Sphaerococcus coronopifolius bromoterpenes as potential cancer stem cell-targeting agents

Con en s lis s a ailable a ScienceDi ec Biomedicine & Pha maco he apy jou nal homepage: www.else ie .com/loca e/biopha O iginal a icle Sphae ococcus co onopi olius b omo e penes as po en ial cance s em cell- a ge ing agen s Celso Al es a, *, Eu ico Se ano b , Joana Sil a a , Ca los Rod igues b,c , Suse e Pin eus a , Helena Gaspa a,d , Luis M. Bo ana e , Ma ia C. Alpoim b , Rui Ped osa a, * a MARE—Ma ine and En i onmen al Sciences Cen e, Ins i u o Poli écnico de Lei ia, 2520-630 Peniche, Po ugal b Cen e o Neu oscience and Cell Biology (CNC), Uni e si y o Coimb a, 3004-517, Coimb a, Po ugal c Depa men o In e nal Medicine, Hospi al o A ei o, Cen o Hospi ala do Baixo Vouga, A ei o, Po ugal d Uni e si y o Lisbon, Facul y o Science, BioISI - Biosys ems and In eg a i e Sciences Ins i u e, 1749-016 Lisbon, Po ugal e Depa amen o Pha macology, Facul y o Ve e ina y, Uni e si y o San iago de Compos ela, 27002 Lugo, Spain ARTICLE INFO Keywo ds: Ma ine na u al p oduc s Red seaweed Lung cance Cance s em cells Mic oen i onmen In e leukin-6 ABSTRACT Cance is one o he majo h ea s o human heal h and, due o dis inc ac o s, i is expec ed ha i s incidence will inc ease in he nex decades leading o an u gen need o new an icance d ugs de elopmen . Ongoing expe imen al and clinical obse a ions p opose ha cance cells wi h s em-like p ope ies (CSCs) a e in ol ed on he de elopmen o lung cance chemo esis ance. As umou g ow h and me as asis can be con olled by umou - associa ed s omal cells, he main goal o his s udy was o access he an i umo po en ial o i e b omo e penes isola ed om Sphae ococcus co onopi olius ed alga o a ge CSCs o igina ed in a co-cul u e sys em o ib oblas and lung malignan cells. Cy o oxici y o compounds (10–500 μM; 72 h) was e alua ed on monocul u es o se e al malignan and non-malignan cells lines (HBF, BEAS-2B, RenG2, SC-DRenG2) and he e ec s es ima ed by MTT assay. Co-cul u es o non-malignan human b onchial ib oblas s (HBF) and malignan human b onchial epi helial cells (RenG2) we e implemen ed and he compounds abili y o selec i ely kill CSCs was e alua ed by sphe e o ming assay. The in e leucine-6 (IL-6) le els we e also de e mined as cy okine is c ucial o CSCs. Rega ding he monocul u es esul s b omosphae ol selec i ely elimina ed he malignan cells. Bo h 12S-hy- d oxy-b omosphae ol and 12R-hyd oxy-b omosphae ol s e oisome s we e cy o oxic owa ds non-malignan b onchial BEAS-2B cell line, IC 50 o 4.29 and 4.30 μM espec i ely. Howe e , none o he s e oisome s induced damage in he HBFs. As o he co-cul u es, 12R-hyd oxy-b omosphae ol e ealed he highes cy o oxici y and abili y o ab oga e he malignan s em cells; howe e i s e ec s we e IL-6 independen . The esul s p esen ed he e a e he i s e idence o he po en ial o hese b omo e penes o ab oga e CSCs opening new esea ch oppo uni ies. The 12R-hyd oxy-b omosphae ol e ealed o be he mos p omising compound o be es in mo e complex li ing models. 1. In oduc ion Despi e he ad ances on biology and he apeu ics achie ed du ing he las decades, cance emains one o hemajo cause o dea h ac oss he wo ld, mos ly due o aging, li es yle changes, widesp ead o smoking habi s and he inc easing accumula ion o a mosphe ic pollu- an s. In ac , he mos ecen s a is ics show 18.1 millions o new cases and 9.6 millions o dea hs in 2018 [1]. Mo eo e , in 2018, lung cance emains as one o he lowes 5-yea ela i e su i al a e pa hologies (18 %) [2]. One o he main ac o s con ibu ing o he high cance mo ali y is he apy ailu e and consequen umou elapse [3,4]. Mechanis ically, esis ance elies ei he on an inapp op ia e pha macological design o he he apeu ic app oach, o mo e equen ly, on he de elopmen o d ug esis ance [5–7]. Se e al mechanisms a e known o be d ug e- sis ance, including d ug e lux, de oxi ica ion, inac i a ion, changes in he d ug a ge s, highly e icien DNA epai mechanisms, apop osis blockage and o ma ion o highly esis an cance s em cells (CSCs) as esul o he apy [8–10]. Mo eo e , umou mic oen i onmen and umou cellula con en we e also iden i ied as impo an playe s in he he apeu ic ou come o umou s [11,12]. In ac , s udies om he las decades e ealed ha a cellula popula ion esiding inside he umou s and designa ed CSCs a e undamen al in umo igenesis, umou main- enance, me as a ic widesp ead and esis ance o con en ional h ps://doi.o g/10.1016/j.biopha.2020.110275 Recei ed 19 Ma ch 2020; Recei ed in e ised o m 9 May 2020; Accep ed 15 May 2020 ⁎ Co esponding au ho s. E-mail add esses: [email p o ec ed] (C. Al es), [email p o ec ed] (R. Ped osa). Biomedicine & Pha maco he apy 128 (2020) 110275 0753-3322/ © 2020 The Au ho s. Published by Else ie Masson SAS. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/BY-NC-ND/4.0/). T he apeu ics [13–15]. CSCs a e umou -associa ed s em cells ha a e esponsible o umou s’ he e ogenei y and boos ed agg essi eness. In ag eemen , i has been showed ha cu en he apeu ic s a egies em- ployed o comba cance a e ex emely e ec i e in a ge ing he apidly di iding bulk o umou cells, bu spa e he slow-di iding quiescen CSCs, ha subsequen ly epopula e a new umou mass wi h mo e e- sis an umou cells [16–19]. The ae iologies o his cellula popula ion emain con o e sial, bu ecen wo k om di e en labo a o ies de i- ni ely con i med ha hey can a ise om dedi e en ia ion o e minally di e en ia ed umou cells, h ough he ac ion o mic oen i onmen - eleased pa ac ine cy okines, pa icula ly IL-6, and Ac i in-A and G-CSF [20–24]. These obse a ions had emendous impac in he scien i ic communi y, as new he apeu ics should no only add ess he umou cell mass, bu also CSCs and he umou mic oen i onmen [15,23,25]. Co obo a ing his idea a e some ecen in silico s udies demons a ing ha he p esence o CSCs bioma ke s was associa ed wi h a poo e pa ien p ognosis [26–28]. Due o he success o na u al p oduc s (NPs) and hei de i a i es in cance ea men s, as well as hei abili y o media e se e al signalling pa hways and cause ewe side e ec s, he e is a g owing in e es o unde s and hei po en ial as an i-CSCs agen s [29]. Nume ous s udies ha e epo ed he g ea po en ial o NPs o in e e e wi h CSCs, in- cluding ex ac s o ma ine na u al p oduc s [30–33]. Fo ins ance, ma ine ex ac o C ambe c ambe sponge inhibi ed he esis ance o apop osis, sel - enewal abili y, and p oli e a ion o panc ea ic cance cells wi h CSCs pheno ype [34]. Mo eo e , i s co-adminis a ion wi h gemci abine d o e o al umou abolishmen on an in i o cance model [34]. Addi ionally, compounds ex ac ed om he ed alga Plocamium co nu um showed selec i e ac i i y o inhibi he de elopmen o MCF-7 sphe e s uc u es, wi hou cy o oxici y on ei he adhe en MCF-7 cells o MCF-12A non- ans o med cells [35]; b own alga Saccha ina japo- nica, ex ac s supp essed sphe e- o ming abili y o glioblas oma s em cells [36]. Finally, ucoxan hinol de i ed om ucoxan hin induced apop osis and supp essed CSCs, as well as in i o umo igenesis in human colo ec al CSCs [37,38]. The e o e, he main goal o his s udy was o unde s and he po en ial o S. co onopi olius b omo e penes o supp ess CSCs on an in i o co-cul u e cellula model o human lung cance , as well as o assess he ole o IL-6 in he o e all supp ession p ocess. Acco ding wi h ou bes knowledge, i is he i s s udy o ac- cess he an i umo po en ial o sphae ococcenol A, 12R-hyd oxy-b o- mosphae ol, 12S-hyd oxy-b omosphae ol, b omosphae ol and sphae - odac ylomelol o a ge CSCs and he umou mic oen i onmen . 2. Ma e ial and me hods 2.1. Ex ac ion and isola ion o Sphae ococcus co onopi olius compounds Fi e b omo e penes (Fig. 1) we e ex ac ed om he ed alga S. co onopi olius collec ed in Be lenga Na u e Rese e, Peniche, Po ugal, acco ding o he p ocedu es desc ibed by Rod igues and co-wo ke s [56]. Subsequen pu i ica ion was accomplished by ch oma og aphic me hods, and s uc u e cha ac e iza ion was pe o med by NMR and MS echniques. Compounds we e dissol ed in DMSO, whose con- cen a ion in all he pe o med expe imen s was lowe han 0.2% o a oid oxici y. Con ols we e always ea ed wi h highe concen a ions o DMSO. 2.2. Cellula sys ems Fou cellula sys ems we e used in his s udy namely, BEAS-2B (immo alized human b onchial epi helial cells), RenG2 (malignan human b onchial epi helial cells), SC-DRenG2 (de i a i e RenG2 cells) and HBF cells (non-malignan human lung ib oblas s). BEAS-2B cells we e acqui ed om he Eu opean Collec ion o Cell Cul u es (ECCAC, Salisbu y, UK; ECCAC no. 95,102,433). RenG2 and SC-DRenG2 cells we e p oduced by he g oup o Alpoim, as p e iously desc ibed by Rod igues and co-wo ke s [24]. BEAS-2B cells we e cul u ed in LHC-9 medium (Gibco, USA). SC-DRenG2 cells we e cul i a ed in cance s em cell p opaga ion media (DMEM:F12) supplemen ed wi h he same concen a ion o bFGF and EGF [24]. HBF cell line was ob ained om non-malignan human lung issue om a pa ien a he Cen o Hospi- ala e Uni e si á io de Coimb a (CHUC) ollowing p o ocols es ab- lished in he labo a o y. App op ia e in o med consen s we e signed acco ding o he e hical p ocedu es app o ed by he E hical Commi ee o he Facul y o Medicine o he Uni e si y o Coimb a. HBF cells we e main ained in DMEM medium supplemen wi h 10 % FBS (Bioch om, Ge many), 20 U/mL penicillin, 20 μg/mL s ep omycin and 50 ng/mL ampho e icin B (Bioch om, Ge many). Cells we e kep a 37 °C in a 95 % ai , 5% CO 2 incuba o . Cul u e lasks we e coa ed wi h a 2% gela in solu ion 2 h be o e use and, cells we e seeded a ecommended ini ial densi y . Subcul u e was pe o med using a 0.25 % ypsin-1 mM EDTA solu ion (Bioch om, Camb idge, UK) whene e cul u es eached 80% con luence. 2.3. Cy o oxic assays using he isola ed compounds To assess he compounds cy o oxic capaci y in monocul u es all he cell lines we e seeded a a densi y o 4 × 10 3 cells/ well in 96-well pla es (SPL-Biosciences®), in he co esponding cul u e medium, and incuba ed o e nigh . Cells we e hen ea ed wi h he compounds a 10, 50 and 500 μM o 72 h. A e ea men , he medium was emo ed and he cells we e washed wice wi h PBS bu e and incuba ed a 37 °C o 1 h wi h a PBS-dissol ed MTT solu ion (1.2 mM). A e washing o he excess o MTT, cells we e disagg ega ed wi h DMSO and he abso bance o he c ys als o o mazan was measu ed a 570 nm using a spec o- pho ome e pla e eade (Bio-Tek Syne gy pla e eade , Bed o dshi e, UK). Con ol expe imen s we e pe o med in pa allel in he absence o he algae compounds. A leas h ee independen expe imen s we e pe o med each in iplica e and esul s exp essed as pe cen age o con ol. 2.4. Expe imen al assays using co-cul u e sys ems To be e mimic lung cance ana omy answell co-cul u e sys ems we e implemen ed as p e iously desc ibed by Rod igues and colla- bo a o s [24]. HBF cells we e seeded as eede laye s in 6-well pla es (SPL-Biosciences®) a a densi y o 1.4 × 10 4 cells/ well. A e 3 days, RenG2 cells we e seeded in T answell®inse s (SPL Li e Sciences, Ko ea) a a densi y o 8 × 10 3 cells/ well. Co-cul u es we e kep in he incuba o a 37 °C in a 95 % ai , 5% CO 2 o wo mon hs, and mediums we e changed e e y 15 days. A e wo mon hs, bo h cell lines we e ea ed wi h he algae compounds o 72 h, a he p e iously de ined concen a ions. The sphe e- o ming assay was subsequen ly employed o e alua e he abili y o he b omo e penes o sup ess CSCs. Con ol expe imen s we e pe o med in pa allel in he absence o he algae compounds. A leas h ee independen expe imen s we e ca ied ou in iplica e and esul s exp essed as pe cen age o con ol. 2.5. Sphe e- o ming assay as a sc een ool o CSCs CSCs we e isola ed using a p o ocol p e iously desc ibed by Rod igues and collabo a o s [24]. 6-well pla es we e coa ed wi h a 2% poli-(2-hyd oxye hyl me hac yla e) (Sigma, USA) solu ion o ensu e low adhe ence condi ions, and CSCs’ isola ion medium wi h app o- p ia e supplemen s was p epa ed con aining a 2% me hylcellulose (Sigma, USA) solu ion (1:1). CSCs isola ion was accomplished a e compounds ea men o 72 h. The cells housed in he uppe com- pa men we e washed wice wi h PBS bu e , de ached using a 0.25 % ypsin-1 mM EDTA solu ion (Bioch ome, Camb idge, UK) and col- lec ed by cen i uga ion a 380 g o 5 min a oom empe a u e. Cells we e hen esuspended in he isola ion medium a a concen a ion o 3 × 10 4 cells/ mL, and 2 mL o his suspension we e added o each well C. Al es, e al. Biomedicine & Pha maco he apy 128 (2020) 110275 2 o he pla e. The isola ion medium was supplemen ed wi h 10 ng/mL o bo h human EGF (E9644, Sigma-Ald ich) and bFGF (100−18B, Pe- p oTech, London) and cells we e main ained a 37 °C in a 95 % ai , 5% CO 2 incuba o . Supplemen s we e eplaced e e y wo days. Whene e sphe e o ma ion was obse ed and sphe es eached a sa is ac o y o- lume (which no mally happens a ound 15 days a e pla ing), hey we e collec ed and analysed using an op ical mic oscope (Axio obse e z1 Ca l Zeiss; Came a AxioCam HR R3; Fiji ImageJ so wa e, Wayne Rasband, Na ional Ins i u es o Heal h, USA). The sphe es’ size was egis e ed and pho os we e aken (Fiji ImageJ so wa e, Wayne Ras- band, Na ional Ins i u es o Heal h, USA). A leas h ee independen expe imen s we e ca ied ou in iplica e. 2.6. IL-6 le els assessmen The IL-6 le els p esen in he supe na an o bo h he uppe and bo om compa men s we e e alua ed by enzyme-linked im- munoso ben assay (ELISA), using he Human IL-6 Quan ikine ELISA Ki (#D6050, R&D Sys ems) acco dingly o he manu ac u e s’ in- s uc ions. A leas h ee independen expe imen s we e ca ied ou in iplica e. 2.7. S a is ical and da a analysis S a is ical analysis was pe o med using one-way analysis o a - iance (ANOVA) wi h Dunne ’s mul iple compa ison o g oup means o de e mine signi ican di e ences ela i ely o con ol ea men . The Tukey's es was applied o he emaining mul iple compa isons. All da a we e checked o no mali y and homoscedas ici y. Resul s a e p esen ed as mean ± s anda d e o o he mean (SEM). Di e ences we e conside ed s a is ically signi ican a le el o 0.05 (p< 0.05). Calcula ions we e pe o med using IBM SPSS S a is ics 24 (IBM Co po a ion, A monk, NY, USA) and G aphPad 5.1 (G aphPad So wa e, La Jolla, CA, USA) so wa e. 3. Resul s 3.1. E alua ion o he cy o oxic ac i i y o he algae-ex ac ed compounds To asce ain he ideal concen a ion o he b omo e penes isola ed om S. co onopi olius o be used in he co-cul u e expe imen s, HBF and RenG2 cells we e monocul u ed o 72 h in he p esence ei he 10, 50 o 500 μM o he isola ed compounds (Fig. 2). The esul s e ealed ha only 10 μM sphae ococcenol A induced a signi ican educ ion (55 %) in HBF cells' iabili y. Mo eo e , all compounds media ed a ma ked cy o oxic e ec on HBF cells a con- cen a ions abo e 10 μM (Fig. 2A). Rega ding RenG2 malignan cells (Fig. 2B), hei iabili y dec eased as he compounds’ concen a ion inc eased, and he s onges cy o oxic e ec was obse ed wi h 12S- hyd oxy-b omopshae ol and b omosphae ol, bo h a a concen a ion o 10 μM. The ea men wi h 50 and 500 μM concen a ions dec eased cells' iabili y in mo e han o 95 %. Toge he , hese esul s e ealed ha d ugs concen a ions highe han 10 μM media ed ma ked non-selec i e cy o oxic e ec s on bo h cell lines. The e o e, only he 10 μM concen a ion p oceed o es s in SC-DRenG2 and BEAS-2B cells (Fig. 3). Unexpec edly, he non-malignan BEAS-2B cells u ned ou o be esis an o bo h b omosphae ol and sphae odac ylomelol, bu 12R- hyd oxy-b omosphae ol (1.01 ± 0.28 % o iable cells), 12S-hyd oxy- b omosphae ol (5.70 ± 2.07 % o iable cells) and sphae ococcenol A (0.82 ± 0.29 % o iable cells) dec eased hei iabili y in mo e han o 90 % (Fig. 3A). Howe e , a a 10 μM concen a ion, all he compounds dec eased signi ican ly SC-DRenG2 cells’ iabili y, wi h b omosphae ol (3.75 ± 0.54 % o iable cells) and sphae ococcenol A (6.16 ± 1.15 % o iable cells) depic ing he highes e ec and sphae odac ylomelol (67.25 ± 7.34 % o iable cells) exhibi ed he lowes e ec (Fig. 3B). Al oge he , he esul s a ained so a show ha 10 μM o 12R-hy- d oxy-b omosphae ol and 12S-hyd oxy-b omosphae ol educe he ia- bili y o bo h BEAS-2B and RenG2 cells bu a e non-cy o oxic owa ds HBF. The e o e, i was decided o pe o m dose- esponse assays on BEAS-2B cells wi h hese wo s e oisome s compounds. D ug con- cen a ions es ed anged om 2 o 10 μM, and he esul s a e depic ed in Fig. 4. T ea men o BEAS-2B cells wi h di e en concen a ions (2−10 Fig. 1. Chemical s uc u es o b omo e penes isola ed om Sphae ococcus co onopi olius collec ed in he Be lenga Na u e Rese e, Peniche, Po ugal (A lan ic coas ). C. Al es, e al. Biomedicine & Pha maco he apy 128 (2020) 110275 3 μM) o 12R-hyd oxy-b omosphae ol and 12S-hyd oxy-b omosphae ol o 72 h e ealed ha he cy o oxic e ec s we e concen a ion-depen- den , wi h an IC 50 o 4.30 μM (Fig. 4A) and 4.29 μM (Fig. 4B), e- spec i ely. As he 2 μM 12S-hyd oxy-b omosphae ol did no a ec BEAS-2B cells’ iabili y, i was decided o s udy he e ec s o 4μM 12R- hyd oxy-b omosphae ol, 12S-hyd oxy-b omosphae ol and b omo- sphae ol on SC-DRenG2 cells’ iabili y (Fig. 5) All he a o emen ioned compounds induced a dec ease in SC- DRenG2 cells' iabili y supe io o 80 %, wi h 12R-hyd oxy-b omo- sphae ol and b omosphae ol showing he s onges cy o oxic e ec (> 90 %) (Fig. 5). I was hen decided o p oceed o he co-cul u e assays wi h only hese wo compounds. 3.2. E ec s o 12R-hyd oxy-b omosphae ol and b omosphae ol on CSCs’ o ma ion and IL-6 le els in co-cul u e sys ems Acco ding o he esul s ob ained in monocul u es, 12R-hyd oxy- b omosphae ol and b omosphae ol we e selec ed o be es ed in answell®co-cul u es o RenG2 and HBF cells. Cells in he co-cul u e sys em we e ea ed wi h 4 μM o he a o emen ioned compounds o 72 h, and he p esence o CSCs, as well as he IL-6 le els on he cell cul u e media we e sc eened h ough he sphe e- o ming assay and ELISA, espec i ely (Figs. 6 and 7). The exposu e o 12R-hyd oxy-b omosphae ol o ally abolished CSCs, while b omosphae ol only sligh ly dec eased he numbe o CSCs. The co-adminis a ion o bo h compounds, on he o he hand, e e ed he e ec s o 12R-hyd oxy-b omosphae ol, as no CSCs’ inhibi ion was obse ed (Fig. 6). P e iously published esul s e idenced ha high le els o IL-6 a e manda o y o CSCs o ma ion [24]. In ligh o such esul s, i was decided o in es iga e whe he he e ec s o b omosphae ol and 12R- hyd oxy-b omosphae ol elied on al e a ions o he IL-6 le els in he co- cul u e sys ems (Fig. 7). IL-6 le els in he bo om and uppe compa men s o co-cul u e did no show signi ican change ollowing he ea men wi h 12R-hyd oxy- b omosphae ol. The ea men wi h b omosphae ol, howe e , exhibi ed a signi ican inc ease in IL-6 le els on he uppe answell®compa - men . Again, he concomi an ea men wi h bo h 12R-hyd oxy-b o- mosphae ol and b omosphae ol did no exhibi signi ican di e ences compa ed o con ol (Fig. 7). 4. Discussion One o he majo challenges in cance diseases is associa ed wi h esis ance o con en ional he apeu ics, being o u mos impo ance o imp o e he cu en s a egies and o de elop new app oaches o igh his bu den [39]. Ad ances in cance biology showed ha his disease is much mo e complex han he simple con inuous uncon olled p o- li e a ion o cance cells, which is sus ained by se e al ac o s ha con ibu e o cance esis ance and elapse [40]. Amongs hem, u- mou he e ogenei y, which elies on he p esence o CSCs and on he umou mic oen i onmen , has been shown key playe s in cance de- elopmen and esis ance [41,42]. The esul s a ained in he p esen wo k e ealed ha despi e he abili y o 12R-hyd oxy-b omosphae ol, 12S-hyd oxy-b omosphae ol, and b omosphae ol a ge CSCs (SC-DRenG2) in monocul u e, only Fig. 2. HBF (A) and RenG2 (B) cells' iabili y ollowing 72 h o exposu e o he Sphae ococcus co onopi olius compounds (10, 50 and 500 μM) exp essed as % o he con ol. The alues co espond o mean ± SEM o a leas h ee independen expe imen s we e ca ied ou in iplica e. Symbols ep esen s a is ically signi ican di e ences (p< 0.05) when compa ed o: * con ol o espec i e concen a ion; # 10 μM ea men ; † 10 and 50 μM ea men . Fig. 3. BEAS-2B (A) and SC-DRenG2 (B) cells' iabili y ollowing 72 h o exposu e o he Sphae ococcus co onopi olius compounds (10 μM) exp essed as % o he con ol. The alues co espond o mean ± SEM o a leas h ee independen expe imen s we e ca ied ou in iplica e. Symbols (*) ep esen s a is ically signi ican di e ences (p< 0.05) when com- pa ed o con ol. C. Al es, e al. Biomedicine & Pha maco he apy 128 (2020) 110275 4 12R-hyd oxy-b omosphae ol e ained ha capaci y in co-cul u es o malignan human b onchial epi helial cells wi h no mal human b on- chial ib oblas s. These esul s, once again, highligh he impo ance o he umou mic oen i onmen in modula ing he he apeu ic esponse, since he p esence o a no mal s oma inhibi ed he ac ion o 12R-hy- d oxy-b omosphae ol and b omosphae ol o e CSCs. They a e in line wi h cu en li e a u e as, o ins ance, he doxo ubicin an i umou ac i i ies we e a enua ed in p os a e cance cells when co-cul i a ed wi h cance -associa ed ib oblas s (CAFs). Acco ding o he au ho s, CAFs blocked doxo ubicin accumula ion in he p os a e cance cells, a oiding ROS p oduc ion and consequen ly DNA damage and apop osis ac i a ion [43]. Simila esul s we e obse ed in co-cul u es o b eas cance cells wi h b eas -cance - issue-de i ed mesenchymal s em cells (BC-MSC) ea ed wi h cispla in [44]. Acco ding o he au ho s he e- sis ance o b eas cance cells in co-cul u e seemed o be associa ed o IL-6 eleased by BC-MSC. To p og ess om bench o bedside, po en ial he apeu ic agen s mus ul il a complex and long lis o c i e ia ha is upda ed along he p ocess [45]. E en hough he mic oen i onmen plays a pi o al ole in he ea ly s eps o he ca cinogenic p ocess, mos o he in i o s udies o es new d ugs neglec ha . In ac mos o he p eclinical s udies o an icance d ugs employ 2D monocul u e o cance cells, whe e no in e cellula communica ions we e conside ed [46]. Elegan co-cul u e sys ems as he one de eloped by Rod igues and collabo a o s [24] and o he s, allow he addi ion o complexi y o p eclinical d ug s udies, pe haps leading o lowe numbe o d ugs ha ail o pe o m in he clinic. Conside ing p e ious obse a ions indica ing IL-6 as a media o o CSCs o ma ion, we hypo hesized ha d ugs a ge ing his cellula po- pula ion may dec ease IL-6 le els in he umou mic oen i onmen . Ou esul s did no co obo a e his hypo hesis, as no signi ican a ia ion was obse ed on IL-6 le els, in he co-cul u e sys ems, ollowing he 12R-hyd oxy-b omosphae ol ea men . None heless, hey a e in line in p e ious obse a ions e ealing ha his cy okine, p esen in he u- mou mic oen i onmen , is in ol ed in umo igenesis by egula ing a ious cance hallma ks and mul iple signalling pa hways, being also in ol ed in chemo esis ance [47,48]. O pa icula in e es is he ailu e o he ea men wi h b omosphae ol in co-cul u es, as compa ed o he esul s achie ed in monocul u es. Appa en ly, he high le els o IL-6 in he co-cul u e expe imen s igge ed some p o-su i al pa hways and ci cum en ed he cy o oxic e ec o some CSCs- a ge ing agen s. Fi- nally, ou indings a e also in ag eemen wi h o he epo s in he li - e a u e suppo ing he po en ial o ex ac s o compounds om ma ine o igin o media e an i-in lamma o y and an i-CSC e ec s[35,37,49–52]. Fo ins ance, di e pene glycosides isola ed om he so co al An illo- go gia elisabe hae we e shown o block NF-κB signalling pa hway in iple-nega i e b eas cance and monocy ic leukaemia cells [53]. Mo eo e , wo polyhalogena ed mono e pene s e eoisome s (RU017 and RU018) and one sesqui e pene (smenospongine) isola ed om he ed alga Plocamium co nu um and he sponge Spongia pe usa espe , e- spec i ely, p e en ed umou sphe e o ma ion in in i o b eas cance models [35,54]. In he speci ic case o smenospongine, i p omo ed cell cycle a es and in insic apop osis, as well as media ed he down- egula ion o speci ic s em cell ma ke s, namely Nanog, Sox2, and Bmi1 [54]. Ou esul s sugges ha 12R-hyd oxy-b omosphae ol is a po en ial CSCs- a ge ing he apeu ic agen , whose cy o oxic ac ion seems o be independen o he IL-6 le els in he umou mic oen i onmen . Al hough, he cy o oxic e ec s o some o hese b omo e penes we e assessed in monocul u es o human lung cance cells [55], o he bes o ou knowledge, his is he i s s udy ha e alua e he capaci y o S. co onopi olius compounds o impac CSCs dynamics in a human lung cance in i o cellula model. Subsequen s udies will be needed o con i m he ep oducibili y o he a ained esul s in mo e complex li ing models, and o assess he po en ial sui abili y o 12R-hyd oxy- b omosphae ol o unde go clinical s udies. The p esen s udy opens new esea ch lines o e alua e he he apeu ic po en ial o hese com- pounds, namely 12R-hyd oxy-b omosphae ol. In o de o unde s and he mechanism o ac ion unde lying he ac i i ies obse ed, he ex- p ession o s emness ac o s (Nanog, oc 4, sox2, STAT3), exp ession o an i-apop o ic p o eins (Su i in, XIAP), analysis o cell cycle and hallma ks linked o apop osis (e.g. mi ochond ial depola iza ion, cas- pases ac i i y, DNA agmen a ion) should be s udied. On he o he hand, he combina ion o hese compounds wi h an icance d ugs (e.g. salinomycin) o imp o e he he apeu ic egimens e icacy as well as hei abili y o sensi ize cance cells and CSCs o adio he apy should be add essed in u u e s udies o unde s and he po en ial o hese com- pounds o inspi e/ c ea e new he apeu ic op ions ha con ibu e o inc ease he an icance ea men s e iciency. Decla a ion o Compe ing In e es The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships ha could ha e appea ed o in lu- ence he wo k epo ed in his pape . All au ho s ha e app o ed he manusc ip o submission and de- cla e ha his manusc ip is o iginal and unpublished, and has no been no will be submi ed o ano he jou nal o conside a ion unless i is ejec ed. Fig. 4. BEAS-2B dose- esponse cu e ollowing 72h exposu e o 12R-hyd oxy- b omosphae ol (A) and 12S-hyd oxy-b omosphae ol (B) a concen a ions be- ween 2 and 10 μM. Values co espond o mean ± SEM o a leas h ee in- dependen expe imen s we e ca ied ou in iplica e. Symbols (*) ep esen s a is ically signi ican di e ences (p< 0.05) when compa ed o con ol. C. Al es, e al. Biomedicine & Pha maco he apy 128 (2020) 110275 5 Fig. 5. SC-DRenG2 cells' iabili y ollowing 72 h o exposu e o 4 μM 12R- hyd oxy-b omosphae ol, 12S-hyd oxy-b omosphae ol and b omosphae ol. Resul s a e exp essed as % o he con ol. The alues co espond o mean ± SEM o a leas h ee independen expe imen s we e ca ied ou in iplica e. Symbols (*) ep esen s a is ically signi ican di e ences (p< 0.05) when compa ed o con ol. Fig. 6. Sphe es a ained ollowing he co-cul u e ea men wi h 4 μM 12R-hyd oxy-b omosphae ol, b omosphae ol o bo h o 72 h. The images a e ep esen a i e o each ea men accomplished (A). Pe ime e analysis (μm) o he a ained sphe es a e 2 weeks in cul u e unde low-adhe ence condi ions (B). Twen y sphe es we e measu ed pe ea men . The esul s we e e ealed by he sphe e- o ming assay. Values co espond o mean ± SEM o a leas h ee independen expe imen s we e ca ied ou in iplica e. Symbols (*) ep esen s a is ically signi ican di e ences (p< 0.05) when compa ed o con ol. Fig. 7. IL-6 le els assessed by ELISA on he bo om (HBF cells) and uppe (RenG2 cells) compa men s o he co-cul u e sys em, ollowing ea men wi h 4 μM 12R-hyd oxy-b omosphae ol, b omosphae ol o bo h o 72 h. The alues co espond o mean ± SEM o a leas h ee independen expe imen s ca ied ou in iplica e. Symbols (*) ep esen s a is ically signi ican di e ences (p< 0.05) when compa ed o espec i e con ol. C. Al es, e al. Biomedicine & Pha maco he apy 128 (2020) 110275 6 Acknowledgmen s This wo k was suppo ed by he Po uguese Founda ion o Science and Technology (FCT) h ough s a egic p ojec UID/MAR/04292/ 2020 and UID/Mul i/04046/2020 g an ed o MARE—Ma ine and En i onmen al Sciences Cen e and BioISI—BioSys ems and In eg a i e Sciences Ins i u e, espec i ely, h ough Red2Disco e y p ojec (PTDC/ MAR-BIO/6149/2014), co- inanced by COMPETE (POCI-01-0145- FEDER-016791), h ough Oncologia de P ecisão: Te apias e Tecnologias Ino ado as p ojec (POINT4PAC) (SAICTPAC/0019/2015 - LISBOA-01-0145-FEDER-016405) and h ough CROSS-ATLANTIC p o- jec (PTDC/BIA-OUT/29250/2017), co- inanced by COMPETE (POCI- 01-0145-FEDER-029250). This wo k was also unded by he In eg a ed P og amme o SR&TD "Sma Valo iza ion o Endogenous Ma ine Biological Resou ces Unde a Changing Clima e" ( e e ence Cen o-01- 0145-FEDER-000018), co- unded by Cen o 2020 P og amme, Po ugal 2020, Eu opean Union, h ough he Eu opean Regional De elopmen Fund. 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