Supplementary Figures (1-6) Deletion of ADAM-9 in HGF/CDK4 mice impairs melanoma development and metastasis

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1 Supplmntary Figurs (1-6) Dltion of ADAM-9 in HGF/CDK4 mic impairs mlanoma vlopmnt an mtastasis Nivs Giblr 1, Alxanr Schönfuß 1, Jnnifr Lansbrg #, Thomas Tüting, Cornlia Mauch an Paola Zigrino*

2 A 25 * ADAM-9 S26 skin tumor ADAM-9 mrna fol chang skin tumor B Tumor-stroma Tumor s t 1µm ADAM-9 ADAM-9 / DAPI Supplmntary Figur 1

3 Supplmntary Figur 1: ADAM-9 in mlanomas from tumor-pron Hgf/Ck4 R24C/R24C mic. Exprssion an localization of ADAM-9 in DMBA-inuc tumors in Hgf/Ck4 R24C/R24C animals. (A) ADAM-9 transcripts tct by RT-PCR in untrat skin (skin) (n=3) an DMBA-inuc tumors (tumor) (n=4) harvst 13 wks aftr DMBA tratmnt. Th graph rprsnts th rlativ intnsity of th bans an shows th avrag of signal intnsitis aftr normalization to S26, us as control. p<.5 (, t-tst). (B) Frozn tumor sctions wr stain for ADAM-9 (grn), an cll nucli (blu). Shown ar rprsntativ picturs of ADAM-9 at th tumor-stroma borr an in intratumoral aras from Hgf/Ck4 R24C/R24C mic post DMBA tratmnt. Th tumor-stroma borr is mark by a ash lin. Th whit arrowhas point to ADAM-9 positiv clls. t, tumor; s, stroma.

4 HGF + 15 Aam-9 mutat/ko HGF + numbr of TRP2+ clls / mm 1 5 Aam-9 mutat/ko H&E K14 / DAPI Loricrin / DAPI Lam β3 / TRP2 / DAPI sc Aam-9 mutat/ko sc 1µ m Supplmntary Figur 2

5 Supplmntary Figur 2: Mic phnotyping. Picturs of control an Aam-9 mutat/ko mic an thir paws showing th HGF inuc black skin phnotyp. Skin sctions from mic at postnatal ay 1 stain by H&E, to arss gnral morphology: cytokratin 14 (r) an loricrin (r) wr us to show pirmal iffrntiation an TRP-2 (grn; tyrosinas rlat protin- 2) to localiz mlanocyts. Cll nucli, stain blu (DAPI). Numbrs of mlanocyts in th skin of postnatal ay 1 mic wr count an hr xprss as an avrag ± SEM (n=3). Th ash lin marks th pirmal-rmal junction., pirmis;, rmis; sc, subcutanous tissu. Th graph isplays th avrag numbrs of mlanocyts in th skin.

6 A B16F1 clls ADAM-9 mrna fol chang [2 Ct ] ** pro ADAM-9 activ ADAM-9 β-actin. shaam-9 B BLM clls ADAM-9 mrna fol chang [2 Ct ] *** pro-adam-9 activ ADAM-9. shaam-9 β-actin C Tumor volum (mm 3 ) * * * shaam Tim (ays) Supplmntary Figur 3

7 Supplmntary Figur 3: Silncing of ADAM-9 in mous an human mlanoma clls an growth of mlanoma in mous. ADAM-9 quantitativ ral-tim PCR, immunoblot analysis of ADAM-9 an cllular prolifration of (A) B16F1 mous mlanoma an (B) BLM human mlanoma clls stably transfct with shrna targt to ADAM-9 (shaam-9) or a scrambl control squnc (). Th graph rprsnts thr inpnnt xprimnts prform in triplicats. Rprsntativ immunoblot analysis to tct ADAM-9 protin xprssion, β-actin was us as a control. p<.1 ( ) an p<.1 ( ), t- tst. (C) Growth of mlanoma aftr injction of control an shaam-9 silnc B16F1 clls in th flank of control animals was monitor ovrtim an hr pict in th graph as avrag ± SEM. Th tabl blow inicats th numbr of tumors visibl as arlir as ay 4 for control an shaam-9 clls. (n=1, control; n=9, shaam-9) *p,3, t- tst

8 wk 13 wk 4 t Aam-9 mutat/ko t % Ki67 positiv clls Aam-9 mutat/ko t Ki67 / cl.casp3 / DAPI t 1µm % Ki67 positiv clls 5 * Aam-9 mutat/ko Supplmntary Figur 4

9 Supplmntary Figur 4: Prolifration an apoptosis of DMBA-inuc tumors grown in control an Aam-9 mutat/ko animals. Frozn tumor sctions from DMBA trat animals wr oubl stain for Ki67 (grn) an clav caspas-3 (r). Th comparison was prform with tumors of comparabl siz (2mm 2 an ~4mm 3, wk 4 an 13, rspctivly ). Cll nucli wr visualiz with DAPI (blu). Th whit arrowhas point to clav casapas-3 positiv clls (r). Th tumor-stroma borr is inicat by th ash lin. t, tumor; s, stroma. Th graphs show th avrag ±SEM numbr of Ki67 positiv clls as % ratio of total count clls (avrag of 3-4 tumors) pr mous. p<.5 (, t-tst) (n=4/6, wk 4 an 13 control/aam-9 mutat/ko ).

10 axillary groin 15 6 Volum [mm³] 1 5 Volum [mm³] 4 2 Aam-9 mutat/ko Aam-9 mutat/ko TRP-1 / CD45 / DAPI TRP-1 / CD45 / DAPI Supplmntary Figur 5

11 Supplmntary Figur 5: Effct of ADAM-9 on lymph no mtastasis. Th volums of axillary an groin lymph nos wr masur an xprss as th avrag volum of th right an th lft lymph no of a singl animal. Each ot rprsnts th avrag siz/mous. Photographs show rprsntativ picturs of th macroscopic apparanc of th lymph nos of both gnotyps. Axillary an groin lymph nos, wr oubl stain for CD45 (lukocyts) an TRP-1 (mlanoma clls) to furthr vrify infiltration of mlanoma clls. scal, 1mm

12 shaam-9 Animals with mtastasis livr lung hart shaam-9 5/5 1 % 3/7 43 % Supplmntary Figur 6

13 Supplmntary Figur 6: Effct of ADAM-9 own-rgulation on mlanoma mtastass. Th numbr an prcntag of mtastass-baring mic ar summariz for control an shaam-9 silnc clls injct intravnously (n=5 in s an n=7 for shaam-9). Th photographs show rprsntativ picturs of mtastatic nouls in th livr, lung an hart.