SUPPLEMENTARY INFORMATION

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1 Prentl doi:.8/nture57 Figure S HPMECs LM Cells Cell lines VEGF (ng/ml) Prentl 7. +/-. LM 7. +/-.99 LM 7. +/-.99 Fold COX induction 5 VEGF: Bevcizum: (µg/ml) Reltive MMP LM mock COX MMP LM+ Bevcizum c Fold induction.5.5 Fold.5.5 MMP Fold.5.5 MMP- Fold.5.5 COX- Hours: Hours: Prentl LM Prentl LM Hours: Prentl LM Hours: Prentl LM Figure S: Expression of, MMP, MMP nd COX- in metsttic tumor cells is independent of incresed VEGF signling., Amounts of VEGF secreted y prentl, LM nd cells were mesured y ELISA., LM cells were either mock treted or incuted with the nti-vegf ntiody Bevcizum ( µg/ml) overnight. Expression of, MMP, MMP nd COX- ws determined y qrt-pcr. To control for the effects of Bevcizum, humn microvsculr endothelil cells were pre-treted with drug nd stimulted with VEGF for hour. The inhiition of COX- expression ws confirmed y qrt-pcr. c, Prentl or LM cells were strved overnight nd treted with 5ng/ml VEGF. RNA ws hrvested t the indicted time points for qrt-pcr. n=; error rs represent 95% confidence intervl for qrt-pcr nd stndrd errors of the men (SEM) for ELISA. Figure S LM derivtives shcox COX Tuulin Figure S: COX- knockdown y Western lot. Western lot of COX- ws performed on protein lystes from the indicted cells lines. Quntifiction of COX- protein for Figure ws determined y using Imge J softwre nd normlizing to tuulin stedy stte levels.

2 doi:.8/nture57 Figure S Secreted MMP (ng/ml) 8 shcon sh sh+ rescue COX Tuulin Secreted MMP (ng/ml) Reltive Tumor volume (mm ) Rescued COX/MMP/ in Dys post-injection 5 ** c Normlized Photon Flux Triple Rescue Dys Post-Injection * d e Reltive ILR ILR Sprc VCAM shcon ILR/Sprc/VCAM /MMP/ Dys post-injection Reltive SPARC 8 5 ILR Sprc VCAM * *** f Reltive VCAM 8 ILR Sprc VCAM ILR/Sprc/VCAM 5 5 Dys Post-Injection ** Figure S: Specificity of, MMP, MMP nd COX- comintoril effects., To generte prtil rescue, cells were super-infected with constructs encoding for MMP, MMP, nd COX- cdnas. LM shcon nd sh lines were lso super-infected with empty vector s controls. All cell lines were sujected to hygromicin selection. Recovered MMP nd MMP protein mounts were mesured vi ELISA, wheres COX- protein ws ssessed y Western lot nlysis. qrt-pcr of epiregulin expression shows tht knockdown ws mintined in the triple MMP/MMP/COX- recovered cell line., Cell lines were injected into the mmmry ft pds of mice nd tumor volumes monitored s in Figure. n=, error rs represent SEM. *, p <.5; **; p <.; ***; p <.; p vlues were clculted using two-tiled Student s t-test for tumor volumes t the lst time point, compred to control. c,, nd triple rescue cell lines were injected intrvenously, nd lung metstsis monitored y ioluminescence. n=5, error rs depict SEM. *, p <.5 sed on two-sided rnk-sum test compred to shcon LM cells. d, LM cells were infected with short hirpin vectors ginst ILR, SPARC, nd VCAM. Comintion knockdown of these three genes ws confirmed y qrt-pcr. e, The growth of ILR/SPARC/VCAM knockdown cells in the mmmry ft pd ws compred to control, /MMP/ nd knockdown cells lines generted s in Figure. n=, error rs represent SEM. *, p <.5; ***, p<.; clculted using two-tiled Student s t-test for tumor volumes t the lst time point, compred to control. f, ILR/SPARC/VCAM knockdown cells were lso injected into the vi til vein of immunocompromised mice, nd lung metstsis monitored y ioluminescence. n=5; error rs represent SEM. **, p <. sed on two-sided Wilcoxon rnk-sum test compred to control LM cells t the lst cquired time point.

3 doi:.8/nture57 Figure S Phospho-Histone COX MMP/ COX/MMP/ /COX /MMP/ Counts per field Apoptosis counts shereg shcox shmmp/ shereg/cox shereg - - No shereg +shereg no shcox +shcox No shmmp/ +shmmp/ no shereg +shereg No shmmp/ +shmmp/ shcox p =. - - No shcox + shcox No shmmp/ +shmmp/ shmmp/ p =. (NS) p =.98 (NS) - no shereg/cox +shereg/cox shereg/cox - - p =.5 (NS) - Figure S: Comintoril effects of, MMP, MMP, nd COX on tumor poptosis., Automted immunohistochemistry for phospho-histone detection ws performed on tumors otined from the vrious comintion knockdown cell lines (Figure ) nd ws quntified using Imge J softwre. n=5; error rs represent SEM. **, p <.; ***; p <.using two-sided Wilcoxon rnk-sum test, compred to levels in control tumors., A sttisticl tle of genetic interctions influencing the rte of primry tumor poptosis. Shown re p vlues ssocited with two-wy ANOVA nlysis on the oserved rtes of poptosis in vrious comintions of knockdown primry tumors. For ny given pir of genetic perturtions, one intersecting cell shows the ssocited p vlue for sttisticl synergy or ntgonism, wheres the other intersecting cell shows grphicl two y two depiction of the genetic comintion. The y-xis of this grph represents rtes of poptosis reltive to the rte oserved in control tumors. Prllel lines indicte n dditive effect. Converging lines indicte sudditive effects, wheres diverging lines specify supr-dditive interctions. A p<.5 sttisticl threshold ws used to signify synergy or ntgonism. Figure S5 CD NG Merge Figure S5: Vessel morphology nd pericyte recruitment in control nd primry tumors., Size mtched control nd tumors were extrcted t pproximtely mm. Frozen sections were stined with nti-cd (red) nd for immunofluorescent detection. Representtive fields were cquired t X. Scle=μm., Frozen sections were lso stined with nti-cd (red) nd nti-ng (yellow) for pericyte coverge immunofluorescent detection. Representtive fields were cquired t X. Merge of stining depicts pericytecovered vessels

4 doi:.8/nture57 Figure S COX MMP MMP MMP/ MMP//COX /MMP/ /COX Predicted (Additivity) Experimentl p =.5 p =. Lung Metstsis Burden (Reltive to s).8... p =.8 shereg +shcox shereg +shmmp/ shcox +shmmp/ Figure S: Confirmtion of genetic inhiition of lung metstsis nd nlysis of genetic synergy versus effects predicted sed on dditivity., Histologicl confirmtion of knockdown effects. Tumor cells in the lungs hrvested weeks fter tumor cell inocultion were identified vi IHC, stining for tumor selective vimentin. Arrows denote smll residul lesions in the comintion knockdown cells, while no lesions could e visulized in sh mice using conventionl histology., Becuse ioluminescent dt from intrvenously injected nimls my not follow Gussin distriution, two-wy ANOVA tests re not pproprite for sttisticl determintion of synergy. For this reson, simulted dt sed on dditive interctions were generted from ll possile comintions of single knockdown (t dy ). The resulting predicted dditive interctions were compiled, nd mens nd SEMs for the vrious knockdown comintions re depicted in the grphs (lue rs). Shown in red re the ctul comintion knockdown dt (tken from Figure ). A two-sided Wilcoxon rnk-sum test ws used to clculte p-vlues for the comprison etween predicted nd ctul dt. A p-vlue less thn.5 indicted sttisticl synergy if the ctul dt yielded greter lung metstsis inhiition thn predicted, or sttisticl ntgonism if the converse were true. Figure S7 8 Trns-endothelil migrtion (Counts/field) 8 Prentl LM shereg shcox shmmpshmmp Figure S7: Effects of single knockdown on trns-endothelil migrtion. Trnsendothelil migrtion of single knockdown LM cells through HUVEC monolyer ws done nd quntified s descried in Methods. n=8; Error rs=sem.

5 doi:.8/nture57 Figure S8 phospho-histone TUNEL CD ph counts per field 57. +/-.8 TUNEL+ nuclei per field 5. +/-. Pimonidzole 7.5 +/-. (NS) Averge Length (µm) 7.7 +/ /-.8 * # of lumen per field.5 +/- 8.9 # of rnch pts per vessel. +/-.9 Figure S8: Immunohistochemicl chrcteriztion of primry tumors treted with cetuxim/celecoxi/gm., Representtive imges (X) of control or cetuxim/celecoxi/gm (three ) treted tumors nlyzed for phosphorylted histone H immunohistochemistry (left pnels), TUNEL immunofluorescence (middle left pnels), CD immunohistochemistry Figure S9 Celecoxi GM Celecoxi/GM GM/Cetuxim Celecoxi/Cetuxim Lung Metstsis Burden (Reltive to s)..8 +/-. * Cetuxim Predicted (Additivity) Experimentl p =.9 (NS). p =.7 NS. Cetuxim +Celecoxi Cetuxim +GM Figure S9: Confirmtion of phrmcologicl inhiition of lung metstsis nd nlysis of phrmcologicl synergy versus effects predicted sed on dditivity., Histologicl confirmtion of drug treted nimls four +/-. **.5 +/-. * (middle right) nd tissue hypoxi using immunofluorescent stining for pimonidzole dducts (fr right pnels)., Quntifiction of phospho-histone, TUNEL nd vessel morphology ws performed s descried in Supplementry Supplementl Figure S8 (--75D) Methods. n=5; error rs represent SEM. Celecoxi +GM Supplementl Figure S9 (--75D) weeks fter tumor cell inocultion ws performed s in Supplementl Figure S., sttisticl determintion of synergy ws performed using individul phrmcologicl gent (t dy 8) dt points s in Supplementl Figure S. 5

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