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Q E C.2F) %# #K9K@& F < P O)?,)'@% Lung Lung Lung flux in liver (p/s)] flux in liver (p/s)] Lung $# Lung Lung flux in bone (p/s)] flux in bone (p/s)] 5 flux in lung (p/s)] flux in lung (p/s)] :) metastases :) metastases metastases A/4 Normalized log [photon metastases789 D)&&).N*789metastases metastasesD)&&).N* D)&&).N* Normalized log [photon Normalized log [photon G) Normalized log [photon Normalized log [photon P.0 P.1 P.2 P.0 P.1 P.2 P.0 P.1 P.2 I)(*#)? I)(*#)? Normalized log [photon P.0I)(*#)? P.1 P.2 P.0 P.1 P.2 P.0 P.1 P.2 PIM013PIM013 E) PIM040 PIM040 PIM073 PIM073 PIM137 PIM137 2014-0045-6-3688 2014-0045-6-3688 e*+,-.) 789;) */012) 789;) *+,-.) 789;) */012) 789;) f f e U !"#$%$="'-!"#$%$ \ T:'% + normalized=,[%. flux in liver _from P.2 lung mice $ Data Set-A % Data Set-A & KD' P0 vs P1 (lung) MG tumor growth ZZZ ZZZ Z 8!1011 6 6 6 R P.0 ;) Q &) ;) 789Q<) Q 'K9 34 34,-) 789 526/,) ZZ789 34,-) 789 526/,) ZZ Legend Legend PIM001-P normalized PIM001-M flux in liver PIM024 from P.2 lung micePIM003 2014-0045-6-3691 54,1.) PIM001-P PIM001-M PIM024P.1 PIM003 2014-0045-6-3691 54,1.) 11 3/ 4 F4 F4 6!10Q F Lung Lung 35P.2 2 2 metastases 2 #K9C 5 5 metastases 5 4 1011 !// F Additional 40 40 Additional 40 metastatic sites: A/4 11 2014-0045-6-3689 metastatic sites: PA14-13PA14-13 PA14-14 PA14-142!10 5 PA14-27 PA14-27 PIM010PIM010 2014-0045-6-3689 E5-2 E5-2 Liver Liver E5-2 ^:()$*#;:(:'*I$"J* >:.&)$,G%?*$:-* >:.&)$,G%?*$:-* Brain
H#;:(:'*I$"J*A#K9CL H#;:(:'*I$"J*A#K9CL 0 -4 -4 Brain >:.&)$,G%?*$:-* -4 4 normalized photon flux in bone normalized photon flux in liver
normalized photon flux in lung EF EF EF Lymph Node H#;:(:'*I$"J*A#K9CL 4 5 6 7 8 9 34***3/**35 34***3/**35Lymph Node 34***3/**35 F**<***Q**]**R**P 7%%89*#:9(E Figure 1. Characterization of metastatic PDX models. (a) BC3_A2 wasp53KD labeledP.0 with bioluminescent and fluorescent markers and implanted in mouse Figure 1.p53KD Characterization P.0 of metastaticp53KD PDX P.0 models. (a) BC3_A2 was labeled with bioluminescent and fluorescent markers and implanted in mouse mammary fat pads; metastases were observed in clinically-relevant sites. (b) BC3_A2 sheds circulating tumor cells%'-.)I(&%'( (CTCs) which are detected with FACS for the mammary fat pads; metastases were observed in clinically-relevantp53KD P.1p53KD lung P.2 lung sites. (b) BC3_A2P53KD sheds P.1p53KD lung circulating P.2 lung tumor cells (CTCs) which are detected with FACS for the mCherry reporter.P53KD0 P.1 (c)p53KD lung BC3_A2 P.2 lung lung metastases were2 passaged to mammary fat pads of1 recipient mice to enrich for metastatic subpopulations. (d) In vivo passaging mCherry reporter. (c) BC3_A2( lungHIM3p53KD metastases were passaged) to mammaryData Set-A fat pads *of recipient micemets to enrich to lung for metastatic subpopulations. (d) In vivo passaging enriches for metastasis to multiple sites. Mammary tumor growth is unaffected by in vivo passaging. Photon flux is normalized to time of euthanasia for each mouse. enriches for metastasis to multiple sites. Mammary tumor growth is unaffected by in vivo passaging.4 Photon flux is normalized to time of euthanasia for each mouse. (e and f) Additional metastatic20 PDX models are being characterized.30 WU-BC3 and PIM001-P PDXsF were both derived from breast tumors that were metastatic in the (e and f) Additional metastatic54 PDX models are being characterized.64 WU-BC3HIM3p53KD and PIM001-P PDXs were bothLegend derived from breastLegend tumors that were metastatic in the patient. patient. 15 52 /< A#K9CL 5420 /410 40 10
AB%%89C /4 <5 E5-2 weeks to euthanasia to weeks I$"J*,'*$"'-* =>*&%(*?%(%@(,:'* 40 40 EF-4 7%%89*(:*%"(;)')9,) 34***3/***35P.0 34****3/***35 >:.&)$,G%?*$:-*H#;:(:'* 34 3/* time to lymph node metastasis (weeks) metastasis node lymph to time Lung P.1 Lung P.2 p53KD P.0 D+3 KD P.1 lungKD P.2 lung p53KD P.0 p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cadherin in KD mg tumors and mets MG set at 1
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BC3--p53KD+ GFP BC3-p53KD + CEACAM5 10.05.2016 CDH1 in KD+GFP and KD+CEA ! B" "# CDH1 10000 ;; 2.5 ;;; 1000 567******** @******A "8!"!9:*******A@ 2.0 100 1.5 10 "8!"!9: 1.0 1 0<=>? "/01*2#3! 0.5 #$%&'()$*+,&-'('.* #$%&'()$*+,&-'('.*
10.05.2016"8!"!9:**2#3! CDH2 in KD+GFP and KD+CEA 0.1 0.0 567 "8!"!9: 567 "8!"!9: relative quantity CDH1 mRNA quantity relative
relative quantity CEACAM5 mRNA CEACAM5 quantity relative CDH2 10.05.2016 vimentin in KD+GFP and KD+CEA /$ 8% vimentin BC3--p53KD+ GFP BC3-p53KD + GFP 1.5 ;;; 1.5 ; BC3-p53KD + CEACAM5 BC3-p53KD + CEACAM5
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250 kDa 250 kDa 150 kDa CEACAM5 150 kDa CEACAM5 100 kDa 100 kDa 75 kDa 75 kDa
50 kDa 50 kDa 37 kDa 37 kDa p-p38 p-p38 25 kDa
50 kDa 50 kDa 37 kDa Total p38 37 kDa Total p38
25 kDa
250 kDa 250 kDa 150 kDa vinculin 150 kDa vinculin 100 kDa 100 kDa 75 kDa 75 kDa
50 kDa 50 kDa 37 kDa p-Smad2/3 37 kDa p-Smad2/3
25 kDa 25 kDa
50 kDa 50 kDa 37 kDa Total Smad 3 Total Smad 3 37 kDa
25 kDa 25 kDa
250 kDa 250 kDa Hsp90 150 kDa 150 kDa Hsp90 100 kDa 100 kDa 75 kDa 75 kDa UncroppedUncropped blots blots from from Figure 4DFig. 4d
c 250 kDa 150 kDa E-cadherin 100 kDa 75 kDa
50 kDa
37 kDa b-tubulin
25 kDa
250 kDa 150 kDa 100 kDa vinculin 75 kDa
50 kDa vimentin 37 kDa
25 kDa
Supplementary Figure 9. Uncropped Western blots. Corresponding figures are indicated, and molecular weight markers are shown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number of lesions JF+2,?"KL"M,=*K-=number of lesions 100 150 /01 50 =DEF6 0 61 /($)6CG:C:H9$S9$=&(&$)+",*&7$=,+6$LMGK$"*7$2&+")+"+,3$5/3,$=&(&$ )6R13
number of lesions =DEF6 /01 Data 1 I =D%$& %$&%&'( G( D$%'"H=D%$&%&'("G( =D%$&%&'("GH ,
E,=*K-"5*4+,.,?" %$&%&'( !7#$P,))1&$)&3+,/*)$/5$01*4)$5(/2$2,3&$,*A&3+&7$=,+6$ lesion size (microns) lesion7+*6?K-=@ size (microns) 1000 1500 2000 2500 500 *+,-.) 54,1.) 34,-) 0 =DEF6 III
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HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION ! ! ! ! HALLMARK_UV_RESPONSE_DN ! ! ! ! HALLMARK_TGF_BETA_SIGNALING ! ! ! ! HALLMARK_HEDGEHOG_SIGNALING ! ! ! ! HALLMARK_HYPOXIA ! ! ! ! HALLMARK_TNFA_SIGNALING_VIA_NFKB ! ! ! ! HALLMARK_ANGIOGENESIS ! ! ! ! HALLMARK_MTORC1_SIGNALING ! ! ! ! HALLMARK_APICAL_JUNCTION ! ! ! ! HALLMARK_MITOTIC_SPINDLE ! ! ! ! HALLMARK_APOPTOSIS ! ! ! ! HALLMARK_KRAS_SIGNALING_UP ! ! ! ! HALLMARK_IL2_STAT5_SIGNALING ! ! ! ! HALLMARK_IL6_JAK_STAT3_SIGNALING ! ! ! ! HALLMARK_GLYCOLYSIS ! ! ! ! HALLMARK_PROTEIN_SECRETION ! ! ! ! HALLMARK_INFLAMMATORY_RESPONSE ! ! ! ! HALLMARK_PI3K_AKT_MTOR_SIGNALING ! ! ! ! HALLMARK_COMPLEMENT ! ! ! ! HALLMARK_HEME_METABOLISM ! ! ! ! NES HALLMARK_NOTCH_SIGNALING ! ! ! ! ! 1.0 HALLMARK_PEROXISOME ! ! ! ! ! 1.5 HALLMARK_ANDROGEN_RESPONSE ! ! ! ! 2.0 HALLMARK_MYOGENESIS ! ! ! ! ! HALLMARK_COAGULATION ! ! ! ! ! 2.5 HALLMARK_PANCREAS_BETA_CELLS ! ! ! ! ! 3.0 HALLMARK_ALLOGRAFT_REJECTION ! ! ! ! Pathways HALLMARK_SPERMATOGENESIS ! ! ! ! pathway up down regulation HALLMARK_CHOLESTEROL_HOMEOSTASIS ! ! ! ! ! neg HALLMARK_BILE_ACID_METABOLISM ! ! ! ! ! pos HALLMARK_KRAS_SIGNALING_DN ! ! ! ! HALLMARK_INTERFERON_GAMMA_RESPONSE ! ! ! ! HALLMARK_UNFOLDED_PROTEIN_RESPONSE ! ! ! ! HALLMARK_XENOBIOTIC_METABOLISM ! ! ! ! HALLMARK_APICAL_SURFACE ! ! ! ! HALLMARK_MYC_TARGETS_V1 ! ! ! ! HALLMARK_ADIPOGENESIS ! ! ! ! HALLMARK_REACTIVE_OXIGEN_SPECIES_PATHWAY ! ! ! ! HALLMARK_E2F_TARGETS ! ! ! ! HALLMARK_P53_PATHWAY ! ! ! ! HALLMARK_WNT_BETA_CATENIN_SIGNALING ! ! ! ! HALLMARK_FATTY_ACID_METABOLISM ! ! ! ! HALLMARK_G2M_CHECKPOINT ! ! ! ! HALLMARK_ESTROGEN_RESPONSE_EARLY ! ! ! ! HALLMARK_MYC_TARGETS_V2 ! ! ! ! HALLMARK_OXIDATIVE_PHOSPHORYLATION ! ! ! ! HALLMARK_INTERFERON_ALPHA_RESPONSE ! ! ! ! HALLMARK_UV_RESPONSE_UP ! ! ! ! HALLMARK_DNA_REPAIR ! ! ! ! HALLMARK_ESTROGEN_RESPONSE_LATE ! ! ! !
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