oi:1.138/nture1138 Supplementl Figure 1 Inflmmtory Monoytes Host ells CCR2 CCL2 Disseminting Tumor Cells Metstsis Assoite Mrophges VEGF Extrvstion & Metstti Seeing Supplementl Figure 1 The t from this stuy support the moel tht isseminting tumor ells n host orgn-erive hemokine CCL2 reruit inflmmtory monoytes, whih filittes effiient tumor ell metstsis seeing n growth in the istnt metstti sites of lung. WWW.NATURE.COM/NATURE 1
oi:1.138/nture1138 Supplementl Figure 2 CCL2 Expression in Fink Brest Groupe y Cner Type CCL2 Expression in Frmer Brest Groupe y Cner Type Supplementl Figure 2 Onomine 27 serh results inite tht CCL2 is overexpresse in oth tumor n tumor strom of invsive rest rinom whih is orrelte with metstti isese n poor prognosis, CCL2 is over-expresse in tumor strom of invsive rest rinom ompre to norml strom. P=3.58E-16, fol hnge 4.77. Mirorry t soure GSE914 28., CCL2 is over-expresse in the sl-like sutype of invsive rest rinom ompre to porine rest rinom n luminl-like sutype of invsive rest rinom. P=8.23E-6. Mirorry t soure GSE1561 29. WWW.NATURE.COM/NATURE 2
oi:1.138/nture1138 Supplementl Figure 3 1 5 1 5 1 5 96.6 1 4 1 4 <PE-Cy7-A> 1 4 CD11 1 2 1 2 1 4 1 5 CD115 Gr1 1 2 1 2 1 4 1 5 Csf-1r-GFP Ly6C Ly6G 1 4 1 5 <APC-A> 1 8 1 CD62L 8 1 MHCII 8 IL4R e 1 5 1 4 6 4 2 % of Mx 6 4 2 1 1 1 2 1 4 1 5 6 4 2 1 1 1 2 1 4 1 5 % of Mx 1 1 1 2 1 4 1 5 <PE-A> Ly-6C- Ly-6C+ Csf-1r-GFP 1 2 1 1 1 1 1 2 1 4 1 5 DAPI Supplementl Figure 3, Flow igrm of CD45+ one mrrow ells lele with CD11 (Y xis) n CD115 (X xis)., Monoytes seprte into Gr1 + IM n Gr1 - RM popultions y FACS n hve the sme level of Csf1r-GFP expression., Virtully ll CD115 + monoytes in the one mrrow re Ly6G - (X xis) n seprte into Ly6C+ n Ly6C (Y xis) popultions whih orrespon to the Gr1 + n Gr1 popultions., Representtive flow histogrm showing tht IMs (re) hs elevte CD62L ell surfe expression (Left pnel)ompre with RMs (Blue). Both popultions express miniml level of ell surfe MHCII n IL4R (mile n right pnels). e, Representtive flow igrm of reovere GFP + onor ells (oxe) from peripherl loo of reipient mie. WWW.NATURE.COM/NATURE 3
oi:1.138/nture1138 Ly6 Supplementl Figure 4 1 5 1 5 1 4 1 4.19 29 1 2 1 2 99.4 69.1 1 1 1 1 1 1 1 2 1 4 1 5 1 1 1 2 1 4 1 5 CD115 Norml Lung Mets Lung (PyMT) Norml Lung Mets Lung 1 5 1 5 1 4 1 4 e Rtio of reruite IM Vs RM per 1^6 CD45+ ells Donor ells per 1^6 CD45+ ells Donor IM per 1^6 CD45+ ells 1..8.6.4.2. 8 6 4 2 Norml Lung RM PyMT Pre-mets Lung 1 ** 6 4 2 IM Gr1.15 1 2 1 2 1 1 1 1 1 1 1 2 1 4 1 5 CD11 12.4 1 1 1 2 1 4 1 5 f Perentge of CD11+ Gr1- in CD45+ ells.8.6.4.2. PBS Ctrl A * Bes nti-ccl2 A Supplementl Figure 4, Representtive flow igrm showing expression of Ly6C (Y xis) n CD115 (X xis) of enogenous monoytes (CD115+CD11+F4/8+) reruite in norml lung (left) n lungs ering spontneous metstsis (right) of lte stge PyMT tumor, n=5., Rtio of lung-reruite IMs vs RMs fter overnight optive trnsfer into 7 week-ol PyMT mie with no etetle lung metstsis n their norml litter mtes. n=3, NS., Numer of RM n IM reruite into estlishe pulmonry metstsis of i.v. injete Met-1 ells. n=4, p=.19., Representtive flow igrm showing the leling of GFP + IMs reovere from norml lung n metstsis-ering lung with Gr1 (Y xis) n CD11 (X xis) 2 ys fter optive trnsfer. Numers shown re perentge of CD11 + Gr1 - ells reltive to totl GFP + ells, n=4. e, Reltive numer of lung reruite IMs fter overnight optive trnsfer into nue mie with PBS or ltex es i.v. injetion. n=3, NS. All r represents men + s.e.m.. f, CD11 + Gr1 - MAMs in lungs hllenge with i.v. injete Met-1 ells for 36 hours with ontrol n nti-mouse CCL2 ntioy tretment. Dt show men + s.e.m., n=3, p=.281. WWW.NATURE.COM/NATURE 4
oi:1.138/nture1138 Supplementl Figure 5 CD3 1 5 1 4 1 2 1 1 DAPI 1 1 1 2 1 4 1 5 1 5 1 4 CD4.53% 1 5 1 4 1 2 1 1 CD8 1 1 1 2 1 4 1 5 Perentge of CD3+CD4+ ells in totl CD45+ ells 15 1 1 5 Ctrl A.23% 1 4 5 Ctrl Mets 36hr 1 2 1 1 1 5 1 4 Ctrl 1 1 1 2 1.52% 4 1 5 1 2 1 1 1 5 1 1 1 Anti-CCL2 2 1 4 1 A 5.2% 1 4.68% 1 2 1 2 CD4 1 1 Mets 36hr 1 1 1 2 1 4 1 5 CD25 1 1.72% 1 1 1 2 1 4 1 5 Foxp3 CCR2 Supplementl Figure 5, Representtive flow igrm showing the gting of CD3 + CD4 + T ells in CD45 + hemtopoieti ells isolte from mouse lung., Reruitment of CD3 + CD4 + T ells in totl CD45 + hemtopoieti ells in ontrol lungs n lungs hllenge with i.v. injete tumor ells for 36 hours, n=4, NS., Representtive flow igrm showing the gting of CD3 + CD4 + Foxp3 + regultory T ells in ontrol lungs n lungs hllenge with i.v. injete tumor ells for 36 hours. Perentge shown is reltive to totl CD45 + hemtopoieti ells, n=4, NS., Representtive flow igrm showing the gting of CD3 + CD4 + T ells lele with CD25 (Y xis) n CCR2 (X xis) in lungs ering estlishe metstses of i.v. injete Met-1 tumor ells with ontrol A or nti-mouse CCL2 A tretment. Perentge shown is reltive to totl CD45 + hemtopoieti ells, n=4. WWW.NATURE.COM/NATURE 5
oi:1.138/nture1138 Supplementl Figure 6 Primry Tumor Lung Mets 2 n A only Anti-CCL2 A e 4 ** f 5 CCR2 mrna level (Fol Vs GAPDH) 3 2 1 Donor IM per 1^6 CD45+ ells 4 3 2 1 RM IM Control Lung Mets Lung Supplementl Figure 6, Representtive mirogrph of CCL2 expression etermine y immunohistohemistry using seonry ntioy lone (,) or nti-ccl2 ntioy (,) in primry PyMT tumors (,) n their spontneous lung metstses (,). Brown olor inites positive signls. Br equls 2 um. e, Reltive CCR2 mrna expression in resient n inflmmtory monoytes etermine y quntittive reltime PCR, normlize ginst housekeeping gene, GAPDH, r represents men + s.e.m., n=3, p=.53. f, Numer of reruite CCR2 knokout IMs into norml lung n lung ering experimentlly-inue metstsis with Met-1 ells in nue mie fter overnight optive trnsfer, n=3 NS. WWW.NATURE.COM/NATURE 6
oi:1.138/nture1138 Supplementl Figure 7 Hu CD14 1 5 1 4 1 2 1 1 1 1 1 2 1 4 1 5 Hu CD16 1 5 e Humn CD45+ ells per 1^6 totl CD45+ ells 1 4 * 1 2 1 1 1 5 1 4 1 2 CD14 lo CD16+ w/o Hu CSF-1 Isotype ontrol CD14 lo CD16+ CD14+CD16- CD14+CD16-1 4 1 1 1 5 w/ Hu CSF-1 1 1 1 2 1 4 1 5 HuCD45 % of Mx 1 2 1 1 1 8 6 4 2 CCR2 5K 1K 15K 2K 25K FSC 1 1 1 2 1 4 1 5 f Donor IM per 1^6 CD45+ ells Hu CD45 5 4 3 2 1 1 4 1 2 1 1 Hu CD14 1 1 1 2 1 4 1 5 Ctrl A ** nti-hu CCL2 A WWW.NATURE.COM/NATURE 7
oi:1.138/nture1138 Supplementl Figure 7, Humn peripherl CD14 + ells were sorte y FACS into CD14 + CD16 - n CD14 lo CD16 + popultions., Flow igrm showing the reovery of humn ells (re ox) fter overnight optive trnsfer into nue mie supplemente with humn CSF-1., Representtive flow histogrm showing CCR2 expression of the ifferent humn monoyte popultions s shown ompre to n isotype ontrol, n=3., Numer of optively trnsferre humn monoytes reruite into lungs ering metstses from 4173 tumors, n=6, p=.239. e, Representtive flow histogrm showing reovery of humn monoytes fter overnight optive trnsfer into mie with s.. injetion of humn CSF-1 (ottom) or ontrol injetion (top), n=3. f, Numer of mouse IM reruite fter overnight optive trnsfer into mie with estlishe pulmonry metstsis from i.v. injete 4173 ells with nti-humn CCL2 A or ontrol A tretment, r represents men + s.e.m., n=5, **p=.39. WWW.NATURE.COM/NATURE 8
oi:1.138/nture1138 Supplementl Figure 8.25 Mets Inex.2.15.1.5. Ctrl A Anti-CCL12 A Supplementl Figure 8 Stereologil quntifition of normlize Mets uren of i.v. injete Met-1 ells with tretment of ontrol A or nti-mouse CCL12 A, r represents men + s.e.m., n=9, NS. WWW.NATURE.COM/NATURE 9
oi:1.138/nture1138 Supplementl Figure 9 CCR2 CCL2 GAPDH Tumor ell Met-1 Enothelil monolyer Monoyte/ ECM BMM BMM Enothelil Cells Cell numer per 2X fiel Trnsmigrte tumor ell per 2X fiel 6 p<.1 p=.4 4 2 4 2 w/o BMM WT w/ BMM w/ BMM + nti-ccl2 A 6 p=.54 w/ BMM + ontrol A CCR2-/- Supplementl Figure 9 Mrophges promote tumor ell in vitro trns-enothelil migrtion., Shemti rwing of the ssy for in vitro tumor ell (re) trnsenothelil (lue) migrtion in Mtrigel (pink) ote invsion hmer with monoytes or one mrrow erive mrophges (BMM, green) loe to the sl-lterl sie of the hmer., Presene of primry BMMs (w/bmm) signifintly promote tumor ell in vitro trns-enothelil migrtion s ompre with the sene of mrophges (w/o BMM). This effet is ompletely inhiite y nti-mouse CCL2 neutrlizing ntioy ut not y isotype mthe ontrol ntioy. Br represents men + s.e.m. of 3 experiments with uplites., RT-PCR shows tht tumor ells (Met-1), mrophges (BMM) n enothelil ells ll express CCL2, ut only mrophges express CCR2 reeptor., Averge numer of trnsmigrte Met-1 ells in the presene of mrophges from WT or CCR2-/- mie. Br represents men + s.e.m. of 3 experiments with uplites, p.54. WWW.NATURE.COM/NATURE 1
oi:1.138/nture1138 Supplementl Figure 1 Reltive expression.25 p=.5.2.15.1.5 Cell Numer 6 Control RNAi CCL2 RNAi 4 2 Mets Inex.5.4.3.2.1 ***. ontrol RNAi Control RNAi CCL2 RNAi hr 24hr CCL2 RNAi 36hr 48hr. Ctrl RNAi CCL2 RNAi e Reltive expression.15.1.5. Control ** CCL2 KD f Normlize Rine 15 1 5 Control *** CCL2 KD Supplementl Figure 1, Reltive CCL2 expression in Met-1 ells infete with retrovirl ontrol (srmle) RNAi n CCL2 RNAi. Dt show men ± s.e.m. n=3., In vitro prolifertion of Met-1 ells infete with retrovirl ontrol RNAi n CCL2 RNAi. Dt show men ± s.e.m. n=3, NS., Representtive mirogrph showing H&E stine trnsverse setions of Met-1 lung metstsis following ontrol or CCL2 sirna tretment. Br equls 1 mm., Mets uren of Met-1 ells with ontrol or CCL2 RNAi. n=6, p=.4. e, Reltive CCL2 expression etermine y quntittive reltime RT-PCR in 4173 ells infete with retrovirl ontrol RNAi n CCL2 RNAi. Br represents men + s.e.m. n=5. **p=.89. f, Normlize ioluminesene (BLI) signl of lung tumor uren of mie inoulte with 4173 ells tht h een trnsfete with luiferse onstrut. The signl intensities were normlize to y, whih ws set ritrrily s 1. n=8, *** p=.1. n, unpire t test with Welh's orretion. WWW.NATURE.COM/NATURE 11
oi:1.138/nture1138 Supplementl Figure 11 Reltive mrna expression 1.5 1..5. RM VEGFA IM ** Mets Numer Inex 5 4 3 2 1 flox VEGF ** flox VEGF ;Csf1r-Cre Donor Ly6+ monoytes per 1^6 CD45+ ells 8 6 4 2 IM flox VEGF VEGF KO IM Supplementl Figure 11, Reltive mrna expression of VEGF y mouse RM n IM y reltime PCR normlize to GAPDH expression. Dt show men + s.e.m., n=5, ** p=.28., Stereologil quntifition of normlize Mets numer (numer of Mets noules per mm^2 of lung re in ross setions) in VEGF flox/flox mie with or without Csf1r-Mer-iCre-Mer trnsgene trete with sme ose of s.. injetion of tmoxifen. n=6, **p=.95., Numer of IMs or VEGF knokout IMs reruite into estlishe pulmonry metstsis of i.v. injete Met-1 ells fter overnight optive trnsfer. n 4, NS. All t shown re men+s.e.m. WWW.NATURE.COM/NATURE 12
oi:1.138/nture1138 Supplementl Movie 1 Representtive 3D reonstrute onfol imges of tumor ells (CFP, lue) n mrophges (GFP, green) 24 hours fter tumor ell til vein injetion in mie trete with ontrol A. Bloo vessels were stine with Alex Fluor 647 onjugte nti-mouse CD31 ntioy (re). Tumor ell ws in the extrvsulr spe with mrophge intertions. Overlpping re of oth re n lue olor ws use y refletion of lser light from the lveolr spe. Supplementl Movie 2 Representtive 3D reonstrute onfol imges of tumor ells (CFP, lue) n mrophges (GFP, green) 24 hours fter tumor ell til vein injetion in mie trete with nti-mouse CCL2 A. Bloo vessels were stine with Alex Fluor 647 onjugte nti-mouse CD31 ntioy (re). Tumor ell stye intrvsulr without mrophge intertion. Overlpping re of oth re n lue olor ws use y refletion of lser light from the lveolr spe. Referenes: 27 Rhoes, D. R. et l. ONCOMINE: ner mirorry tse n integrte t-mining pltform. Neoplsi 6, 1-6 (24). 28 Fink, G. et l. Stroml gene expression preits linil outome in rest ner. Nture meiine 14, 518-527 (28). 29 Frmer, P. et l. Ientifition of moleulr porine rest tumours y mirorry nlysis. Onogene 24, 466-4671 (25). WWW.NATURE.COM/NATURE 13