Supporting Information: Figure Legends (Kudo-Saito Et Al

Supplementary Figure Legends (Kudo-Saito et al.)

Fig. S1

Gating of cells for flow cytometric analysis. Before analysis of specific molecular expressions, cells were firstly gated except debris and dead cells using FSC and SSC. DCs were analyzed after gating leukocyte fraction (blue range) including almost of all leukocytes such as T cells, neutrophils, monocytes and DCs. T cells including CD4+FOXP3+ Treg cells were analyzed after gating lymphocyte fraction (green range). The distribution pattern of murine immune cells was basically similar to that of human PBMCs.

Fig. S2

Human pancreatic cancer Panc1 cells acquire EMT-like characteristics including low-adhesive but high- invasive abilities accompanied by decreased epithelial markers and increased mesenchymal markers after human snail cDNA transduction (D6, D10, F3 and F5 clones). a, The mRNA expression was analyzed by RT-

PCR (NC, negative control), and the intensity of the representative EMT markers was normalized to GAPDH expression as a control. b, These tumor cells were stained with mAb specific for Snail, E-cadherin or

Fibronectin (scale bar = 0.5 mm). Microscopic observation shows the shape of the Snail transfectants are spindle and dendritic. c, The tumor proliferation, adhesion and invasion were evaluated (n=3, mean ± SD).

Data shown in each panel are representative of three independent experiments.

Fig. S3

CCL2 and LCN2 are also involved in murine Snail-induced mechanisms. a, CCL2 and LCN2 are increased in murine melanoma B16-F10 cells after murine snail cDNA transduction (E9 and H6 clones). b, LCN2 production is regulated by CCL2. H6-snail+ tumor cells were transfected with siRNA specific for murine snail or ccl2 (CCL2 #1 or #2), or Control siRNA. The mRNA expression was analyzed by RT-PCR. CCL2 and

LCN2 in the 3-day-cultured supernatant were measured by ELISA (n=3, mean ± SD). c, Treg cells are induced by Snail+ tumor-derived CCL2. Naive SPCs were stimulated with tumor supernatant for 5 days, and

1 were analyzed for Foxp3 expression in the CD4+ cells by flow cytometry. Data shown in each panel are representative of three independent experiments.

Fig. S4

Anti-tumor efficacy of siRNA-ccl2 injection on Snail-/low B16-F10 tumors. C57BL/6 mice were both s.c. and i.v. implanted with B16-F10 parental tumor cells, and were i.t. injected with PEI-complexed siRNA specific for murine snail or ccl2, or Control siRNA on day 7 after tumor implantation (n=5 per experiment). On day

17, tumor volume was evaluated, and the s.c. tumors and spleens were harvested for assays. a, Suppression of tumor growth by siRNA-ccl2 injection (n=5, mean ± SD). b, Anti-tumor effector cells are increased in the siRNA-ccl2-injected tumors. Tumor-infiltrating cells were analyzed by flow cytometry. The percentage of each cell fraction is indicated in dot plot. Histogram shows the percentage of Foxp3+ cells in the CD4+ cells, or the percentage of gp70-tetramer+ cells in the CD8+ cells. In parentheses, the intensity (MFI) of Foxp3 in the

CD4+Foxp3+ T cells, or of the gp70 tetramer-binding in the CD8+ T cells is indicated. Each flow cytometric data is one of the data. c, CTL activity is enhanced in the mice receiving the siRNA-ccl2 injection. Splenic

CD8+ T cells were stimulated with gp70 peptide in the presence of APCs for 24 hrs, and the IFN-γ in the supernatant was measured by ELISA (n=3, mean ± SD). Data shown in each panel are representative of three independent experiments.

Fig. S5

Increase of anti-tumor effector cells in the siRNA-ccl2-injected Snail+ tumors. C57BL/6 mice were both s.c. and i.v. implanted with F10-mock or H6-snail+ tumor cells, and the H6-snail+ tumors were i.t. injected with

PEI-complexed siRNA specific for murine snail or ccl2, or Control siRNA on day 7 after tumor implantation

(n=5 per experiment). The s.c. tumors were harvested, and the tumor-infiltrating cells were analyzed by flow cytometry on day 17 (n=3, pooled). The percentage of each cell fraction is indicated in dot plot. Histogram shows the percentage of Foxp3+ cells in the CD4+ cells, or the percentage of gp70-tetramer+ cells in the CD8+ cells. In parentheses, Foxp3 MFI in the CD4+Foxp3+ T cells, or the gp70 tetramer-binding MFI in the CD8+ T

2 cells is indicated. Each flow cytometric data is one of the data. Data shown in each panel are representative of three independent experiments.

Fig. S6

A new mechanism induced by CCL2 produced from Snail+ tumor cells undergoing EMT. Tumor cells release

CCL2 after Snail acquisition during EMT. The Snail+ tumor-derived CCL2 expands EMT cascade in the tumor microenvironment, and also induces LCN2 autocrinely. Combination of CCL2 and LCN2 generates immunosuppressive CD11c+HLA-DRlow DCreg cells, and subsequently induces CD4+Foxp3+ Treg cells.

CD4+Foxp3+CCR2+ Treg cells are the first runner in response to CCL2 in the tumor microenvironment. Other

CCR2+ cells such as MDSCs and TAMs also take part in this mechanism. All of these events lead to impairment of tumor-specific CTL induction, and finally promote tumor progression including tumor metastasis.