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Supplementary Figure 1
Supplementay Figure 1. Acquired CAF phenotype by conditioned media of gastric cancer cell was
maintained after removal of CM. The expressions of CAF markers in gastric normal fibroblasts (A)Hs738 and
(B)NF#19 were induced by incubation with conditioned media derived from SNU668, gastric cancer cell line, for 48 hours.
Then conditioned media was removed and cultured for more 5 days.RT-PCR analysis showed that CAF phnotype was
maintained even after removal of CM. (-), negative control. CM, incubation in conditioned media of SNU668 for 2days.
CM->(-), Removal of CM after 2days then incubation with normal cell culture condition for 5days. *P<0.05; **P<0.01;
***P<0.001
Supplementary Figure 2
1.5
NF#5
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+ IL6 50ng/ml +IL6 100ng/ml
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+IL6
+STAT inhibitor
10nM 20nM
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CAF#32
Supplementary Figure 2. Densitometric analysis of western blots. Densitometric analysis of three
independent experiments was performed with Image J software. The graph demonstrates results of western blot
in Figure 1, 3, and 4. (A) densitometric analysis of Fig.1F, (B) densitometric analysis of Fig.1G, (C)
densitometric analysis of Fig.1H, (D) densitometric analysis of Fig.3A and (E) densitometric analysis of Fig.4B.
*P<0.05; **P<0.01; ***P<0.001
Supplementary Figure 3
Supplementary Figure 3. The IL-6 cytokine induces the generation of Twist1 positive CAFs in vivo.
Stomach cancer cells MKN74 were transduced with GFP and GFP/IL-6 lentivirus. (A) IL-6 expression was confirmed by
western blot after lentivirus infection. IL-6 induced tumor growth compared with that of control cells (n=8 mice per group).
(*),p<0.01 versus control (B) Immunofluorescence staining of Twist1 positive stromal cells (red) co-localized with IL-6
expressing GFP+ cancer cells (green) in graft models. Twist1 positive cells (red) secreted tenascin-c (green) (right panel).
Nuclei were visualized by staining with Dapi (blue). (C) Immunofluorescence intensity analysis(right) of Twist1(red) and
Tenascin-C(green).
Supplementary Figure 4A
Supplementary Figure 4A. Overexpression of Twist1 induced the attributes of CAFs. Proliferation of stomach
cancer cells, MKN28, MKN74,after incubation with the CM from GFP and Twist1 expressing NFs and CaFs. The data are
shown as mean ± SD of the number of cells invaded or migrated in n>3 separate experiments.
Supplementary Figure 4B
Supplementary Figure 4B. H&E staining of xenograft of two gastric cancer cell lines (SNU668 and
SNU638) to immune compromised mice.
A. The microscopic feature of tumor formed by SNU668 only, SNU668 with control gastric fibroblast (SNU668 + Hs738GFP), SNU668 with Twist1 expressing fibroblast (SNU668 + Hs738-Twist1)
B. The microscopic feature of tumor formed by SNU638 only, SNU638 with control gastric fibroblast (SNU638 + Hs738GFP), SNU638 with Twist1 expressing fibroblast (SNU638 + Hs738-Twist1)
Supplementary Figure 5
Supplementary Figure 5. Correlation between Twist1 and CXCL12 expression in various cancers using
mRNA expression data from TCGA. Significance correlations were present in most tumors.
Supplementary Figure 6
Supplementary Figure 6. Survival of patients with CXCL12 expression in cancer cells in gastric cancer.
CXCL12 expression in cancer cells has no significant effect on gastric cancer patients’ prognosis. There was no significant
difference in survival rate between patients with high expression of CXCL12 in cancer cells and those with low expression
of CXCL12.
Supplementary Figure 7
Supplementary Figure 7. Suppression of CXCL12 attenuates ability of tumor progression during Twist1induced activation in gastric normal fibroblasts. (A) Invasion, migration and (B)proliferation of gastric cancer cell
MKN28, MKN74 and SNU668 after incubation with the conditioned media from NF-GFP, NF-Twist1 and NF-Twist1 cells
expressing shCXCL12-1, ShCXCL12-2 or ShNS. *P<0.05; **P<0.01