Oxidative damage and response to Bacillus Calmette-Guérin in bladder cancer cells expressing sialyltransferase ST3GAL1

RESEARCH ARTICLE Open Access Oxida i e damage and esponse o Bacillus Calme e-Gué in in bladde cance cells exp essing sialyl ans e ase ST3GAL1 Paulo F. Se e ino 1,3† , Ma iana Sil a 1† , Mylene Ca ascal 1 , Nadia Malagolini 3 , Ma iella Chi icolo 3 , Giulia Ven u i 3 , Robe o Ba ba o Fo leo 3 , Annalisa As ol i 4 , Ma iangela Ca e a 3 , Paula A. Videi a 1,2* and Fabio Dall’Olio 3* Abs ac Backg ound: T ea men wi h Bacillus Calme e-Gué in (BCG) is he gold s anda d adju an immuno he apy o non- muscle in asi e bladde cance (NMIBC), al hough i ails in one hi d o he pa ien s. NMIBC exp esses wo umo - associa ed O-linked ca bohyd a es: he disaccha ide (Galβ1,3GalNAc) Thomsen-F ieden eich (T) an igen, and i s sialyla ed coun e pa (Siaα2,3Galβ1,3GalNAc) sialyl-T (sT), syn hesized by sialyl ans e ase ST3GAL1, whose oles in BCG esponse a e unknown. Me hods: The human bladde cance (BC) cell line HT1376 s ongly exp essing he T an igen, was e o i ally ansduced wi h he ST3GAL1 cDNA o wi h an emp y ec o , yielding he cell lines HT1376 sT and HT1376 T , ha exp ess, espec i ely, ei he he sT o he T an igens. Cells we e in i o challenged wi h BCG. Whole gene exp ession was s udied by mic oa ay echnology, cy okine sec e ion was measu ed by mul iplex immune-beads assay. Human mac ophages de i ed om blood monocy es we e challenged wi h he sec e ome o BCG-challenged BC cells. Resul s: The sec e ome om BCG-challenged HT1376 sT cells induced a s onge mac ophage sec e ion o IL-6, IL-1β, TNFαand IL-10 han ha o HT1376 T cells. T ansc ip omic analysis e ealed ha ST3GAL1 o e exp ession and T/sT eplacemen modula ed hund eds o genes. Se e al genes p ese ing genomic s abili y we e down- egula ed in HT1376 sT cells which, as a consequence, displayed inc eased sensi i i y o oxida i e damage. A e BCG challenge, he ansc ip ome o HT1376 sT cells showed highe suscep ibili y o BCG modula ion han ha o HT1376 T cells. Conclusions: High ST3GAL1 exp ession and T/sT eplacemen in BCG challenged-BC cance cells induce a s onge mac ophage esponse and al e he gene exp ession owa ds genomic ins abili y, indica ing a po en ial impac on BC biology and pa ien ’s esponse o BCG. Keywo ds: Bacillus Calme e-Gué in, Glycosyla ion, Sialyl T an igen, Sialyl ans e ase, Thomsen-F ieden eich an igen Backg ound The in a esical inocula ion wi h he Bacillus Calme e- Gué in (BCG) is he mos e ec i e adju an he apy o non-muscle in asi e bladde cance s (NMIBC) a e ansu e h al esec ion. Howe e , one hi d o he pa ien s ail o espond and expe ience ecu ence a e ea men . The easons why BCG he apy ail a e s ill unclea , al hough i is well es ablished ha he an i- umo ac i i y o BCG depends on i s abili y o elici an e ec i e local immune esponse [1–3]. Glycosyla ion, one o he mos equen pos - ansla ional modi ica ion o p o eins, unde goes p o ound changes in all ypes o cance [4], including bladde cance (BC) [5–8]. The abe an exp ession o glycoconjuga es is o en caused by he de anged egula ion o hei biosyn he ic enzymes: he glyco- syl ans e ases [9]. The Thomsen-F ieden eich (T) an igen is a disaccha ide (Galβ1,3GalNAc) O-linked o se ine o h eo- nine esidues o glycop o eins (Addi ional ile 1A), whose * Co espondence: [email p o ec ed];[email p o ec ed] † Equal con ibu o s 1 Cen o de Es udos de Doenças C ónicas, CEDOC, NOVA Medical School/ Faculdade de Ciências Médicas, Uni e sidade NOVA de Lisboa, Campo dos Má i es da Pá ia, 130, 1169-056 Lisbon, Po ugal 3 Dipa imen o di Medicina Specialis ica, Diagnos ica e Spe imen ale, Sede di Pa ologia Gene ale, Uni e si à di Bologna, Via S. Giacomo 14, 40126 Bologna, I aly Full lis o au ho in o ma ion is a ailable a he end o he a icle © The Au ho (s). 2018 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Se e ino e al. BMC Cance (2018) 18:198 h ps://doi.o g/10.1186/s12885-018-4107-1 abe an exp ession in cance has been associa ed wi h ma- lignancy [10–13] and used as a possible a ge o he apy [14–16]. I s sialyla ed coun e pa , he sialyl-T (sT) (Siaα2,3- Galβ1,3GalNAc-O-Se /Th ) s uc u e and i s main biosyn- he ic enzyme, he sialyl ans e ase ST3GAL1, a e also abe an ly exp essed in a a ie y o cance s [17,18] [ e iewed in [19,20]]. In BC, he exp ession o T/sT an igens is also abe an and i in luences in asion and immune ec- ogni ion [21–23]. In a p e ious wo k [24], we ha e shown ha he mRNA o ST3GAL1 was o e exp essed in NMIBC bu no in muscle in asi e BC o in benign bladde umo s and ST3GAL1 plays he majo ole in he sialyla ion o he T an igen in BC. The T an igen has been sugges ed as a use ul ma ke o BCG esponse [23], e en hough he ela ionship be ween ST3GAL1/sT and BCG esponse has ne e been es ablished. In his s udy, we in es iga ed he e ec s o he al e na- i e exp ession o he T o sT an igens on he abili y o BC cells o ac i a e mac ophages in esponse o BCG challenge and on he ansc ip ome o BC cells, u ilizing he HT1376 cell line in which he T an igen was eplaced by he sT an igen, by e o i al ansduc ion wi h he ST3GAL1 cDNA. This cell line was chosen because o i s low ST3GAL1 exp ession and i s high and homogenous eac i i y wi h he T an igen-speci ic lec in PNA [24]. The gene exp ession and cy okine p o iles o he cell lines exp essing ei he he T o he sT an igens a e BCG challenge and he abili y o hei sec e ome o s imula e cy okine elease by mac ophages was s udied. Me hods Gene a ion o ST3GAL1-exp essing cell lines The HT1376 cell line was es ablished om a p ima y in- asi e ansi ional cell cance o he bladde [25]. Cells we e g own in DMEM (4.5 g/L glucose, Sigma), con ain- ing 10% oe al cal se um (FCS, Sigma), 2 mM L-glu am- ine (Sigma) and 100 μg/mL penicillin/s ep omycin (Sigma). HT1376 cells exp essing ST3GAL1 we e gene - a ed by ansduc ion wi h a e o i al ec o ob ained wi h he Vi aPowe Len i i al Exp ession Sys em (In i- ogen), acco ding o manu ac u e ’s ins uc ions. The cDNA o he whole coding egion o human ST3GAL1 was ob ained by PCR ampli ica ion o he cDNA o he colon cance cell line HT29 wi h he ollowing p ime pai : o wa d p ime : 5’-CACCATGGTGACCCTGCG- GAAGAGG-3′; e e se p ime : 5’-TCATCTCCCCTT- GAAGATCCGG-3′. Ampli ica ion was pe o med o 35 cycles o he ollowing p og am: dena u a ion 94 °C 1 min; annealing 60 °C 1 min; ex ension 72 °C 1 min. The PCR p oduc was gel isola ed and cloned in o he pLen i6/V5 Di ec ional TOPO cloning ec o (In i o- gen) which d i es he exp ession o inse ed genes unde he con ol o he cy omegalo i us p omo e . A nega i e con ol e o i al ec o was p epa ed wi h an emp y plasmid. A e ansduc ion wi h nega i e con ol- o ST3GAL1-exp essing ec o s, HT1376 cells we e selec ed wi h 4 μg/mL o blas icidin. The eplacemen o he T an igen wi h he sT an igen was e alua ed as loss o cell eac i i y wi h he luo escen labeled lec in om A achis hypogea (peanu agglu inin, PNA), conjuga ed wi h luo - escein iso hiocyana e (PNA-FITC). Al hough selec ed cells we e mainly nega i e o PNA-FITC as de ec ed by FACS analysis, a small popula ion o PNA-FITC posi i e cells was s ill p esen . To ob ain a popula ion o cells homoge- neously nega i e o he T an igen, abou 100 ST3GAL1- ansduced HT1376 cells we e seeded in a 10 cm Pe i dish and a e one mon h, PNA-FITC nega i e colonies we e selec ed and pooled. This polyclonal cell popula ion homogeneously nega i e o T an igen exp ession is he e- a e e e ed o as HT1376 sT . The polyclonal cell popula- ion ob ained a e ansduc ion wi h he nega i e con ol e o i al ec o ollowed by blas icidin selec ion is e- e ed o as HT1376 T . Flow cy ome y Cells we e incuba ed wi h PNA-FITC o 30 min a 4 °C in he da k, washed and analyzed by low cy ome y. Si- alidase ea men was pe o med wi h 20 mU o Clos- idium pe ingens sialidase (Roche Diagnos ics), o 90 min a 37 °C. Real ime RT-PCR To al RNA was isola ed using he GenElu e Mammalian To al RNA Pu i ica ion ki and DNase ea men (Sigma), acco ding o he manu ac u e ’s ins uc ions. One mic og am o o al RNA was e e se ansc ibed, using he andom-p ime s based High Capaci y cDNA A chi e Ki (Applied Biosys ems). The exp ession le el o ST3GAL1 (Hs00161688_m1; NM_173344.2 and NM_003033.3) was e alua ed wi h he TaqMan assay sys em in a 7500 Fas Real-Time PCR Sys em (Applied Biosys ems) using he TaqMan Uni e sal PCR Mas e Mix Fas , as p e iously desc ibed [24,26,27]. The e i- ciency o he ampli ica ion eac ion o each p ime -p obe was abo e 95% (as de e mined by he manu ac u e ). No - malized mRNA exp ession was compu ed as he numbe o mRNA molecules o he gene o in e es pe 1000 mRNA molecules o he endogenous con ol β-ac in gene, calcula ed using he 2 -ΔCT ×1000 o mula [28]. Sialyl ans e ase ac i i y assay Cell pelle s we e homogenized in wa e and he p o ein concen a ion o he homogena es was de e mined by he Low y me hod. The ac i i y o ST3GAL1 was measu ed in he homogena es in he ange o ime and subs a e concen a ion linea i y in a 25 μL olume con- aining: 50 mM o 2-(N-mo pholino)e hanesulphonic acid (MES) bu e pH 6.5, 0.5% T i on X-100, 23.5 μgo Se e ino e al. BMC Cance (2018) 18:198 Page 2 o 9 Galβ1,3GalNAcα1-O-benzyl (benzyl-T; Sigma) as ac- cep o subs a e, 15 μM (640 Bq) o CMP-[ 14 C]Sia (Ame sham) and 50 μg o homogena e p o eins. Reac- ions we e incuba ed a 37 °C, o 2 h and he p oduc s we e hen isola ed by hyd ophobic ch oma og aphy in SepPak C18 Classic Ca idge (Wa e s). The columns we e washed wi h wa e and elu ed wi h 1 mL ace o- ni ile, which was coun ed in a liquid scin illa ion coun- e . The inco po a ion on endogenous subs a es, in he absence o he accep o subs a e, was sub ac ed. BCG challenge o HT1376 cells Comme cial Connaugh BCG (ImmuCys , Sano i Pas- eu SA, F ance) was suspended in PBS con aining 0.05% Tween 80 and s o ed a −80 °C. Be o e each assay, BCG agg ega es we e disca ded by cen i uga ion (300 × g o 5 min). To assess BCG in e naliza ion, bac- e ia we e s ained wi h 2 μg/mL o 5-(and-6-)(((4-chlo - ome hyl)benzoyl)amino) e ame hyl hodamine (CMTR, In i ogen) o 2 h in cul u e medium, incuba ed wi h HT1376 T o HT1376 sT cells in a 1:10 cell/bac e ia a io o 2 h a 37 °C and analyzed by low cy ome y. To as- sess cy okine sec e ion, HT1376 T o HT1376 sT cells we e challenged wi h uns ained BCG o 2 h a 37 °C, he medium was emo ed and he cells we e washed wice wi h PBS and incuba ed wi h esh medium o 16 h. Condi ioned media we e used o cy okine analysis and o challenge mac ophages, while cell pelle s we e used o RNA ex ac ion and ansc ip omic analysis. De e mina ion o cy okine concen a ion The concen a ion o cy okines IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IFN-γand TNF-αwas mea- su ed in a 96-well s ip pla e om a comme cial MIBA ki (Bio-Rad), as ecommended by manu ac u e ’s in- s uc ions. Fluo escence was ead in a Luminex 100 Bio- Plex Liquid A ay Mul iplexing Sys em eade (Bio-Rad) and he da a analyzed wi h he Bio-Plex Manage 5 so wa e (Bio-Rad). Mac ophage p epa a ion and s imula ion Mononuclea cells we e isola ed by Ficoll-Hypaque densi y g adien cen i uga ion (GE Heal hca e) om he pe iphe al blood o heal hy blood-dono s. Fo his use, no s udy app o al was necessa y. The only au ho iza ion equi ed was ha ob ained om he Blood Collec ion Se ice o he Pizza di Hospi al in Bologna, I aly, which keeps he igh s on dono s’blood samples. Mac ophages we e ob ained by di e en ia ion o monocy es by cul u e in RPMI 1640 (Sigma) medium supplemen ed wi h 20% FCS, 2 mM L-glu amine and 100 μg/mL penicillin/ s ep omycin. A e 7 days, monocy e-de i ed mac o- phages we e de ached wi h a cell sc ape and dispensed in 24 well pla es a a cell densi y co esponding app oxima ely o 50% o con luence. One day la e , mac- ophages we e incuba ed wi h s anda d uncondi ioned cul u e medium o wi h he media condi ioned by HT1376 T o HT1376 sT cells ei he BCG-challenged (as desc ibed abo e) o mock challenged. A e 2 h, he con- di ioned media we e eplaced by esh medium, which was collec ed 24 h la e and s o ed a −80 °C o he de- ec ion o cy okines sec e ed by mac ophages. H 2 O 2 ea men Cells in exponen ial g ow h phase we e incuba ed in se um- ee medium con aining 5 mM H 2 O 2 o 1 h. The medium was hen eplaced wi h esh comple e medium and he cells we e ha es ed and analyzed 24 h la e . Mock- ea ed cells we e incuba ed as abo e wi hou H 2 O 2 . The cy o oxic e ec o H 2 O 2 was de e mined by coun ing he numbe o cells in six eplicas seeded in 6- wells pla es. Rep esen a i e ields we e pho og aphed wi h an in e ed phase con as mic oscope. Whole ansc ip ome analysis by exp ession mic oa ay To al RNA was isola ed by he guanidinium hiocyana e- me hod [29] and con e ed o labelled single s and cDNA (ssDNA) by he comme cial Whole T ansc ip Exp ession ki (Ambion), acco ding o he manu ac- u e ’s ins uc ions. Labelled ssDNA agmen s we e hy- b idized in a Human T ansc ip ome A ay 2.0 o e nigh . A e s aining wi h phycoe y h in-s ep a idin, luo es- cence was ead in a GeneChip Scanne 3000 7G (A y- me ix). A e s a is ical analysis (see below), a ay da a we e unc ionally analyzed by he A ayS a 2.0 so - wa e (DNASTAR) and h ough a li e a u e sea ch o he biological oles o modula ed genes. Gene nomencla u e ollowed he HUGO Gene Nomencla u e Commi ee ules (h ps://www.genenames.o g/) in i alic uppe case le e s. Wi h excep ion o cy okines, p o eins had he same name as he gene, ep esen ed in egula uppe case. S a is ical me hods Mic oa ay aw da a we e backg ound-sub ac ed, no - malized and summa ized wi h he obus mul i-a ay a e age (RMA) algo i hm implemen ed in he A y pack- age o Bioconduc o (www.bioconduc o .o g), which u i- lizes R so wa e. Di e en ially exp essed genes be ween que y and con ol assay we e selec ed by applica ion o he wo ail ANOVA, ollowed by he Benjamini- Hochbe g alse disco e y a e es wi h a po q≤0.05 cu -o and by he log 2 exp ession a io, conside ing only a ia ions ≥0.5. MIBA da a we e analyzed by ANOVA, ollowed by Tukey mul iple compa ison es . H 2 O 2 ox- ici y da a we e analyzed by he S uden ’s es . The so - wa e used was G aphpad P ism, e sion 7.0. Se e ino e al. BMC Cance (2018) 18:198 Page 3 o 9 Resul s ST3GAL1-exp ession leads o eplacemen o T wi h sT an igen in HT1376 cells The mock- ansduced cell line HT1376 T , like he wild ype HT1376, exp essed high and homogeneous T ex- p ession, while he ST3GAL1- ansduced cell line HT1376 sT displayed an homogeneous low PNA eac i - i y which could be e e ed o high eac i i y a e sialid- ase ea men (Addi ional ile 1B). Bo h he ST3GAL1 ac i i y (Addi ional ile 1C) and he ST3GAL1 mRNA measu ed by eal ime PCR (Addi ional ile 1D) we e e y high in HT1376 sT al hough almos unde ec able in HT1376 T . The le el o ST3GAL1 ac i i y and PNA e- ac i i y eached by HT1376 sT cells was simila o ha displayed by he wild ype BC cell line 5637 s ongly ex- p essing ST3GAL1 (da a no shown [24]). This is consis - en wi h he no ion ha , in BC cells, ST3GAL1-media ed α2,3 sialyla ion o galac ose (Addi ional ile 1A) masks he Tan igen[24]. IL-6 and IL-8 sec e ion by HT1376 T and HT1376 sT cells To assess he e ec o ST3GAL1 o e exp ession and he consequen eplacemen o he T wi h he sT an igen on cy okine p oduc ion ollowing BCG s imula ion, we challenged he wo HT1376 cell lines wi h BCG. Among he se e al cy okines es ed (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IFN-γand TNF-α), only IL-8 was de- ec able in media condi ioned by unchallenged HT1376 T o HT1376 sT cells. A e BCG challenge, IL-6 became de ec - able in bo h HT1376 sT and HT1376 T cells, while he IL-8 sec e ion showed a clea endency o up- egula ion a e BCG challenge, especially in HT1376 T cells (Fig. 1a). The p alues a e epo ed in Addi ional ile 2. To es ablish whe he he di e en IL-8 p oduc ion by HT1376 sT a e BCG challenge was due o a highe BCG up ake by hese cells, we measu ed BCG in e naliza ion by he wo HT1376 cell popula ions by low cy ome y. As shown in Fig. 1b, he e we e no signi ican di e - ences in he up ake o luo escen BCG by HT1376 T and HT1376 sT cells, uling ou he possibili y ha di e ences in cy okine p oduc ion by BCG-s imula ed HT1376 T and HT1376 sT was dependen on di e en BCG in e naliza- ion a e. HT1376 sT cells induce a s onge mac ophage sec e o y esponse a e BCG s imuli To in es iga e he ole played by ST3GAL1 exp ession and he consequen T/sT eplacemen on he s imula- ion o inna e immuni y by BCG-challenged BC cells, he sec e ion o cy okines by human mac ophages s im- ula ed wi h he sec e ome o BCG-challenged BC cells was measu ed. Uns imula ed mac ophages sec e ed high le els o IL-8 and e y low le els o TNF-α, IL-6, IL-1β and IL-10. This pa e n o sec e ion was no signi ican ly changed by s imula ion wi h he sec e ome o BCG- unchallenged HT1376 T o HT1376 sT cells (Fig. 2). In con as , he sec e ome om bo h BCG-challenged cell lines, in pa icula ha om HT1376 sT , signi ican ly in- c eased he sec e ion o IL-6, IL-1β, TNF-αand IL-10 whe eas IL-8 sec e ion was ha dly a ec ed by he sec e- ome o BCG-challenged cells, ega dless o hei ST3GAL1 exp ession (Fig. 2). I should be no ed ha he le el o sec e ion o IL-8 and IL-6 by mac ophages was abou 10 and 3- old highe , espec i ely, han he le el o ei he in HT1376 cells (Fig. 1), uling ou he possibili y o a signi ican con amina ion o cy okines se- c e ed by mac ophages wi h hose sec e ed by HT1376 cells. The cy okines IL-2, IL-4, IL-12 and IL-17 we e no sec e ed by mac ophages in any o he es ed condi ions (da a no shown). The pand q alues a e epo ed in Addi ional ile 2. HT1376 sT cells display down- egula ion o genes p ese ing genomic s abili y The impac o ST3GAL1 o e exp ession and o T e- placemen by sT on he global ansc ip ional ac i i y o HT1376 cells was e alua ed by exp ession mic oa ay echnology. O he 254 genes, which displayed ST3GAL1-dependen modula ion by a leas a log 2 ex- p ession a io ≥1.0 (meaning a old change o a leas 2) wi h po q alues ≤0.05, 106 we e up- egula ed and 148 we e down- egula ed. The comple e lis o genes BA Fig. 1 BCG-s imula ion o HT1376 cells. a: Sec e ion o IL-6 and IL-8. HT1376 T , (whi e ci cles) and HT1376 sT (black ci cles) cells we e challenged wi h BCG o 2 h, hen hey we e washed and cul u ed in medium o 16 h. Cy okine concen a ion was de e mined as desc ibed in Ma e ials and Me hods sec ion. Da a o h ee independen expe imen s a e epo ed. S a is ical analysis is epo ed in Addi ional ile 2.b:Timecou se o BCG up ake. HT1376T (whi e ci cles) and HT1376sT (black ci cles) cells we e challenged wi h CMTMR-labelled BCG o 2 and 6 h, as desc ibed in he Me hods sec ion. BCG in e naliza ion was es ima ed as mean luo escence in ensi y o he cells, measu ed by low cy ome y Da a o h ee independen expe imen s a e epo ed Se e ino e al. BMC Cance (2018) 18:198 Page 4 o 9 showing signi ican modula ion (changes equal o highe han 2- old) in HT1376 sT s. HT1376 T is epo ed in Addi ional ile 3. A leas 29 genes pu a i ely egula ing malignancy because o hei in ol emen in he con ol o apop osis, cell g ow h, angiogenesis, in lamma ion o p o eolysis, we e di e en ially exp essed in HT1376 sT cells, compa ed wi h HT1376 T cells (Addi ional ile 4). A sea ch in he li e a u e e ealed ha 18 genes we e mod- ula ed owa ds inc eased malignancy and 14 owa ds de- c eased malignancy (Addi ional ile 4). Rema kably, among he 18 genes modula ed owa ds inc eased malig- nancy in HT1376 sT cells, 11 down- egula ed genes can be collec i ely e e ed o as “ca e ake genes”because o hei in ol emen in DNA epai and mi o ic ideli y (Addi ional ile 4). Mo eo e , i h eshold was lowe ed o include genes down- egula ed abo e 1.3- old, he num- be o down- egula ed ca e ake genes ose o 34 (Table 1). As a unc ional alida ion o gene exp ession da a, cells we e exposed o he geno oxic e ec s o he oxidizing agen H 2 O 2 . As expec ed o a cell line wi h impai ed DNA epai mechanisms, H 2 O 2 - ea men Fig. 2 Cy okine sec e ion by human mac ophages ea ed wi h condi ioned media o BC cell lines. Cy okine sec e ion was measu ed in cul u e media condi ioned by uns imula ed mac ophages (MΦ, g ey ci cles) o s imula ed wi h condi ioned media om BCG- challenged o unchallenged BC cell lines: HT1376 T , (whi e ci cles); HT1376 sT (black ci cles). Cells we e challenged wi h BCG o 2 h, hen hey we e washed and allowed o condi ion he cul u e medium o 16 h. The condi ioned cul u e media o BCG-challenged o unchallenged cells was used o s imula e human monocy e-de i ed mac ophages o 24 h. MØ: cy okines eleased by uns imula ed mac ophages; MØ + unchallenged: cy okines eleased by mac ophages s imula ed wi h he condi ioned medium o unchallenged HT1376 cells; MØ + BCG- challenged: cy okines eleased om mac ophages s imula ed wi h he condi ioned medium o BCG-challenged cells. Da a o h ee independen expe imen s a e epo ed. S a is ical analysis is epo ed in Addi ional ile 2 Table 1 Genes in ol ed in main aining ch omosomal s abili y and/o DNA epai showing down- egula ion in HT1376 sT cells Genes showing a old change ≥2 a e boxed in g ay Se e ino e al. BMC Cance (2018) 18:198 Page 5 o 9 esul ed in a educed iabili y in HT1376 sT han in HT1376 T cells (Fig. 3a and b). HT1376 sT cells display inc eased ansc ip omic changes a e BCG challenging To in es iga e he e ec o a di e en ial T/sT exp ession on BCG challenging, he global ansc ip ional ac i i y was also compa ed in BCG-challenged HT1376 T and HT1376 sT cell lines. While in HT1376 sT cells a g oup o 38 genes showed signi ican modula ion by BCG (pand q alues ≤0.05) wi h a log 2 exp ession a io ≥0.5 ( old change a leas 1.4) (Addi ional in o ma ion 5), he en- dency o modula ion showed by a ew genes in HT1376 T cells ne e eached s a is ical signi icance (da a no shown). Modula ed genes in HT1376 sT cells ell in o se - e al b oad unc ional ca ego ies (Addi ional in o ma ion 5). In pa icula , a se o genes ela ed o “aminoacid and p o ein biosyn hesis”we e p e alen ly down- egula ed in BCG-challenged HT1376 sT cells (Addi ional ile 5). This unc ional ca ego y includes genes in ol ed in he biosyn- hesis and anspo o aminoacids, a achmen o aminoa- cids o RNAs and egula ion o ansla ion. O he ca ego ies o modula ed genes include “cell g ow h”and “in lamma o y and immune esponse”(Addi ional ile 5). Discussion The o e exp ession o ST3GAL1 has been widely s ud- ied in cellula models o b eas cance and ound o be esponsible o inc eased malignancy [30–32]. In con- as , e y li le is known on he ole o his enzyme in BC biology. In his wo k, we ha e pe o med an exhaus - i e analysis o a BC expe imen al sys em in which h ough he o e exp ession o ST3GAL1, he cons i u- i ely exp essed T an igen was eplaced by i s sialyla ed coun e pa , he sT ca bohyd a e s uc u e. Bo h s uc- u es a e biologically ac i e, being ecep o s ei he o galec ins o siglecs, as well as o suga binding ecep- o s o mic oo ganisms [33]. Thus, he pheno ypic changes we epo a e ST3GAL1 o e exp ession could be a ibu able ei he o he lack o T an igen o exp es- sion o sT o bo h. The modula ion o he immune e- sponse, in pa icula , he sec e ion o cy okines, is a well known unc ional consequence o BCG in e ac ion wi h BC cells [1,34,35]. Ou s udy con i ms ha BCG in- duces IL-6 and IL-8 sec e ion by BC cells and shows a endency o highe IL-8 sec e ion by BCG-s imula ed cells exp essing sT. In BC pa ien s, plasma [36] and is- sue [37] IL-6 le els a e ele a ed and associa ed wi h umo p og ession and poo p ognosis. P e ious s udies ha e indica ed ha BCG s imula es IL-6 p oduc ion in u ine o pa ien s and BC cell lines [38], inducing non apop o ic cell dea h [39]. Inc eased u ina y IL-8 le el also co ela es wi h p og ession in BC pa ien s [40], al- hough a high u ina y IL-8 le el a e BCG ins illa ion is a p ognos ic ac o o success ul ou come [41,42]. Ou da a indica e ha a e BCG challenge, only he sec e- ion o IL-6 and, o a lesse ex en , IL-8 we e s imula ed while he o he cy okines emained unde ec able. This is consis en wi h clinical obse a ions epo ing ha a ew cy okines, including IL-6 and IL-8, we e de ec able in u ine a e a i s in a esical BCG adminis a ion, while o he cy okines equi ed mul iple BCG ins illa ions [43]. A e BCG challenge, he sec e ome o BC cells ex- p essing he sT s uc u e showed highe capaci y o s imula e cy okine sec e ion by mac ophages, u he suppo ing he no ion ha he sT an igen po en ia es he BC esponse o BCG. An in lamma o y en i onmen can ei he p omo e o inhibi umo p og ession, consis en wi h he no ion ha in lamma ion is a double edge swo d which can bo h e adica e he umo bu also uel i s g ow h. Mac- ophages a e di e en ia ed ei he as p o-in lamma o y M1 o an i-in lamma o y M2 pheno ypes, e en i in many cases hey a e in an in e media e condi ion. While a a iable capaci y o sec e e IL-6, IL-1βand TNF-αis sha ed by di e en ially pola ized mac ophages, an IL-12 A B Fig. 3 Cy o oxic e ec o H 2 O 2 on HT1376 T and HT1376 sT cells. a: cells we e ea ed wi h 5 mM H 2 O 2 o mock- ea ed as desc ibed in Me hods and coun ed. Da a o six expe imen s a e epo ed. The HT1376 sT cells a e mo e suscep ible o he oxic e ec o H 2 O 2 (**p= 0.005 acco ding o S uden ’s es ). b: phase con as mic og aph o ep esen a i e ields o he wo cell popula ions ea ed as desc ibed abo e, showing he s onge cy o oxic e ec s o H 2 O 2 on HT1376 sT . Scale ba : 100 μm Se e ino e al. BMC Cance (2018) 18:198 Page 6 o 9 low/IL-10 high pheno ype is he hallma k o M2 mac o- phages [44], which a e known o p omo e umo g ow h [45]. The mac ophages used in his s udy sec e ed IL-6, IL-8, IL-1βand TNF-αbu no IL-12 and li le IL-10 and a e p obably ep esen a i e o an in e media e condi ion be ween he wo ex eme pheno ypes. The na u e o he mac ophages associa ed wi h BC is indeed a iable as in- dica ed by he di e en le el o exp ession o he M2- speci ic ma ke CD163 among pa ien s [46]. In e es - ingly, he p edominance o M2 mac ophages is associ- a ed wi h highe s age and g ade [46] and wi h a wo se esponse o BCG [47]. The mos p ominen ansc ip omic change we ob- se ed because o ST3GAL1 exp ession and he conse- quen eplacemen o he T wi h he sT an igen in HT1376 cells was he dec eased exp ession o se e al genes in ol ed in di e en mechanisms o DNA epai and in he accu acy o ch omosomal seg ega ion. This esul ed in inc eased sensi i i y o HT1376 sT cells o he cy o oxic e ec o H 2 O 2. This is o in e es i one con- side s ha he gene a ion o eac i e oxygen species by BCG is a c ucial mechanism o BCG-induced damage o BC cells [48]. In e es ingly, also in glioma cells, ST3GAL1 exp ession esul ed in ansc ip omic changes a ec ing malignancy and he cell cycle [49], suppo ing he no ion ha a ca bohyd a e s uc u e on he cell su - ace can gene a e an “ou side in” low o in o ma ion modula ing gene exp ession [20]. BCG challenge esul ed in a deepe modula ion o he ansc ip ome in HT1376 sT han in HT1376T, sugges - ing ha he eplacemen o he T wi h he sT an igen on he cell su ace changes he de elopmen o he gene ic p og am igge ed by BCG con ac . E en hough genes in ol ed in in lamma o y and im- mune esponse we e ound o be modula ed by BCG challenge in HT1376sT cells, li le o no changes we e obse ed in genes encoding cy okines, including hose whose exp ession was s imula ed by BCG-challenge. Pos- sible disc epancies be ween gene and p o ein exp ession can be explained conside ing ha mul iple mechanisms op- e a ing a pos ansc ip ional and pos ansla ional le els (in- cluding non-coding RNAs, ansla ion egula o y mechanisms, p o easomal ac i i y) egula e p o ein exp es- sion. In addi ion, as shown o IL-1β, cy okine sec e ion can be egula ed by he elease o p e o med molecules om in acellula s o es, a he han a he le el o gene ansc ip ion [50]. The soluble ac o s esponsible o he s imula ion o cy okine sec e ion by mac ophages a e con- cei ably a e y complex cock ail o bioac i e compounds o p o ein and non-p o ein na u e, including biologically ac- i e molecules (e.g. p os aglandins, glycosaminoglycans) which a e no p ima y gene p oduc s bu p oduc s o mul- iple enzyma ic eac ions. Fo hese easons, he na u e o he molecules sec e ed by BC cells esponsible o he e ec on mac ophages may no be di ec ly ela ed o he gene ex- p ession p o ile. Conclusions In conclusion, ou da a show ha ST3GAL1 exp ession and he consequen eplacemen o he T by he sT an i- gen in BC cells induce ansc ip omic changes wi h a pu a i e impac on mul iple cellula unc ions associa ed wi h inc eased malignancy and suscep ibili y o oxida i e damage and s eng hens he in lamma o y esponse o mac ophages. This indica es ha ST3GAL1 and T/sT ca bohyd a e s uc u es can be ac o s wi h mul iple clinical implica ions in BC. Addi ional iles Addi ional ile 1: Figu e S1. ST3GAL1-o e exp ession in HT1376 cells (PPTX 130 kb) Addi ional ile 2: p alues calcula ed wi h ANOVA, ollowed by Tukey mul iple compa ison es o da a epo ed in Fig. 1and Fig. 2(PPTX 84 kb) Addi ional ile 3: GENES MODULATED IN HT1376sT CELLS AS COMPARED TO HT1376T CELLS. (XLSX 47 kb) Addi ional ile 4: CANCER-ASSOCIATED GENES MODULATED IN HT1376sT CELLS AS COMPARED TO HT1376T CELLS. (XLS 43 kb) Addi ional ile 5: GENES MODULATED BY BCG IN HT1376 sT (XLS 49 kb) Abb e ia ions BC: bladde cance ; BCG: Bacillus Calme e-Gué in; CMTMR: 5-(and-6)-(((4- chlo ome hyl)benzoyl)amino) e ame hyl hodamine; DMEM: Dulbecco’s modi ied Eagle medium; FCS: Foe al cal se um; FITC: Fluo escein iso hiocyana e; Gal: Galac ose; GalNAc: N-ace ylgalac osamine; GlcNAc: N-ace ylglucosamine; MES: (N-mo pholino)e hanesulphonic acid; MIBA: Mul iplex immune-beads assay; NMIBC: Non-muscle in asi e bladde cance ; PBS: Phospha e-bu e ed saline; PNA: Peanu (A achis hypogea) agglu inin; RPMI: Roswell Pa k Memo ial Ins i u e; Sia: sialic acid Acknowledgemen s We hank D . F ancesca Bo se i and D . Enzo Spisni, BIGEA Depa men o he Uni e si y o Bologna o he help wi h he mul iplex immune-beads assays, D . Ch is ine M. Be s o he c i ical eading o he manusc ip and D . Ma ia Le izia Bacchi-Reggiani (DIMES) o ad ices in s a is ical analysis. Funding This wo k was suppo ed by: Po uguese Founda ion o Science and Technology (FCT) PhD g an s SFRH/BD/45120/2008 (Paulo F. Se e ino), SFRH/ BD/81860/2011 (Ma iana Sil a) and SFRH/BD/100970/2014 (Mylène Ca ascal), Liga Po uguesa Con a o Canc o 2011 (Mylène A. Ca ascal); P émio Bluepha ma Ino ação Uni e sidade de Coimb a and he San ande To a/ Uni e sidade NOVA de Lisboa p izes (Paula Videi a). G an s om he Uni e si y o Bologna (Fabio Dall’Olio). Ma iangela Ca e a and Giulia Ven u i a e PhD s uden s suppo ed by g an s om he Uni e si y o Bologna. A ailabili y o da a and ma e ials The da ase s gene a ed du ing he cu en s udy a e a ailable om he co esponding au ho on easonable eques . Au ho s’con ibu ions P.F.S and M.S. pe o med he bulk o he expe imen al wo k, analysed da a and helped w i ing he manusc ip . M. Ca , G.V., M. Ca and R.B.F. pe o med pa o he expe imen al wo k. N.M. and M. Chi . con ibu ed o he es ablishmen o ansduced cell lines. A.A. pe o med mic oa ay expe imen s and analysed mic oa ay da a. P.A.V. and F.D concei ed he wo k, analysed he da a and w o e he manusc ip . All au ho s e ised c i ically he manusc ip , ha e gi en inal app o al o he e sion o be Se e ino e al. BMC Cance (2018) 18:198 Page 7 o 9 published and ag ee o be accoun able o all aspec s o he wo k in ensu ing ha ques ions ela ed o he accu acy o in eg i y o any pa o he wo k a e app op ia ely in es iga ed and esol ed. E hics app o al and consen o pa icipa e This s udy does no use samples om diseased pe sons. The blood used o he p epa a ion o monocy e-de i ed mac ophages was om heal hy blood dono olun a ies. Fo his, no s udy app o al was necessa y. The only au ho iza ion equi ed was ha ob ained om he Blood Collec ion Se ice o he Pizza di Hospi al in Bologna, I aly, which keeps he igh s on dono s’ blood samples. This au ho iza ion is a ailable o he Edi o . Consen o publica ion No applicable. Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Publishe ’sNo e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a ilia ions. Au ho de ails 1 Cen o de Es udos de Doenças C ónicas, CEDOC, NOVA Medical School/ Faculdade de Ciências Médicas, Uni e sidade NOVA de Lisboa, Campo dos Má i es da Pá ia, 130, 1169-056 Lisbon, Po ugal. 2 UCIBIO, Depa amen o Ciências da Vida, Faculdade de Ciências e Tecnologia, Uni e sidade NOVA de Lisboa, 2829-516 Capa ica, Po ugal. 3 Dipa imen o di Medicina Specialis ica, Diagnos ica e Spe imen ale, Sede di Pa ologia Gene ale, Uni e si à di Bologna, Via S. Giacomo 14, 40126 Bologna, I aly. 4 Cen o In e dipa imen ale Rice che sul Canc o “Gio gio P odi”, Uni e si à di Bologna, Bologna, I aly. Recei ed: 3 Ap il 2017 Accep ed: 8 Feb ua y 2018 Re e ences 1. Be e s RF, Ku h KH, Schamha DH. Role o u o helial cells in BCG immuno he apy o supe icial bladde cance . B J Cance . 2004;91(4): 607–12. 2. Videi a PA, Calais FM, Co eia M, Ligei o D, C espo HJ, Calais F, T indade H. E icacy o bacille Calme e-Gue in immuno he apy p edic ed by exp ession o an igen-p esen ing molecules and chemokines. U ology. 2009;74(4):944–50. 3. Ca e e o R, Cab e a T, Gil H, Saenz-Lopez P, Maleno I, Ap siau i N, Coza JM, Ga ido F. Bacillus Calme e-Gue in immuno he apy o bladde cance induces selec ion o human leukocy e an igen class I-de icien umo cells. In J Cance . 2011;129(4):839–46. 4. Dall'Olio F, Malagolini N, T inche a M, Chi icolo M. Mechanisms o cance - associa ed glycosyla ion changes. F on Biosci. 2012;17:670–99. 5. Ohyama C. Glycosyla ion in bladde cance . In J Clin Oncol. 2008;13(4): 308–13. 6. Yang G, Tan Z, Lu W, Guo J, Yu H, Yu J, Sun C, Qi X, Li Z, Guan F. Quan i a i e Glycome analysis o N-glycan pa e ns in bladde cance s no mal bladde cells using an in eg a ed s a egy. J P o eome Res. 2015; 14(2):639–53. 7. Fe ei a JA, Videi a PA, Lima L, Pe ei a S, Sil a M, Ca ascal M, Se e ino PF, Fe nandes E, Almeida A, Cos a C, e al. O e exp ession o umou -associa ed ca bohyd a e an igen sialyl-Tn in ad anced bladde umou s. Mol Oncol. 2013;7(3):719–31. 8. Lima L, Se e ino PF, Sil a M, Mi anda A, Ta a es A, Pe ei a S, Fe nandes E, C uz R, Ama o T, Reis CA, e al. Response o high- isk o ecu ence/ p og ession bladde umou s exp essing sialyl-Tn and sialyl-6-T o BCG immuno he apy. B J Cance . 2013;109(8):2106–14. 9. B ockhausen I. Pa hways o O-glycan biosyn hesis in cance cells. Biochim Biophys Ac a. 1999;1473(1):67–95. 10. Glinsky VV, Glinsky GV, Ri enhouse-Olson K, Hu lej ME, Glinskii OV, Deu sche SL, Quinn TP. The ole o Thomsen-F ieden eich an igen in adhesion o human b eas and p os a e cance cells o he endo helium. Cance Res. 2001;61(12):4851–7. 11. Zhao Q, Ba clay M, Hilkens J, Guo X, Ba ow H, Rhodes JM, Yu LG. In e ac ion be ween ci cula ing galec in-3 and cance -associa ed MUC1 enhances umou cell homo ypic agg ega ion and p e en s anoikis. Mol Cance . 2010;9(1):154. 12. Glinsky VV, Hu lej ME, Glinsky GV, Deu sche SL, Quinn TP. E ec s o Thomsen-F ieden eich an igen-speci ic pep ide P-30 on β-galac oside- media ed homo ypic agg ega ion and adhesion o he endo helium o MDA-MB-435 human b eas ca cinoma cells. Cance Res. 2000;60(10):2584–8. 13. Engels aed e V, Fluegel B, Kunze S, May D, F iese K, Jeschke U, Be gaue F. Exp ession o he ca bohyd a e umou ma ke Sialyl Lewis a, Sialyl Lewis X, Lewis Y and Thomsen-F ieden eich an igen in no mal squamous epi helium o he u e ine ce ix, ce ical dysplasia and ce ical cance . His ol His opa hol. 2012;27(4):507–14. 14. Almog en A, Abdullah J, Ghapu e K, Fe guson K, Glinsky VV, Ri enhouse- Olson K. An i-Thomsen-F ieden eich-ag (an i-TF-ag) po en ial o cance he apy. F on Biosci (Schol Ed). 2012;4:840–63. 15. Fe guson K, Yada A, Mo ey S, Abdullah J, H ysenko G, Eng JY, Sajjad M, Kou y S, Ri enhouse-Olson K. P eclinical s udies wi h JAA-F11 an i- Thomsen-F ieden eich monoclonal an ibody o human b eas cance . Fu u e Oncol. 2014;10(3):385–99. 16. Heimbu g J, Yan J, Mo ey S, Glinskii OV, Huxley VH, Wild L, Klick R, Roy R, Glinsky VV, Ri enhouse-Olson K. Inhibi ion o spon aneous b eas cance me as asis by an i-Thomsen-F ieden eich an igen monoclonal an ibody JAA-F11. Neoplasia. 2006;8(11):939–48. 17. B ockhausen I, Yang JM, Bu chell J, Whi ehouse C, Taylo -Papadimi iou J. Mechanisms unde lying abe an glycosyla ion o MUC1 mucin in b eas cance cells. Eu J Biochem. 1995;233(2):607–17. 18. Whi ehouse C, Bu chell J, Gschmeissne S, B ockhausen I, Lloyd KO, Taylo - Papadimi iou J. A ans ec ed sialyl ans e ase ha is ele a ed in b eas cance and localizes o he medial/ ans-Golgi appa a us inhibi s he de elopmen o co e-2-based O-glycans. J Cell Biol. 1997;137(6):1229–41. 19. Dall'Olio F, Chi icolo M. Sialyl ans e ases in cance . Glycoconj J. 2001; 18(11–12):841–50. 20. Dall'Olio F, Malagolini N, T inche a M, Chi icolo M. Sialosignaling: Sialyl ans e ases as engines o sel - ueling loops in cance p og ession. Biochim Biophys Ac a. 2014;1840(9):2752–64. 21. Langkilde NC, Wol H, Clausen H, Kjeldsen T, O n o TF. Nuclea olume and exp ession o T-an igen, sialosyl-Tn-an igen, and Tn- an igen in ca cinoma o he human bladde . Rela ion o umo ecu ence and p og ession. Cance . 1992;69(1):219–27. 22. Langkilde NC. T-An igens in p ima y non-in asi e and supe icially in asi e human u ina y bladde umo s: he co ela ion o umo ecu ence and umo p og ession. A mini- e iew. Scand J U ol Neph ol Suppl. 1995;172:45–9. 23. Dow JA, di San 'Agnese PA, Cocke AT. Exp ession o blood g oup p ecu so T an igen as a p ognos ic ma ke o human bladde cance ea ed by bacillus Calme e-Gue in and in e leukin-2. J U ol. 1989;142(4): 978–81. 24. Videi a PA, Co eia M, Malagolini N, C espo HJ, Ligei o D, Calais FM, T indade H, Dall'Olio F. ST3Gal.I sialyl ans e ase ele ance in bladde cance issues and cell lines. BMC Cance . 2009;9(1):357. 25. Rasheed S, Ga dne MB, Rongey RW, Nelson-Rees WA, A ns ein P. Human bladde ca cinoma: cha ac e iza ion o wo new umo cell lines and sea ch o umo i uses. J Na l Cance Ins . 1977;58(4):881–90. 26. Ca ascal MA, Se e ino PF, Guadalupe CM, Sil a M, Fe ei a JA, Calais F, Quin o H, Pen C, Ligei o D, San os LL, e al. Sialyl Tn-exp essing bladde cance cells induce a ole ogenic pheno ype in inna e and adap i e immune cells. Mol Oncol. 2014;8(3):753–65. 27. C espo HJ, Cab al MG, Teixei a AV, Lau JT, T indade H, Videi a PA. E ec o sialic acid loss on dend i ic cell ma u a ion. Immunology. 2009;128(1 Suppl): e621-e631. 28. Meije ink J, Mandige s C, an de Loch L, Tonnissen E, Goodsaid F, Raemaeke s J. A no el me hod o compensa e o di e en ampli ica ion e iciencies be ween pa ien DNA samples in quan i a i e eal- ime PCR. J Mol Diagn. 2001;3(2):55–61. 29. Chomczynski P, Sacchi N. Single-s ep me hod o RNA isola ion by acid guanidinium hiocyana e- phenol-chlo o o m ex ac ion. Anal Biochem. 1987;162(1):156–9. 30. Picco G, Julien S, B ockhausen I, Bea son R, An onopoulos A, Haslam S, Mandel U, Dell A, Pinde S, Taylo -Papadimi iou J, e al. O e -exp ession o ST3Gal-I p omo es mamma y umo igenesis. Glycobiology. 2010;20(10): 1241–50. 31. Sp o ie o D, Julien S, Bu o d B, Taylo -Papadimi iou J, Bu chell JM. Cyclooxygenase-2 enzyme induces he exp ession o he α-2,3- Se e ino e al. BMC Cance (2018) 18:198 Page 8 o 9 Sialyl ans e ase-3 (ST3Gal-I) in b eas cance . J Biol Chem. 2012;287(53): 44490–7. 32. Ma cos NT, C uz A, Sil a F, Almeida R, Da id L, Mandel U, Clausen H, Mensdo -Pouilly S, Reis CA. Polypep ide GalNAc- ans e ases, ST6GalNAc- ans e ase I, and ST3Gal- ans e ase I exp ession in gas ic ca cinoma cell lines. J His ochem Cy ochem. 2003;51(6):761–71. 33. Bu in d, Rozie s N, Chadebech P, Bodi i G, Guincha d E, B uneel A, Dup e T, Che e L, Jugie M, Gallon P, Bie ling P, e al. Red blood cell Thomsen- F ieden eich an igen exp ession and galec in-3 plasma concen a ions in S ep ococcus Pneumoniae-associa ed hemoly ic u emic synd ome and hemoly ic anemia. T ans usion. 2015;55(6 P 2):1563–71. 34. Be e s RF, de Boe EC, Ku h KH, Schamha DH. BCG-induced in e leukin-6 up egula ion and BCG in e naliza ion in well and poo ly di e en ia ed human bladde cance cell lines. Eu Cy okine Ne w. 1998;9(2):181–6. 35. Chen FH, C is SA, Zhang GJ, Iwamo o Y, See WA. In e leukin-6 p oduc ion by human bladde umo cell lines is up- egula ed by bacillus Calme e- Gue in h ough nuclea ac o -kappaB and Ap-1 ia an immedia e ea ly pa hway. J U ol. 2002;168(2):786–97. 36. And ews B, Sha ia SF, Kim JH, Wheele TM, Slawin KM, Le ne SP. P eope a i e plasma le els o in e leukin-6 and i s soluble ecep o p edic disease ecu ence and su i al o pa ien s wi h bladde cance . J U ol. 2002; 167(3):1475–81. 37. Chen MF, Lin PY, Wu CF, Chen WC, Wu CT. IL-6 exp ession egula es umo igenici y and co ela es wi h p ognosis in bladde cance . PLoS One. 2013;8(4):e61901. 38. Esu a ana han K, Alexand o AB, McIn y e M, Jackson AM, P esco S, Chisholm GD, James K. In e leukin-6 p oduc ion by bladde umo s is up egula ed by BCG immuno he apy. J U ol. 1995;154(2 P 1):572–5. 39. Chen F, Zhang G, Cao Y, Hessne MJ, See WA. MB49 mu ine u o helial ca cinoma: molecula and pheno ypic compa ison o human cell lines as a model o he di ec umo esponse o bacillus Calme e-Gue in. J U ol. 2009;182(6):2932–7. 40. She yka E, Wheele MA, Hausladen DA, Weiss RM. U ina y in e leukin-8 le els a e ele a ed in subjec s wi h ansi ional cell ca cinoma. U ology. 2003;62(1): 162–6. 41. Sagnak L, E soy H, Ozok U, Sen u k B, E cil H, Baha G, Oz u k E. P edic i e alue o u ina y in e leukin-8 cu o poin o ecu ences a e ansu e h al esec ion plus induc ion bacillus Calme e-Gue in ea men in non-muscle- in asi e bladde umo s. Clin Geni ou in Cance . 2009;7(2):E16–23. 42. Kuma A, Dubey D, Bansal P, Mandhani A, Naik S. U ina y in e leukin-8 p edic s he esponse o s anda d and low dose in a esical bacillus Calme e-Gue in (modi ied Danish 1331 s ain) o supe icial bladde cance . J U ol. 2002;168(5):2232–5. 43. Jackson AM, Alexand o AB, Kelly RW, Skibinska A, Esu a ana han K, P esco S, Chisholm GD, James K. Changes in u ina y cy okines and soluble in e cellula adhesion molecule-1 (ICAM-1) in bladde cance pa ien s a e bacillus Calme e-Gue in (BCG) immuno he apy. Clin Exp Immunol. 1995; 99(3):369–75. 44. Balkwill F, Cha les KA, Man o ani A. Smolde ing and pola ized in lamma ion in he ini ia ion and p omo ion o malignan disease. Cance Cell. 2005;7(3): 211–7. 45. Man o ani A, Alla ena P, Sica A, Balkwill F. Cance - ela ed in lamma ion. Na u e. 2008;454(7203):436–44. 46. Takeuchi H, Tanaka M, Tanaka A, Tsunemi A, Yamamo o H. P edominance o M2-pola ized mac ophages in bladde cance a ec s angiogenesis, umo g ade and in asi eness. Oncol Le . 2016;11(5):3403–8. 47. Lima L, Oli ei a D, Ta a es A, Ama o T, C uz R, Oli ei a MJ, Fe ei a JA, San os L. The p edominance o M2-pola ized mac ophages in he s oma o low-hypoxic bladde umo s is associa ed wi h BCG immuno he apy ailu e. U ol Oncol. 2014;32(4):449–57. 48. Shah G, Zielonka J, Chen F, Zhang G, Cao Y, Kalyana aman B, See W. H2O2 gene a ion by bacillus Calme e-Gue in induces he cellula oxida i e s ess esponse equi ed o bacillus Calme e-Gue in di ec e ec s on u o helial ca cinoma biology. J U ol. 2014;192(4):1238–48. 49. Chong YK, Sandana aj E, Koh LW, Thanga eloo M, Tan MS, Koh GR, Toh TB, Lim GG, Holb ook JD, Kon OL, e al. ST3GAL1-associa ed ansc ip omic p og am in glioblas oma umo g ow h, in asion, and p ognosis. J Na l Cance Ins . 2016;108(2) 50. S anley AC, Lacy P. Pa hways o cy okine sec e ion. Physiology (Be hesda). 2010;25(4):218–29. • We accep p e-submission inqui ies • Ou selec o ool helps you o ind he mos ele an jou nal • We p o ide ound he clock cus ome suppo • Con enien online submission • Tho ough pee e iew • Inclusion in PubMed and all majo indexing se ices • Maximum isibili y o you esea ch Submi you manusc ip a www.biomedcen al.com/submi Submi you nex manusc ip o BioMed Cen al and we will help you a e e y s ep: Se e ino e al. BMC Cance (2018) 18:198 Page 9 o 9