro growth and lung colonization. a. Change in confluence relative to day 0 of MG63.3 (top) and MNNG (bottom) cells transduced with F3 shRNA constructs grown in standard culture conditions over 108hrs in the presence or absence of 5ug/ml doxycycline. Values represent averages from 6 plates +/- SD. b. Representative 2.5x images of from day 21 of ex vivo lung culture sections of GFP+ MG63.3 (left) and MNNG (right) cells transduced with F3 shRNA constructs untreated (top) or treated with 5ug/ml doxycycline (bottom). Lung sections outlined with dashed white line. Scale bar = 500µm. c. Quantification of metastatic burden at day 21 of ex vivo lung culture sections of GFP+ MG63.3 (left) and MNNG (right) cells transduced with F3 shRNA constructs untreated (red) or treated (blue) with 5ug/ml doxycycline. Values represent averages +/-SEM from 8 sections per condition (4 sections per mouse x 2 mice) normalized to the same section at day 0. P-Value calculated by Mann-Whitney Test. d. Representative 2.5x images of in vivo metastatic burden in untreated (left) or doxycycline-treated (right) mice receiving tail vein injection of 5x105 GFP+ MG63.3 cells transduced with shF3B construct. Scale bar = 500µm. e. Quantification of in vivo metastatic burden in untreated (red) or doxycycline-treated (blue) mice receiving tail vein injection of 5x105 GFP+ MG63.3 cells transduced with shF3B construct. Values represent log2 of total GFP+ area per 2.5x field, black bars represent average +/- SEM (N= 5 mice per condition, 5 images per mouse). P-Value calculated by Mann-Whitney Test.
Nature Medicine: doi:10.1038/nm.4475
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Nature Medicine: doi:10.1038/nm.4475
Supplementary Figure 13: Assessment of lung metastatic burden at experimental end point of orthotopic spontaneous metastasis experiment with F3 knockdown. a. All images used for quantification of in vivo metastatic lesions in lungs 21 days after measureable tumor formation in untreated mice receiving orthotopic injection of 8x105 MG63.3 cells transduced with shF3B construct (5 mice per condition, 5 images per mouse). Scale bar = 250µm. b. All images used for quantification of in vivo metastatic lesions in lungs 21 days after measureable tumor formation in doxycycline-treated mice receiving orthotopic injection of 8x105 MG63.3 cells transduced with shF3B construct (5 mice per condition, 5 images per mouse). Scale bar = 250µm.
Nature Medicine: doi:10.1038/nm.4475
Gene Inclusion Criteria Metastasis Specific?BACH2 Enriched Motif NDOCK4 Common Gained Met-VEL N
Gained Met-VEL Cluster (MG63.3)F3 Gained Met-VEL Cluster (MG63.3) YFBXO42 Gained Met-VEL Cluster (MNNG/143B) YFLNA Common Gained Met-VEL Y
Gained Met-VEL Cluster (MNNG/143B)FOS Enriched Motif YFOSL1 Enriched Motif YFOXO3 Gained Met-VEL Cluster (MNNG/143B) YGHR Gained Met-VEL Cluster (MG63.3) NJUN Enriched Motif NJUNB Enriched Motif NRASSF2 Common Gained Met-VEL NTMEM230 Common Gained Met-VEL N
Nature Medicine: doi:10.1038/nm.4475
Supplementary Table 1: Candidate metastasis dependency genes identified by in vivo high-throughput RNAi functional assay.
Nature Medicine: doi:10.1038/nm.4475