Supporting Information O’Connell et al. 10.1073/pnas.1109493108 SI Materials and Methods software. For quantification of microvessel density, regions of Cells and Transgenic Mice. 4T1 mammary carcinoma cells and CT26 highest vessel density were first identified at low magnification colorectal cancer cells were grown in DMEM supplemented with (magnification of 10×). Then, under high magnification (mag- 10% (vol/vol) FBS and 100 U/mL penicillin/streptomycin. The nification of 40×), microvessel density was determined by + S100A4-GFP (1), S100A4-tk (2), Tenascin-C KO (3), S100A4-Cre counting CD31 blood vessels in 10 or more visual fields. All flox/flox (4), VEGF-A (5), and Rosa-lox-Stop-lox-YFP (6) transgenic patient samples were processed according to institutional mice have been described elsewhere. guidelines and an institutional review board-approved protocol. + Immunostaining. Harvested tumors and lungs were fixed in 10% Microarray Analysis. S100A4 stromal cells were isolated by (vol/vol) neutral buffered formalin, dehydrated, and embedded fluorescence-activated cell sorting (FACS) from normal lung in paraffin. Antigen retrieval was performed on deparaffinized tissue and metastatic lung tissue of S100A4-GFP mice. RNA was sections with 10 mM citrate buffer (pH 6.0) before im- isolated using the Qiagen RNeasy Plus Micro Kit following munostaining. Alternatively, harvested tumors and lungs were the manufacturer’s instructions and was then amplified with the embedded in O.C.T. medium (TissueTek) and immunostaining NuGen WT-Ovation Pico Kit. Microarray hybridization to the was performed on frozen sections. Anti-tk antibody was obtained Illumina Mouse Ref-8 Expression BeadChip was performed by from William C. Summers (Yale University, New Haven, CT). the Molecular Genetics Core Facility at Children’s Hospital in Anti-S100A4 antibody was a gift from Eric G. Neilson (Van- Boston. Microarray data have been deposited in National Center derbilt University, Nashville, TN). Anti-CD31 antibody was ac- for Biotechnology Information’s Gene Expression Omnibus quired from Santa Cruz Biotechnology, anti-BrdU antibody was (GEO) and are accessible through GEO Series accession no. acquired from Roche, anti-Ki67 antibody was acquired from GSE31711. Microarray targets were sorted for ECM molecules, AbCam, anti-CK8 antibody was acquired from the National In- chemokines, and growth factors using the public gene ontology stitutes of Health (NIH) Hybridoma Bank, anti–Tenascin-C identification numbers from the Gene Ontology Consortium antibody was acquired from AbCam, and anti–VEGF-A anti- [GO:0005604 (basement membrane), GO:0008009 (chemokine body was acquired from NeoMarkers. TUNEL staining was activity), and GO:0008083 (growth factor activity)]. performed using an In Situ Cell Death Detection Kit (Roche). Cytospins were also prepared from the bone marrow of trans- FACS. FACS was performed on single-cell suspensions prepared planted mice at death and stained for Y chromosome using from lung tissue. To prepare a single-cell suspension, lung tissue StarFISH reagents (Cambio, Ltd.) per the manufacturer’s in- was cut into 1 mm3 pieces and incubated in 1 mg/mL collagenase/ structions to determine the percentage of bone marrow re- dispase solution (Roche) for 1 h in a 37 °C shaker. The resulting constitution by donor bone marrow (Fig. S9). For quantification suspension was filtered through a 40-μm cell strainer, treated of immunostaining, the number of positively stained cells was with ammonium chloride-potassium (ACK) lysis buffer for re- counted in 10 or more visual fields at a magnification of 40×, moval of red blood cells, and resuspended in a 2% (vol/vol) FBS whereas relative expression was quantified using NIH ImageJ in PBS plus propidium iodide solution before FACS analysis. 1. Xue C, Plieth D, Venkov C, Xu C, Neilson EG (2003) The gatekeeper effect of epithelial- 4. Bhowmick NA, et al. (2004) TGF-beta signaling in fibroblasts modulates the oncogenic mesenchymal transition regulates the frequency of breast cancer metastasis. Cancer potential of adjacent epithelia. Science 303:848e851. Res 63:3386e3394. 5. Dong J, et al. (2004) VEGF-null cells require PDGFR alpha signaling-mediated stromal 2. Iwano M, et al. (2001) Conditional abatement of tissue fibrosis using nucleoside fibroblast recruitment for tumorigenesis. EMBO J 23:2800e2810. analogs to selectively corrupt DNA replication in transgenic fibroblasts. Mol Ther 3: 6. Srinivas S, et al. (2001) Cre reporter strains produced by targeted insertion of EYFP and 149e159. ECFP into the ROSA26 locus. BMC Dev Biol 1:4. 3. Talts JF, Wirl G, Dictor M, Muller WJ, Fässler R (1999) Tenascin-C modulates tumor stroma and monocyte/macrophage recruitment but not tumor growth or metastasis in a mouse strain with spontaneous mammary cancer. J Cell Sci 112:1855e1864. O’Connell et al. www.pnas.org/cgi/content/short/1109493108 1of6 ABCMet S Met S D Met E F S Met S S Met S Met Met S Met Fig. S1. S100A4+ stromal cells are recruited to metastatic lung tissue in human cancers. Sections of lung tissue from patients with cancer were stained with an antibody specific for S100A4 (brown or red). Metastatic areas and stromal regions are separated by dotted lines (Met, metastasis; S, stromal region). (Scale bars: 20 μm.) No specific staining for S100A4 is observed in normal lung tissue from an uninvolved area (A), whereas S100A4+ cells are observed in the stromal regions associated with metastases (B–E). Patient samples represent a variety of cancer types, including renal cell carcinoma (B), colorectal cancer (C, D, and F), and breast cancer (E). A S100A4-GFP Ki67 DAPI Merged 30 cells + **** 20 10 0 per x400 visual field Normal Lung # S100A4-GFP Normal Metastatic Lung Lung + 100 75 50 **** cells /S100A4-GFP + 25 0 Normal Metastatic Metastatic Lung % Ki67 Lung Lung B tk tk 2.5 2.0 cells + 1.5 1.0 * 0.5 0 Control S100A4-tk GCV # S100A4-tk per x400 visual field Control S100A4-tk Tumor Tumor GCV tk tk 12.5 10.0 cells + 7.5 5.0 2.5 Control S100A4-tk GCV 0 ** # S100A4-tk per x400 visual field Control S100A4-tk Metastatic Lung Metastatic Lung GCV Fig. S2. Proliferating S100A4+ stromal cells in the metastatic microenvironment can be targeted for selective ablation on GCV treatment of S100A4-tk mice. (A) Frozen sections of normal and metastatic lung tissue from S100A4-GFP mice were immunostained for Ki67 (red) with DAPI nuclear counterstain (blue) to assess proliferating S100A4+ stromal cells (green). White arrows point to S100A4-GFP+ single-positive cells; yellow arrowheads point to Ki67+/S100A4-GFP+ double-positive cells. (Scale bars: 20 μm.) The total number of S100A4-GFP+ cells and proliferating Ki67+/S100A4-GFP+ cells as a percentage of total S100A4-GFP+ cells was quantified on an average of 10 high-powered visual fields (magnification of 400×). (B) Representative photomicrographs of immunostaining for tk (red) with DAPI nuclear counterstain (blue) in 4T1 mammary tumors and metastatic lung lesions of control and GCV-treated S100A4-tk mice (n = 6 in each group). Arrows point to S100A4-tk+ cells. (Scale bars: 20 μm.) (A and B) Bar graphs present a quantitative assessment of S100A4-tk+ stromal cells in the tumor and metastatic microenvironments. Mean ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001. O’Connell et al. www.pnas.org/cgi/content/short/1109493108 2of6 AB 3000 2 ) 3 2500 1.5 2000 1500 1 1000 0.5 500 % Metastatic area Tumor volume (mm Tumor 0 0 WT WT WT WT PBS GCV PBS GCV Fig. S3. GCV treatment of tumor-bearing WT mice. 4T1 cancer cells were orthotopically implanted into the mammary fat pads of PBS-treated WT mice (WT PBS; n = 7) and GCV-treated WT mice (WT GCV; n = 7). Primary tumor volume (A) and percent metastatic area in lung tissue (B) were assessed 24 d after cancer cell implantation. Mean ± SEM. A S100A4-GFP S100A4-GFP 40 *** cells + 30 20 10 per x400 visual field # S100A4-GFP 0 Normal Met Normal Liver Metastatic Liver B 25 20 15 10 ** 5 % Metastatic area 0 Control S100A4-tk Control S100A4-tk GCV GCV Fig. S4. Ablation of S100A4+ stromal cells attenuates liver metastasis of CT26 colorectal cancer cells. (A) Quantification of S100A4+ stromal cells in normal liver tissue vs. metastatic liver tissue from S100A4-GFP mice (8 visual fields per group). (Scale bars: 50 μm.) (B) CT26 cancer cells were also injected intrasplenically into control (n = 8) and GCV-treated S100A4-tk (n = 4) mice. Representative photographs of H&E-stained liver section are shown. (Scale bars: 40 μm.) Percent metastatic area in the liver was quantified. Metastatic lesions are encircled by dotted lines. Mean ± SEM. **P < 0.01; ***P < 0.001. S100A4 CD45 Fig. S5. S100A4 and CD45 expression in metastatic lung tissue of patients with breast cancer. Representative photomicrographs of S100A4 and CD45 immunostaining (brown) with hematoxylin nuclear counterstain (purple) in adjacent tissue sections of metastatic lung tissue from patients with breast cancer are shown. O’Connell et al. www.pnas.org/cgi/content/short/1109493108 3of6 A B Selective Expression Profiling of Fibroblast-Derived Top 20 Basement Extracellular Matrix Proteins Membrane / ECM Proteins Rank Gene Fold-Change Normal Control S100A4-tk GCV Lung Metastatic Lung Metastatic Lung 1 Timp1 1.6 2 Lama2 1.3 Collagen I + +++ +++ 2 Col18a1 1.3 Collagen III + +++ +++ 2 Col7a1 1.3 Collagen XVIII + +++ +++ 2 Acan 1.3 Entactin +++ ++ ++ 6 Fras1 1.2 Fibronectin ED-A + +++ + 6 Smoc1 1.2 Laminin-1 (α1) + + + 6 Col4a6 1.2 Laminin-10 (α5) +++ ++ ++ 6 Lamc3 1.2 Perlecan +++ ++ ++ SPARC + ++ + 6 Lamc2 1.2 Tenascin-C - +++ + 6 Col4a5 1.2 TSP1 + +++ +++ 6 Col4a4 1.2 Vitronectin + ++ + 6 Lama4 1.2 6 Tnc 1.2 6 Col17a1 1.2 6 Fbln1 1.2 C TN-C TN-C 17 Col28a1 1.1 17 Col8a2 1.1 17 Lad1 1.1 17 Lamc1 1.1 Wildtype TN-C KO D TN-C/CD31 H&E Wildtype Wildtype Fig.