doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 1 c CFU-F colonies per 1 5 stroml cells 14 12 1 8 6 4 2 Mtrigel plug Neg. MCF7/Rs MDA-MB-231 * * MCF7/Rs-Lung MDA-MB-231-Lung MCF7/Rs-Kidney MDA-MB-231-Kidney DME DME-1% MCF7/Rs-Tumor MDA-MB-231-Tumor MCF-7/Rs MDA-MB-231 Supplementry Figure 1 MSCs home to developing rest cncer xenogrfts., GFP-leled MCF7/Rs or MDA-MB-231 sucutneous tumors were hrvested 4 weeks fter implnttion, nd GFP-negtive mouse strom purified using FACS. The stroml cells were counted nd plted in MesenCult (Stem Cell Technologies) for 14 dys for the detection of mouse MSCs. Murine CFU-F colonies (figure inset) were counted, nd normlized to the totl numer of plted stroml cells. Mtrigel plugs or norml skin tissue (negtive control) were used s controls., 5, humn MSCs were plted in triplictes on the top of Boyden chmer memrnes in trnswell ssys nd incuted for 24 hours. Chemotxis ws set up y llowing cells to migrte from.5% IFS/DME to DME, DME with 1% IFS, or DME/1% IFS derived from MCF7/Rs or MDA-MB-231 cells. For motility ssys, complete medi ws dded to oth top nd ottom chmers. Imges re representtive of multiple independent experiments. c, One million humn GFP-leled MSCs were introduced into the circultion of tumor-ering mice 2 weeks fter tumor cell implnttion. Ten dys lter, nimls were scrificed, nd tumors, lungs, nd kidneys were excised nd exmined under fluorescence microscopy for the presence of green-leled MSCs. Mouse lungs exhiit distriution of GFP-MSCs which re physiclly entrpped. Leled MSCs locte to the interior s well s the edges of developing tumors. www.nture.com/nture 1
doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 2 MDA-MB-231 MDA-MB-231+MSC Ki-67-positive cells/field 35 3 25 2 15 1 5 MSC - + MDA-231 Supplementry Figure 2 MSCs do no imprt prolifertive dvntge to the co-mixed MDA- MB-231 cells. The prolifertive indices of MDA-MB-231 nd MDA-MB-231+MSC tumors in Figure 1 were ssessed y immunohistochemicl stining of prffin-emedded tumor sections for the prolifertion mrker Ki-67 using mouse monoclonl PP-67 ntiody (6526, Acm)., Representtive sections of MDA-MB-231 nd MDA-MB-231+MSC tumors re shown. Mgnifiction 4X., Ki-67- positive nuclei were counted in rndom fields (n=24 for MDA-MB-231; n=29 for MDA-MB-231+MSC) tken of 4 representtive tumors in ech group. Dt shown represent mens ± s.e.m. p=.27 in Student s t-test nd indictes no sttisticl significnce. www.nture.com/nture 2
doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 3 Tumor Volume (mm 3 ) 6 5 4 3 2 1 MDA 231 lone MDA 231+WI-38 26 32 38 44 Time (dys) Tumor mss (g).7.6.5.4.3.2.1 WI-38 - + MDA MB-231 c d MDA-MB-231 MDA-MB-231+WI-38 Met index (fold over control) 1.8.6.4.2 WI-38 - + MDA MB-231 Supplementry Figure 3 WI-38 firolsts do not promote the metstsis of commingled MDA- MB-231 cells to the lung. 5, GFP-leled MDA-MB-231 cells were injected sucutneously with or without 1.5 x 1 6 WI-38 firolsts (1:3 rtio) in NOD/SCID mice., Tumor growth kinetics of the indicted groups of mice (n=8 for MDA-MB-231 injected lone; n=9 for MDA-MB-231+MSC) were monitored for ~6.5 weeks. Dt points represent mens of tumor volume in mm 3 ± s.e.m., Men tumor weights ± s.e.m. of groups in. p=.28 in Student s t-test nd indictes no sttisticl significnce. c, Representtive lung imges of mice ering MDA-MB-231 or MDA-MB-231+WI-38 tumors. Cncer colonies re in green. d, Metstsis indices of the indicted groups were clculted s in Figure 1. www.nture.com/nture 3
doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 4 hmsc/231-4wks hmsc/231-5wks hmsc/231-6wks Primry tumor Lung/Green chnnel Lung/Red chnnel hmsc (til vein)-6 wks Supplementry Figure 4 MSCs do not disseminte with cncer cells to sites of metstsis., Redleled MSCs (1.5 million cells) were dmixed to GFP-leled MDA-MB-231 (5,) cells nd implnted sucutneously in recipient mice. Animls were scrificed 4, 5, nd 6 weeks lter nd their lungs exmined under fluorescence microscopy. After 6 weeks, red-mscs re still present in primry tumors (inset)., The lung environment is hospitle to MSCs s GFP-MSCs tht lodge in the lungs of recipient nimls fter til vein infusion survive in tht environment for ~6 wks post-injection. www.nture.com/nture 4
doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 5 c Aritrry Units 7 6 5 4 3 2 1 Rtio: BCC:MSC=2 BCC:MSC=6 BCC:MSC=12 BCC:MSC=5 MSC MDA-231 MSC+MDA-231 (fold increse-elisa) 45 4 35 3 25 2 15 1 5 MDA-231 MSC MDA-231 + MSC + (fold increse-mrna) 14 12 1 8 6 4 2 MDA-231 MSC MDA-231 + MSC + Supplementry Figure 5 Cncer cells induce de-novo expression of in co-cultured MSCs, nd its levels re proportionl to the rtio of MDA-MB-231 to MSCs., MDA-MB-231 cells were co-cultured with MSCs t the indicted rtios nd clered culture medi ssyed for using xmap Bio-Plex technology. Brs represent mens ± s.d. of triplictes., GFP-leled MDA-MB-231 cells were co-cultured with MSCs t rtio of 1:3 BCC:MSC. The two cell types were seprted 3 dys lter using FACS, fter which 2, cncer cells (MDA-231+) or MSCs (MSC+) were cultured seprtely in triplictes. As controls, 2, MDA-MB-231 cells nd MSCs which were cultured seprtely were lso sujected to sorting. The levels of in the culture medi of MDA-231+ cells, MSC+ cells, nd MSCs were ssyed 24 hrs lter, nd normlized to the levels oserved in the medi of MDA-231 cells. Dt points indicte fold induction. c, Cells in were hrvested immeditely fter sorting nd processed for stndrd RNA/cDNA preprtions. The levels of mrna expression in the indicted smples were ssyed using semi-quntittive RT-PCR using primers 5 -tgcgggtcgcgc-3 (left) nd 5 -ggccttgccctggtgt-3 (right). Levels were normlized over those oserved in MDA-MB-231 controls. www.nture.com/nture 5
doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 6 1.4 Fold (ELISA) 1.2 1.8.6.4.2 si Luc si.1 si.2 si.3 si.4 si.5 Supplementry Figure 6 Identifiction of sirna ginst., shrna inhiition of ws sed on the plko-puro system (W.C. Hhn, Dn-Frer Cncer Institute). Five hirpins were tested nd lentivirl prticles of the indicted shrna were infected into MDA-MB-231 cells. Cells infected with siluc were used s controls. Levels of were ssyed in the culture medi using ELISA. Brs represent mens ± s.d. of triplictes. www.nture.com/nture 6
doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 7 (-f) c d levels (pg/ml) 1 8 6 4 2 control Cell numer (fold increse) 4 35 3 25 2 15 1 5 (.5%) (.5%) (2%) (2%) (1%) (1%) d1 d3 d5 d7 Colonies (soft gr) 12 1 8 6 4 2 5, 1, Cell numer (x 1 5 ) 6 5 4 3 2 1 d1 d2 d3 d4 e vehicle Doxoruicin f /vehicle /Dox Dys 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 CC3 PARP /vehicle /Dox Actin Supplementry Figure 7 does not promote cellulr growth or protect from stress-induced poptosis., Expression levels of ectopic in the culture medi of MDA-MB-231/ cells compred to control MDA-MB-231 cells., Growth curves of MDA-MB-231 controls compred to MDA- MB-231/ cells grown for 7 dys in complete medi (1% IFS), 2%IFS-contining medi, or.5%ifscontining medi. Dt points represent mens ± s.d. of triplictes. c, Anchorge-independent growth of MDA-MB-231 cells compred to cells overexpressing. Cells were plted t two different densities. Brs represent mens of colony numers per plte ± s.d. of triplictes. d, The viility of suspended MDA- MB-231 control cells nd MDA-MB-231/ cells (i.e. their ility to withstnd noikis) ws followed for 4 dys using Trypn lue exclusion. Dt points indicte mens ± s.d. of triplictes. Representtive experiment is shown. e, The levels of CC3 nd PARP in the lystes of MDA-MB-231 controls or MDA-MB- 231/ cultures treted for four dys with 1 μg/ml doxoruicin (Sigm) or vehicle were proed y Western lotting. β-ctin ws used s loding control. f, MDA-MB-231 or MDA-MB-231/ cells were treted with doxoruicin (1 μg/ml) or vehicle for 36 hours, fter which the cultures were fixed in methnol, nd processed for TUNEL determintions. Percentge cell deth ws clculted s the numer of FITC positive cells normlized over the totl numer of DAPI-positive nuclei nd ws 22.5% nd 24.4% for doxoruicin-treted control MDA-MB-231 nd MDA-MB-231/ cells, respectively. www.nture.com/nture 7
doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 7 (g,h) g h Ki-67 CC3 Ki-67-positive BCC/field 12 1 8 6 4 2 # ## 12 1 8 6 4 2 CC3-positive BCC/ field Supplementry Figure 7 does not promote cellulr growth or protect from stress-induced poptosis. g, Immunohistochemistry of prffin-emedded MDA-MB-231 or MDA-MB-231/ tumor sections using Ki-67 (B126.1, cm) nd cleved cspse-3 (CC3, 9664, Cell Signling) ntiodies. Imges were tken t 2X mgnifiction. h, Quntifiction of Ki-67- nd CC3-positive cncer cells in rndom sections of representtive tumors (n=2 per group). Brs represent mens ± s.e.m. # nd ## re p vlues of.49 nd.23 in Student s t-test, respectively, nd indicte no sttisticl significnce. www.nture.com/nture 8
doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 8 Positive αsma MECA-32 F4/8 per field 14 12 1 8 6 4 # ## 8 7 6 5 4 3 2 per field 2 1 Vessels Mcrophges Supplementry Figure 8 Ectopic expression of in MDA-MB-231 cells does not ffect tumor stromliztion., Immunohistochemistry of prffin-emedded MDA-MB-231 or MDA-MB-231/ tumor sections using αsma (1A4, Dko), MECA-32 (12951, BioLegend), nd F4/8 (CI:A3-1, 664, Acm) ntiodies. Imges were tken t 2X mgnifiction., Quntifiction of the lood vessel density nd mcrophge infiltrtion in rndom sections of representtive tumors (n=2 per group). Brs represent mens ± s.e.m. # nd ## re p vlues of.15 nd.27 in Student s t-test, respectively, nd indicte no sttisticl significnce. www.nture.com/nture 9
doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 9 /DAPI /DAPI /Phlloidin /Phlloidin c MCF7/Rs 231-vector 231- N-Cdherin Vimentin β-actin Supplementry Figure 9 Ectopic expression of in MDA-MB-231 cells is not ssocited with n epithelil-to-mesenchyml trnsition., Representtive right field imges of control MDA-MB- 231 nd MDA-MB-231/ cultures. 2X mgnifiction., or -overexpressing MDA- MB-231 cells were proed with TRITC-phlloidin (for filmetous ctin distriution; in red) nd DAPI (for nuclei stining; in lue). c, The levels of N-cdherin nd vimentin were proed in the lystes of MDA-MB-231 control (231-vector) or MDA-MB-231/ (231-) cells y Western lotting. MCF7/Rs ws used s negtive control. β-ctin ws used s loding control. www.nture.com/nture 1
doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Figure 1 (fold increse-elisa) 8 7 6 5 4 3 2 1 MSC MDA-435 MDA-435+MSC Met Index (fold of control) 4 3 2 1 MDA-435 MDA-435+MSC.1 MDA-435+MSC.c Supplementry Figure 1 is importnt in the MSC-induced metstsis of MDA-MB-435 cells., MDA-MB-435 cncer cells (2,) were cultured lone or together with humn MSCs (6,) in 2%IFS/DME for 48 hrs, fter which the culture medi ws hrvested nd clered of cell deris. levels were determined in the indicted conditioned medi using ELISA. Dt points represent men ± s.d. of triplictes., 5, MDA-MB-435 cells were implnted lone or co-mixed with 1.5 x 1 6 MSCs crrying the control shrna (MSC.c) or.1 shrna (MSC.1) nd injected sucutneously in Nude mice. Mice were scrificed when tumors reched 1 cm in dimeter nd the metstsis index clculted for ech cohort (n=4 per group) s in Figure 1. Results represent mens ± s.e.m. www.nture.com/nture 11
doi: 1.138/nture6188 SUPPLEMENTARY INFORMATION Supplementry Tle 1 Supplementry Tle S1 Incidence of metsttic spred in mice ering MDA-MB-231 tumors with or without dmixed MSCs. Sttistics shown re derived from two representtives of multiple experiments. ND, not determined. Primry Tumors MDA MB-231 lone MDA-MB-231+MSC Metstsis incidence to: Lung 8/19 (42%) 12/17 (7.5%) Lymph nodes 1/19 (5.2%) 4/17 (23.5%) Mmmry glnds /19 (%) 5/17 (29.4%) Liver /19 (%) 1/17 (5.8%) Brin /19 (%) /17 (%) Bone ND ND www.nture.com/nture 12