Phenotypic differences of proliferating fibroblasts in the stroma of lung adenocarcinoma and normal bronchus tissue

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1 Phenotypi ifferenes of proliferting firolsts in the strom of lung enorinom n norml ronhus tissue Noko Nkmur, Ttsuo Iijim, Kentro Mse, 2 Shuihiroh Furuy, 4 Junko Kno, Yukio Morishit 3 n Msyuki Noguhi, 5 Deprtment of Pthology, Institute of Bsi Meil Sienes, University of Tsuku, -- Tennoi, Tsuku, Irki ; 2 Deprtment of Surgery, Mito Generl Hospitl, Hithi, Lt., 2- Ishikw-ho, Hithink, Irki 32-57; 3 Deprtment of Clinil Pthology, Institute of Clinil Meiine, University of Tsuku, -- Tennoi, Tsuku, Irki ; n 4 Lortory of Clinil Pthology, University Hospitl of Tsuku, 2-- Amkuo, Tsuku, Irki (Reeive Septemer 22, 23/Revise Jnury 2, 24/Aepte Jnury 25, 24) Firolsts in tumor tissue re thought to intert with tumor ells iretly n/or iniretly n to hve importnt roles in tumor invsion n metstsis. To hrterize the phenotype of proliferting firolsts in pulmonry enorinom, we estlishe short-term firolst ell lines from oth norml ronhus n enorinom tissues otine from the sme ptients n ompre the gene expression profiles. Four sets of firolst ell lines (eight ell lines in totl) were use in the nlysis. Totl RNA ws extrte from eh ell line n hyriize with 55 nerrelte RNAs lotte on DNA filter rry. Five up-regulte genes n 2 own-regulte genes (totl of 7 genes) were etete in the firolst ell lines from the tumor tissues ompre with those from norml ronhus. Using rel-time quntittive RT-PCR methos, the expression profile of eh gene ws exmine; five genes, one up-regulte (MLH) n four own-regulte (Cox, FGFR4, p2, n Sm3), were onfirme. Furthermore, the protein expression levels of the five genes in the nerous n norml tissues were exmine immunohistohemilly, n the up-regultion of MLH n the own-regultion of Cox in nerous tissue were onfirme in vivo. These results inite tht the proliferting firolsts in pulmonry enorinoms re phenotypilly ifferent from firolsts in norml ronhus tissues. (Cner Si 24; 95: ) R eent vnes in moleulr iology hve le to the estlishment n eptne of the onept of multi-step rinogenesis. The si onept of multi-step rinogenesis for pulmonry enorinom ws first propose y Miller et l. in 988, n ws nlogous to the proess esrie for olon enorinom. ) Lter, typil enomtous hyperplsi (AAH) ws lssifie s pre-invsive enorinom lesion y the Worl Helth Orgniztion (WHO). 2) In 995, we propose lssifition system for smll-size enorinoms, inluing well-ifferentite lolize ronholveolr rinom (LBAC). 3) We ivie peripherl smll-size enorinoms into two types, se on their growth pttern: lveolr struture replement or expnsion n estrution of the originl lveolr struture. The former group inlues LBAC (type A), LBAC with lveolr ollpse (type B), n LBAC with firolsti prolifertion (type C), representing the mjority of erly peripherl-type enorinoms of the lung. The 5-yer survivl rtes of type A n B LBAC re % ut tht of type C is out 75%. Therefore, type A n B tumors re thought of iologilly s in situ enorinoms tht mimi AAH, while type C tumors re thought of s erly ut invsive rinoms. The importnt morphologil ifferene etween type A n B tumors n type C tumors is the prolifertion of firolsts in the tumor tissue. The morphology of the proliferting firolsts in invsive ner is ifferent from tht of firolsts in norml tissue: proliferting firolsts in invsive ner hve lrge nulei n usully ontin nuleoli. 3) Mny reports hve presente importnt orreltions etween stroml firolsts n ner ells in invsive ner tissue. For exmple, stroml firolsts re thought to minly express mtrix metlloproteinse-2 (MMP-2), n memrne-type MMP (MT-MMP) expresse y ner ells tivtes MMP ) Ativte MMP-2 is key enzyme for issolving the extrellulr mtrix. Further, onitione meium of humn firolst ell lines elertes the invsiveness of severl ner ell lines in vitro. 8, 9) These reports inite tht either stroml firolsts re strongly ffete y invsive ner ells or invsive ner ells re iologilly influene y stroml firolsts. 2) In this stuy, we estlishe severl sets of short-term firolst ell lines from invsive ner n norml ronhus tissues otine from the sme ptients to hrterize the ifferenes etween stroml firolsts in erly ut invsive lung enorinom n those in norml ronhus tissue. The expression profiles of the ell lines were then exmine using filter rrys. Mterils n Methos Estlishment of ell lines. A setion of tumor tissue n the resete en of the ronhus were exise from surgilly resete speimens. n inute in Duleo s moifie Egle s meium (DMEM) ontining ollgense ( unit/ml), ispse (, units/ml), peniillin ( units/ml), n streptomyin ( µg/ml) without growth ftors or serum for 2 h t 37 C. The issoite ells were then filtere using FAL- CON -µm ell striner (Beton Dikinson Lwre, Frnklin Lkes, NJ) n entrifuge for 5 min t 9 rpm. The ell pellet ws trnsferre to DMEM n F2 with L-glutmine, % fetl ovine serum, peniillin ( units/ml) n streptomyin ( µg/ml) n ulture on ollgen-ote ishes t 37 C in humi tmosphere of 5% CO 2 /95% ir. After three or four pssges, we onfirme tht the ulture ells were nonepithelil ells, ut not enoytes, using immunohistohemistry. Anti-vimentin (Ventn Meil Systems, In., Tuson, AZ), nti-pnkertin (Ventn Meil Systems, In.), n nti- CD3 (DACO A/S, Denmrk) ntioies were use to ientify the ells. An enorinom ell line, A549, n n enothelil ell line, HUVEC, were lso stine s ontrol ell lines. RNA extrtion n DNA leling. RNA extrtion from the firolsts ws performe one the ells h rehe out 7% onfluene (etween 3 ell pssges). Totl ellulr RNA ws prepre using TRIzol Regent (Life Tehnologies, In., 5 To whom orresponene n reprint requests shoul e resse. E-mil: nmsyuk@m.tsuku..jp Cner Si Mrh 24 vol. 95 no. 3 Nkmur et l.

2 Rokville, MD) oring to the mnufturer s iretions. mrna ws isolte from the totl RNA trete with DNse I using the MgExtrtor mrna isoltion kit (Toyoo Co., Osk). A DNA synthesis retion ws perfome using 3 µg of mrna n ReverTrAe (Toyoo); the DNA ws then lele with iotin y inserting iotin-6-eoxyuriine triphosphte (Rohe Dignostis, GmH, Mnnheim, Germny). The omplete DNA ws use s proe. Hyriiztion n signl etetion. A Gene Nvigtor DNA Arry Filter (Toyoo) ontining 55 frgments of known ner-relte humn genes n frgments of housekeeping genes ws use for the ssy. 3, 4) Lele DNA ws hyriize with this filter in 2 ml of PerfetHy (Toyoo) overnight t 68 C fter prehyriiztion for 3 min t 68 C. After hyriiztion, the filter ws wshe three times with 2 SSC,.% SDS t 68 C for 5 min n three times with. SSC,.% SDS t 68 C for 5 min. The speifi signls on the filter were oserve using the Imging High (Toyoo) hemiluminesene etetion kit, n the hemiluminesene signl intensity ws etete using STORM Snning Systems (Amershm Tle. Sequene of TqMn primers n proes use in this stuy Gene nme Lotion Sequene Size PCR prout Aession No. Bl-x-FP omp CACCAAATACCTGCATCTCCTTGT-3 75 p U72398 Bl-x-RP omp CGCATTGTGGCCTTTTTCTC-3 Bl-x-T omp TACGCTTTCCACGCACAGTGCCC-3 CD4 Assy-on Dem Gene Expression Prouts (Applie Biosystems) Assy ID: hs827_ml: Inluing 5 -CAGCTTCCCAGAAGAAGAGCATACA-3 M287 Cox-FP 6 5 -CAATACCGCAACCGCATTG-3 77 p M59979 Cox-RP ACCTTGAAGGAGTCAGGCATGA-3 Cox-T TGGAGTTCAACCATCTCTACCACTGGCAC-3 Crk-FP GGTGAGCTGGTAAAGGTTACGAA-3 2 p D656 Crk-RP 6 5 -CATCGGGATTCTGTTGATCCA-3 Crk-T CGAGGTCACTTCCCATTCACACATGTCC-3 E2F2-FP CATTTACCTCCTGAGCGAGTCA-3 49 p L22846 E2F2-RP GGATGTTGTTCTTGGCCTTCTT-3 E2F2-T ATCACCAACGTGCTGGAAGGCATCC-3 Eph-FP TGCATGTCAATGGCCTTGA-3 28 p M839 Eph-RP CTCTGCATGCCCCATGCT-3 Eph-T CCAYGCCAGCACCTCAGTCAGCA-3 FAS-FP AACTTGGAAGGCCTGCATCAT-3 4 p M67454 FAS-RP CAGTCTGGTTCATCCCCATTG-3 FAS-T CTGCCATAAGCCCTGTCCTCCAGGT-3 FGFR-4-FP CGCATGGAGAAGAAACTGCAT-3 p X5725 FGFR-4-RP CCTTAAGCCAGCGGATGGT-3 FGFR-4-T AACACCGTCAAGTTCCGCTGTCCAG-3 Flt--FP TCAAGGAACCTCGGACAAGTCT-3 92 p AFO63657 Flt--RP AGAGGGTTAATAGGAGCAGAAGAG-3 Flt--T TCTGGAGCTGATCACTCTAACATGCACCTG-3 Mt-FP CCAGCCACTGCAGATAGAGACATA-3 26 p X92669 Mt-RP GAAGAAGTATAGCCTCCAGCAAGGT-3 Mt-T AACCATGTGTCAGAGCTGCCTCACCACA-3 MLH-FP omp-8 5 -GCTTGGTGGTGTTGAGAAGGTATA-3 78 p U7343 MLH-RP omp CCTTCGTGGGCTGTGTGAA-3 MLH-T omp TTGATGCTGTGCCAAGGCCCAC-3 p2-fp GGAATTTCTCTGAGGACACTGTCA-3 4 p AF62324 p2-rp AATACCCTGTGTCTCTCGAAGCTT-3 p2-t CAACGAGGTTATCGCTGAG-3 Tlin Assy-on Dem Gene Expression Prouts (Applie Biosystems) Assy ID: hs967757_ml: Inluing 5 -CCAGAAGACTTCATCCGAATGACCA-3 AF7798 Sm3-FP GTCGGTCAACCAGGGCTTT-3 86 p U76622 Sm3-RP CCGCTCCCCAGCCTTT-3 Sm3-T CTGTCTACCAGTTGACCCGAATGTGCAC-3 TGF-RI-FP TGCATCTCACTCATGTTGATGGT-3 93 p L695 TGF-RI-RP GCGATCTAATGAAGGGTCCTCTT-3 TGF-RI-T CAACCGCACTGTCATTCACCATCGA-3 TPA-FP omp TCAGCCTGCGGTTCTTCAG-3 75 p M558 TPA-RP omp GCAAACATAATTACTGCCGGAAT-3 TPA-T omp CAGGGCTTGGCATCCCCATCAG-3 VWF Assy-on Dem Gene Expression Prouts (Applie Biosystems) Assy ID: hs69795_ml: Inluing 5 -CTGTCTCATCGCAGCAAAAGGAGCC-3 M25865 GAPDH-FP AATTCCATGGCACCGTCAA-3 9 p NM246 GAPDH-RP CCAGCATCGCCCCACTT-3 GAPDH-T CCATCACCATCTTCCAGGAGCGAGA-3 FP, forwr primer; RP, reverse primer; T, TqMn proe; omp, omplementl sequene. Bol: Those genes were onfirme y reltime RT-PCR. Nkmur et l. Cner Si Mrh 24 vol. 95 no

Biosienes Corp., Pistwy, NJ); the imge intensities were quntifie using ImGene (BioDisovery, In., LA). Gene expression ws onverte into reltive numer se on the intensity of the ontrol gene.

DNA ws prepre using Tq- Mn Reverse Trnsription Regents (PE Applie Biosystems, In., Foster City, CA) in the mnufturer s uffer ontining 5 µm eh NTP, 5.5 mm MgCl 2, 2.5 µm rnom hexmers,.

Clinio-pthologil informtion on the suessfully estlishe short-term ell lines of enorinom ses Cse No.

IA 74/F mo IIA n 66/F poor IA 2 68/F well IIA n 3 75/F well IA 4 66/F well IA 5 7/M poor IIB n 6 74/M poor IB 7 52/M well IA 8 58/F mo IIA n2 9 73/M well IA mo, moerte; n, positive for pulmonry hilr

3 Biosienes Corp., Pistwy, NJ); the imge intensities were quntifie using ImGene (BioDisovery, In., LA). Gene expression ws onverte into reltive numer se on the intensity of the ontrol gene. The expression profiles shown y the two filters were sttistilly ompre using E-Gene Nvigtor Anlysis (GenetiL, Spporo). Rel-time quntittive RT-PCR. DNA ws prepre using Tq- Mn Reverse Trnsription Regents (PE Applie Biosystems, In., Foster City, CA) in the mnufturer s uffer ontining 5 µm eh NTP, 5.5 mm MgCl 2, 2.5 µm rnom hexmers,.4 units of RNse inhiitor, n.25 units of MuLV reverse trnsriptse. Rel-time quntittive RT-PCR nlysis Tle 2. Clinio-pthologil informtion on the suessfully estlishe short-term ell lines of enorinom ses Cse No. Age/gener Histologil ifferentition Pthologil stge Lymph noe metstsis 5/F well IA 2 62/M mo IA 3 65/M poor IV n2 4 67/M poor IB 5 66/M mo IA 6 64/M well IA 7 58/F well IA 8 72/M poor IIIB 9 8/F well IA 74/F mo IIA n 66/F poor IA 2 68/F well IIA n 3 75/F well IA 4 66/F well IA 5 7/M poor IIB n 6 74/M poor IB 7 52/M well IA 8 58/F mo IIA n2 9 73/M well IA mo, moerte; n, positive for pulmonry hilr lymph noes; n2, positive for meistinl lymph noes. Cses tht were use in this stuy. ws performe in triplite using the ABI PRISM 57 Sequene Detetion System instrument n softwre (PE Applie Biosystems). The primers n proes for the TqMn system were esigne using Primer Express softwre (Perkin Elmer, Foster City, CA) n synthesize using PE ABI (Weiterstt, Germny). The 5 -en nuleotie of the proe ws lele with reporter ye (FAM). The sequenes of the PCR primer sets n proes use for eh gene re shown in Tle. The retion onitions n PCR yle were set oring to the mnufturer s iretions. Immunostining of MLH, p2, Cox, Sm3, n FGFR4. Six ses of type C enorinoms were use in the stuy. The speimens were fixe in uffere formlin n emee in prffin. Prffin-emee loks were then ut into 3-µm setions n mounte on poly-l-lysine-ote slies. Immunohistohemil stining ws performe using the following primry ntioies: nti-mlh (mouse monolonl IgG; BD Biosienes Phrmingen, CA), nti-p2 (mouse monolonl IgG; Zyme Lortories, In., CA), nti-cox (got polylonl IgG; Snt Cruz Biotehnology, In., CA), nti-sm3 (mouse monolonl IgG; Trnsution Lortories, KY), n nti-fgfr4 (rit polylonl IgG; Snt Cruz Biotehnology, In). Enogenous peroxise tivity ws inhiite y treting the setions with 3% hyrogen peroxie for 3 min. Bloking ws performe with 2% norml swine serum (DAKO A/S). For p2 n Sm3, the setions were pretrete in n utolve t 2 C for min in. M itrte uffer (ph 6). For MLH, p2 n FGFR4, Ventn Meil Systems (Ventn Meil Systems, In., Tuson, AZ) ws use. For Cox n Sm3, iotinylte seonry ntioy n streptviin-peroxise onjugte were use to etet the signl (LSAB+ kit/hrp, universl; DAKO A/S). Results Estlishment n immunohistohemil stining of short-term ell lines. We ttempte to estlish two sets of short-term ulture ell lines from the sme ptient using norml n nerous ells from more thn 3 ptients with lung ner; in the en, we sueee in estlishing oth ell lines in 28 ses. Of Fig.. Histology finings (HE stin). ) Cse 4 (golet ell-type enorinom), ) se 9 (Clr ell n ronhil-surfe epithelil ell-type enorinom), ) se 2 (Clr ell n ronhil-surfe epithelil ell-type enorinom), n ) se 6 (poorly ifferentite enorinom). 228 Nkmur et l.

these 28 ses, 9 ses were smll-size enorinoms with mximum imeter of 2 m or less (Tle 2).

All tumors h een gre s pthologil stge I; the tumor in se 4 ws golet ell-type enorinom, while the others were Clr ell-type n/or ronhil

The eight ell lines were positive for nti-vimentin n negtive for ntipnkertin n nti-cd3 (Fig. 2).

We nlyze the up-regulte n own-regulte genes in ll four sets of firolst ell lines n etete 7 genes whose expression ptterns iffere in tumor

As Tle 3 shows, E2F2, MLH, Tlin, TGFβRI, n TPA were up-regulte in firolst ell lines estlishe from tumor tissues, while Bl-x, CD4, Crk,

To onfirm the hrteristis of the expression profiles, we nlyze the expression levels of the 7 genes using rel-time RT-PCR.

4 these 28 ses, 9 ses were smll-size enorinoms with mximum imeter of 2 m or less (Tle 2). We use the four sets of short-term ulture ell lines from ses 2, 4, n 9 (type C enorinom) n se 6 (type D enorinom). All tumors h een gre s pthologil stge I; the tumor in se 4 ws golet ell-type enorinom, while the others were Clr ell-type n/or ronhil surfe epithelil ell (BSE)-type enorinoms (Fig. ). The eight ell lines were positive for nti-vimentin n negtive for ntipnkertin n nti-cd3 (Fig. 2). Thus, we onlue tht the eight ell lines were firolst ell lines. Gene expression profiles of firolst ell lines. We nlyze the up-regulte n own-regulte genes in ll four sets of firolst ell lines n etete 7 genes whose expression ptterns iffere in tumor n norml ronhus tissues. A representtive expression pttern for one set of firolst ell lines (se 4) is shown in Fig. 3. As Tle 3 shows, E2F2, MLH, Tlin, TGFβRI, n TPA were up-regulte in firolst ell lines estlishe from tumor tissues, while Bl-x, CD4, Crk, Cox, Eph, FAS, FGFR4, Flt-, Mt, p2, Sm3, n VWF were ownregulte. To onfirm the hrteristis of the expression profiles, we nlyze the expression levels of the 7 genes using rel-time RT-PCR. Two own-regulte genes, Cox n FGFR4, were onfirme in ll four sets of the ell lines (Tle 3 n Fig. 4). One up-regulte gene (MLH) n two own-regulte genes (p2 n Sm3) were lso onfirme in three sets of the ell lines. e f Fig. 2. Immnohistohemil stining of short-term ell lines.,, e) Firolsts in norml ronhus.,, f) Stroml firolst from tumors. The firolst ell lines were positive for nti-vimentin (, ) n negtive for nti-pnkertin (, ) n nti-cd3 (e, f). Nkmur et l. Cner Si Mrh 24 vol. 95 no

Expression of MLH/ GAPDH 3 2.5 2.5.5 Cse 4 Cse 9 Cse 2 Cse 6 Expression of Cox / GAPDH.9.8.7.6.5.4.3.2. Cse 4 Cse 9 Cse 2 Cse 6 Fig. 3. Representtive expression profiles of DNA filter rrys for one set of firolst ell lines.

5 Expression of MLH/ GAPDH Cse 4 Cse 9 Cse 2 Cse 6 Expression of Cox / GAPDH Cse 4 Cse 9 Cse 2 Cse 6 Fig. 3. Representtive expression profiles of DNA filter rrys for one set of firolst ell lines. A uplite set of 55 frgments of humn ner-relte genes n frgments of housekeeping genes, inluing the positive n negtive ontrols, were spotte on the filters. ) Expression profile of firolsts from norml ronhus. ) Expression profile of firolsts from ner strom. The empty rrowhes show high expression level in firolsts from norml ronhus tissue, while the lose rrowhes show high expression level in firolsts from ner strom tissue. Immunohistohemil nlysis. To onfirm the protein expression levels of the five genes in the nerous n norml lung tissues, n immunohistohemil nlysis ws performe using 6 ses of type C tumors. The up-regultion of MLH expression n the own-regultion of Cox expression were etete in the stroml firolsts of the rinoms in ll 6 ses (Fig. 5). However, the protein expression levels of the other three genes were not own-regulte in the stroml firolsts of the rinom. Disussion In this stuy, we exmine the expression profiles of proliferting firolsts in tumor strom n norml ronhus tissues n foun five genes tht were ifferently expresse. The min etetion strtegy use in this stuy ws the mro-rry system. Although the expression levels of hunres of genes n e esily n quikly estimte using this powerful tool, n urte interprettion of the expression level of eh gene is iffiult ue to the numerous rtifts tht re present. 5) As shown in Tle 3 n Fig. 4, the results of the rry nlysis of some genes, suh s Sm3, were opposite to tht otine using rel-time RT-PCR nlysis. Therefore, when we estimte the results of the rry nlysis, the limittions of this system were kept in min n rel-time RT-PCR ws use to onfirm the results. Furthermore, we lso exmine the protein expression profiles of the five selete genes in the resete tissue speimens using immunohistohemistry. Finlly, the up-regultion of e Expression of FGFR4 / GAPDH Expression of p2 / GAPDH Expression of Sm3 / GAPDH Cse 4 Cse 9 Cse 2 Cse 6 Cse 4 Cse 9 Cse 2 Cse 6 Cse 4 Cse 9 Cse 2 Cse 6 Fig. 4. Results of rel-time quntittive PCR. ) hmlh, ) Cox, ) FGFR4, ) p2, e) Sm3. All mesurements re shown reltive to the expression levels of the GAPDH housekeeping gene. Brs show the men n stnr evition (SD). P<.5 versus norml, Stuent s n Welh s t tests. the MLH gene n the own-regultion of the Cox gene were onfirme using in vivo speimens. MLH ws up-regulte in firolsts otine from the tumor strom. MLH expression hs een reporte to e up-regulte in proliferting ells, suh s sl ells in the skin n reserve ells in the limentry trt. Therefore, the up-regultion in the firolsts from the tumor strom my inite 23 Nkmur et l.

4/N 2.6/N.3/.5 MLH Tumor suppressor gene/poptosis.8/n.6/n.4/n./n Tlin Cell hesion protein 2.8/6. 6.7/9.4 6.4/N 24.3/5.3 TGFRI Memrne reeptors 2.5/.2./N 2.9/.3.6/N TPA Signling Intermeites 2./.5.2/N.

4 N/2.5 2./4.4 Eph Memrne reeptors.6/3.8 N/.7 N/3.8 N/.7 FAS Tumor suppressor gene/apoptosis N/.4 N/.3 N/2.6 N/.8 FGFR-4 Memrne reeptors N/2.4./2.2 N/.2 N/. Flt Memrne reeptors N/.6 N/.7 N/2.3 N/2.3 Mt Cell yle proteins 2.

6 t-firolst, stroml firolsts from tumors; n-firolst, firolsts in norml ronhus; N, no numeril vlue. Bol: Chnges in expression of these genes were onfirme y rel-time RT-PCR. Fig. 5.

6 Tle 3. Reltive rtio of up-regulte n own-regulte gene expression in t-firolst/n-firolst speimens Gene nme Funtion t-firolst/n-firolst Cse Cse 2 Cse 3 Cse 4 Up-regultion E2F2 Regultory trnsription ftors.7/. 4/N 2.6/N.3/.5 MLH Tumor suppressor gene/poptosis.8/n.6/n.4/n./n Tlin Cell hesion protein 2.8/6. 6.7/ /N 24.3/5.3 TGFRI Memrne reeptors 2.5/.2./N 2.9/.3.6/N TPA Signling Intermeites 2./.5.2/N.6/.7.9/N Down-regultion Bl-x Tumor suppressor gene 2.4/2.6 N/.3 N/.7./4.3 CD4 Lymphoyte signling N/.9 N/.6./4.7 N/.3 Crk Signling Intermeites.3/5.7.3/3. 2./7.7.4/2. Cox Signling Intermeites.5/5. N/3.4 N/2.5 2./4.4 Eph Memrne reeptors.6/3.8 N/.7 N/3.8 N/.7 FAS Tumor suppressor gene/apoptosis N/.4 N/.3 N/2.6 N/.8 FGFR-4 Memrne reeptors N/2.4./2.2 N/.2 N/. Flt Memrne reeptors N/.6 N/.7 N/2.3 N/2.3 Mt Cell yle proteins 2./3. N/. N/. N/N p2 Signling Intermeites N/. N/2.3 N/2.3 N/.6 Sm3 Regultory trnsription ftors N/3.8.2/.8.2/2.9 4./.4 VWF Cell hesion protein N/5.3 N/..6/.9 N/.6 t-firolst, stroml firolsts from tumors; n-firolst, firolsts in norml ronhus; N, no numeril vlue. Bol: Chnges in expression of these genes were onfirme y rel-time RT-PCR. Fig. 5. Results of immunohistohemil nlysis ( 4) for MLH (, ) n Cox (, )., ) Firolsts in norml tissue., ) Stroml firolst from tumors. Arrows inite representtive firolsts tht re positive for the two ntigens. high prolifertive tivity, ompre to tht in the firolsts from norml ronhus tissue. Cox expression erese in the stroml firolsts. Cox is representtive enzyme ssoite with eiosnoi iosynthesis n is expresse in ll humn tissues, inluing the lungs. Its tivity is thought to e responsile for prouing ytoprotetive prostglnins, suh s prostylin n PGE2. Therefore, this enzyme is thought to e ritil to mintining the integrity of the gstri muos. 6 8) Reently, Petkov et l. exmine the expressions of Cox n Cox2 in iiopthi pulmonry firosis (IPF) using immunohistohemil methos n reporte tht oth expression levels were reue in the ronhiolr epithelium; furthermore, the group i not etet ny positive retions for expression of these genes in proliferting Nkmur et l. Cner Si Mrh 24 vol. 95 no. 3 23

7 firolsts. 9) Although the ext role of Cox in stroml firolsts ontinues to e ete, we speulte tht the own-regultion of the Cox gene in the stroml firolsts of enorinoms my e funtionlly similr to tht whih ours in the stroml firolsts in IPF. This stuy ws fouse on the expression profiles of stroml firolsts in pulmonry enorinom. We foun tht stroml firolsts from erly invsive enorinom tissue n firolsts from norml ronhus tissue h ifferent hrteristi phenotypes. Severl explntions for these results re possile. First, invsive rinom ells my require speifi environmentl onitions, n invsive tumor ells my selet only the most suitle firolsts for their strom. Alterntively, the selete firolsts my quire ifferent stle hrters, ompre with firolsts in norml ronhus tissue. The finl possiility is tht the tumor ell environment my ffet the firolsts n lter their phenotype in suh wy tht 2, 2) they eome stle in ulture. To eluite the mehnism of tumor invsion, proliferting firolsts in the tumor strom must e nlyze more extensively. If tumor ells require speifi stroml firolsts to exhiit invsive growth, therpeuti interventions trgete t eliminting stroml firolsts my e effetive for reuing tumor invsion.. Miller RR, Nelems B, Evns KG, Muller NL, Ostrow DN. Glnulr neoplsi of the lung. A propose nlogy to oloni tumors. Cner 988; 6: Trvis WD, Coly TV, Corrin B et l. Interntionl Histologil Clssifition of Tumors: Histologi typing of lung n pleurl tumors. 3r e. Germny: Springer; Noguhi M, Morikw A, Kwski M, Mtsuno Y, Ym T, Hirohshi S, Kono H, Shimosto Y. Smll enorinom of the lung. Histologi hrteristis n prognosis. Cner 995; 75: Brown PD, Bloxige RE, Sturt NS, Gtter KC, Crmihel J. Assoition etween expression of tivte 72-kilolton geltinse n tumor spre in non-smll-ell lung rinom. J Ntl Cner Inst 993; 85: Azzm HS, Arn G, Lippmn ME, Thompson EW. Assoition of MMP-2 tivtion potentil with metstti progression in humn rest ner ell lines inepenent of MMP-2 proution. J Ntl Cner Inst 993; 85: Crwfor HC, Mtrisin LM. Tumor n stroml expression of mtrix metlloproteinses n their role in tumor progression. Invsion Metstsis ; 4 : Sto H, Tkino T, Ok Y, Co J, Shingw A, Ymmoto E, Seiki M. A mtrix metlloproteinse expresse on the surfe of invsive tumour ells. Nture 994; 37: Bln JF, Bisson C, Frnkenne F, Noel A, Munut C, Colige A, Collette J, Rosenum J, Foirt JM. Heptorinom ell lines own-regulte mtrix metlloproteinse-2 expression in humn hepti myofirolsts. Int J Onol 22; 2: Lng SH, Stower M, Mitln NJ. In vitro moelling of epithelil n stroml intertions in non-mlignnt n mlignnt prosttes. Br J Cner 2; 82: Swhney N, Grrhn N, Dougls-Jones AG, Willims ED. Epithelil-stroml intertions in tumors. A morphologi stuy of firoepithelil tumors of the rest. Cner 992; 7: Iozzo RV. Tumor strom s regultor of neoplsti ehvior. Agonisti n ntgonisti elements emee in the sme onnetive tissue. L Invest 995; 73: Yshiro M, Chung YS, Nishimur S, Inoue T, Sow M. Firosis in the peritoneum inue y sirrhous gstri ner ells my t s soil for peritonel issemintion. Cner 996; 77: Okuno K, Ysutomi M, Nishimur N, Arkw T, Shiomi M, Hi J, Ue K, Minmi K. Gene expression nlysis in oloretl ner using prtil DNA rry filter. Dis Colon Retum 2; 44: Gemm A, Tkenk K, Hosoy Y, Mtu K, Seike M, Kurimoto F, Ono Y, Uemtsu K, Tke Y, Hiino S, Yoshimur A, Shiuy M, Kuoh S. Altere expression of severl genes in highly metstti supopultions of humn pulmonry enorinom ell line. Eur J Cner 2; 37: Simon R, Rmher MD, Doin K, MShne LM. Pitflls in the use of DNA mirorry t for ignosti n prognosti lssifition. J Ntl Cner Inst 23; 95: Allison MC, Howtson AG, Torrne CJ, Lee FD, Russell RI. Gstrointestinl mge ssoite with the use of nonsteroil ntiinflmmtory rugs. N Engl J Me 992; 327: Soll AH, Weinstein WM, Kurt J, MCrthy D. Nonsteroil nti-inflmmtory rugs n pepti uler isese. Ann Intern Me 99; 4: Miller TA. Protetive effets of prostglnins ginst gstri muosl mge: urrent knowlege n propose mehnisms. Am J Physiol 983; 245: G Petkov DK, Clelln CA, Ronn JE, Lewis S, Knox AJ. Reue expression of ylooxygense (COX) in iiopthi pulmonry firosis n sroiosis. Histopthology 23; 43: Elens B, Weinerg RA. Heterotypi signling etween epithelil tumor ells n firolsts in rinom formtion. Exp Cell Res 2; 264: Tlsty TD, Hein PW. Know thy neighor: stroml ells n ontriute onogeni signls. Curr Opin Genet Dev 2; : Nkmur et l.

Title of Experiment: Author, Institute and address:

Title of Experiment: Author, Institute and address: Title of Experiment: Trsfetion of murine mrophge RAW264.7 ells with METAFECTENE PRO. Author, Institute n ress: Ptrizi Pellegtti n Frneso Di Virgilio. Deprtment of Experimentl n Dignosti Meiine, Setion