Kudo-Saito, et al.

F10-mock F10-snail+

R1R1

(N t )

F10-mock F10-snail+

Mice implanted with (s.c.+i.v.)

SCbo

nes Naive mice

r (s

.c.)

57.56 80.73

A

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FL1-Height

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R1

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R1

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R1

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(No tumor) SS

Leg

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2.31

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7.03

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5.02

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R1

GFP+ t ll

3.170.29

Spl

een

Lung

Spl

een

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GFP+ tumor cells

TU SP LN LG BM KD

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Mice mRNA

B

F10-mock F10-snail+

s.c. tumor

Gp100GAPDH

Gp100GAPDH

Gp100GAPDH

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#1

#2

#3

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25.6020.62

Naive F10-mock F10-snail+

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#2

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SS

C

Tum

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-

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Gp100GAPDH

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#3

#4

#5

Figure S1. Snail+ tumor cells frequently metastasize into bone marrow. A Snail+ tumor cells are highly metastatic particularly to bone marrow (BM) Murine melanoma GFP+ F10-snail+ cells or GFP+ F10-mock cells were implanted

GFP- cells(no tumor)

A, Snail tumor cells are highly metastatic, particularly to bone marrow (BM). Murine melanoma GFP F10 snail cells or GFP F10 mock cells were implanted both subcutaneously (s.c., 5x105) and intravenously (i.v., 1x105) into C57BL/6 mice (n=5 per experiment). Two weeks later, cells isolated from the s.c. tumors, BM, spleen, and lung were microscopically observed (scale bar = 100 µm), and were also analyzed for the GFP+ tumor cells by flow cytometry (n=3, pooled). Photos show the representative mice having black melanoma metastases in bones, inguinal and popliteal lymph nodes (arrows), and lungs. B, Snail+ tumor cells spontaneously migrate into BM even after s.c. implantation. F10-snail+ or F10-mock cells were implanted only s.c. into mice (n=5). Three weeks later, cells isolated from the s.c. tumors (TU), spleen (SP), inguinal lymph node (LN), lung (LG), BM and kidney (KD) were analyzed for gp100 mRNA expression by RT-PCR. C, CD45- mesenchymal cells are increased in the Snail+ tumor-implanted mice. The isolated cells obtained from the same mice used in A were analyzed by flow cytometry (n=3, pooled). This data is shown after gating GFP- cell fraction to eliminate tumor cells. Data in each panel are representative of three independent experiments.

A FABP4+ adipocytes

Kudo-Saito, et al.

CD45- ALCAM+ DIP2A+

A FABP4+ adipocytes

CD146+ CD271+ IL10RB+

CD45 ALCAM+ DIP2A+

B Osteocalcin+ osteoblasts

CD45-

CD146+ CD271+

ALCAM+ DIP2A+

IL10RB+

Figure S2. Differentiation activity of the specific subpopulations isolated from the Snail+ tumor-stimulated BMCs. BMCs were stimulated with F10-snail+ supernatant for 10 days, and the sorted subpopulations were tested for differentiation activity into FABP4+ adipocytes (A) or Osteocalcin+ osteoblasts (B). Scale bar = 200 µm. Data are representative of two independent experiments.

Kudo-Saito, et al.

Tumor cells + BMCs treated withA

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20.97 1.87 2.795.05 57.72 78.62

59.88 7.44 7.51

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#1 #2 #3 #4 #5 #6 #7

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GAPDHF10-snail+

Figure S3. The Snail+ tumor-induced MSCs promote tumor metastatic seeding into bone. BMCs obtained from C57BL/6-CAG-EGFP mice were stimulated with none or F10-mock or F10-snail+ supernatant for 10 days. The sorted CD45- cells (3x105) were mixed with F10-mock tumor cells (3x105), and were s.c. injected into mice (n=7 per experiment). Twenty-five days later,cells (3x10 ), and were s.c. injected into mice (n 7 per experiment). Twenty five days later, spleen volume was measured (mean ± SD), and tumor-infiltrating cells (A) and spleen cells (B) were analyzed by flow cytometry. According to the data, the number of EGFP+ BMCs and the CD45- MSCs were calculated (mean ± SD). As another method to confirm the spontaneous bone metastasis, a part of BMCs were analyzed for gp100 mRNA expression by RT-PCR, and another part of BMCs were cultured in 10% FCS-supplemented DMEM for 14 days (C). Data in each panel are representative of three independent experiments.

Kudo-Saito, et al.

A

Snail

FSTL1

DIP2A

Mo

ck

Sna

il+A

Sna

il+B

B16-F10

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3

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FSTL1

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GAPDH

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M M U K K M

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Figure S4. Increase of fstl1 and its receptor dip2a mRNA expressions in Snail+ tumor cells. A, Snail transduction induces fstl1 and its receptor dip2a in murine and human melanoma cells. Two Snail transfectants (Snail+ A and Snail+ B) and the mock transfectant were used. B, Endogenous fstl1and dip2a are also increased in various human Snail+ tumor cells. Data in each panel are representative of two independent experiments.

Kudo-Saito, et al.

rol

TL1

#1

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0 20 40 60

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MDA231 supernatant

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CD271

ALC

AM

8.76 1.14

0.53

12.01 11.64

10.08

9.75 4.64

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13.18 7.52

3.90

GFP+ MDA231 tumor cells + ALCAM+ human PBMCs

Figure S5. FSTL1-knockdown suppresses metastatic seeding of human tumor cells into bone. Human breast cancer Snail+FSTL1+ MDA231 cells were transfected with siRNA-fstl1 or control siRNA, and the mRNA expression was analyzed by RT-PCR (A). FSTL1 in the cultured supernatant was measured by ELISA (n=3 mean ± SD) and CCR2 CXCR4 and RANKL expressions were analyzed by flow cytometry (B) These(n=3, mean ± SD), and CCR2, CXCR4 and RANKL expressions were analyzed by flow cytometry (B). These cells were tested for matrigel invasive ability and proliferative ability in vitro, and also for metastatic ability in vivo (n=3, mean ± SD; C). In the assay in vivo, GFP+ MDA231 cells were intracardinally implanted into BALB/c nu/nu mice, and 3 weeks later, BMCs were analyzed for the GFP+ tumor cells by flow cytometry. In D, human PBMCs were stimulated with MDA231 supernatant or FSTL1 for 7 days, and the isolated ALCAM+ cells (1x106) were mixed with GFP+ MDA231 cells (1x106) and s.c. injected into BALB/c nu/nu mice (n=5 per experiment). Sixty days later, BMCs were microscopically observed, and were analyzed for ALCAM+ cells by flow cytometry. CD271 is also a human MSC marker reported in literature. Data in each panel are representative of two independent experiments.

Kudo-Saito, et al.

Primary tumor

CCR2CXCR4RANKRANKL

FSTL1 Tumor hominginto bone marrow

Escalation ofbone metastasis

+ CCL2

G ti f i i d CD8l T ll

Expansion of CD45-ALCAM+ sMSCs

Bone marrow

Periphery

& sMSC expansion

sMSC dissemination

Generation of impaired CD8low T cells

Figure S6. FSTL1-induced cancer bone metastasis mechanism. In cancer bone metastasis, FSTL1 that is produced from tumor cells undergoing Snail-induced EMT promotes immune dysfunction utilizing CD45-ALCAM+ MSC-like cells (sMSCs). FSTL1 is a trigger to cause tumor metastatic seeding to bone where is rich in BMCs as a source of sMSCs through elevation of various chemokine receptors i th ti l t d t ll FSTL1 l lif t d id t MSC ithi thin the stimulated tumor cells. FSTL1 also proliferated resident sMSCs within the primary tumor microenvironment. These sMSCs disseminate all over the body, and induce not only de novo tumor bone metastasis that is followed by further increase of sMSCs, but also CTL dysfunction via generation of impaired CD8low T cells. All these events accelerate cancer bone metastasis.