IMPACT OF STROMAL CELL COMPONENTS OF TUMOR MICROENVIRONMENT ON EPITHELIAL-MESENCHYMAL TRANSITION IN BREAST CANCER CELLS

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1 72 Experimentl Oncology 36, 72 78, 214 (June) Exp Oncol , 2, IMPACT OF STROMAL CELL COMPONENTS OF TUMOR MICROENVIRONMENT ON EPITHELIAL-MESENCHYMAL TRANSITION IN BREAST CANCER CELLS N. Bezdenezhnykh 1,*, N. Semesiuk 1, O. Lykhov 1, V. Zhylchuk 2,3, Y. Kudryvets 1 1 R.E. Kvetsky Institute of Experimentl Pthology, Oncology nd Rdioiology, NAS of Ukrine, Kyiv 322, Ukrine 2 Rivne Region Oncology Hospitl, Rivne 331, Ukrine 3 Dnylo Hlytsky Lviv Ntionl Medicl University, Lviv 791, Ukrine Bckground: Cell nd tissue homeostsis results from the dynmic lnce of cell cell nd cell extrcellulr component crosstlk tht regultes prolifertion, differentition, nd poptosis of s well s secretion nd ctivtion of solule fctors nd/or deposition of extrcellulr mtrix (ECM) components. Aim: The im of the work ws to study the crosstlk etween tumor nd stroml cell components using noncontct co-cultivtion in vitro system. Mterils nd Methods: Humn nd rt rest cncer (BC) cell lines, norml humn firolsts (NHF) nd endothelil, nd spirtes of one mrrow (BM) of BC ptients with different clinicl course of the disese (groups Remission (BM-R) nd Progression (BM-P)) were used in noncontct co-cultivtion system in vitro. The cell growth, expression of epithelil-mesenchyml trnsition (EMT) nd tumor stem cell mrkers (E-cdherin, vimentin, CD44), Ki-67, p21 nd Slug were investigted using immunocytochemicl nlysis. Results: Anlysis of expression of E- nd N-cdherin, vimentin nd Slug in BC hs shown tht T-47D nd - possess mesenchyml phenotype, while MCF-7 nd possess mostly epithelil phenotype with prt of with mesenchyml ptterns. Upon noncontct co-cultivtion of firolsts with Т-47D or -Т5, BC cquired higher prolifertive ctivity compred to the control (р <.5) or MCF-7 nd co-cultivted with firolsts. Upon noncontct co-cultivtion of Т-47D with norml firolsts nd BM from BC ptients from group Progression there were oserved incresed quntity of CD44 + Т-47D (y 26%), decresed quntity of Е-cdherin + Т-47D, nd ppernce of vimentin-positive. In co-cultivtion vrint Т-47D + NHF + BM-R ( Remission ) the quntity of CD44 + Т-47D significntly decresed (р <.5) nd E-cdherin expression remined unltered compred to control. At the sme time, in NHF cell popultion (co-cultivtion vrint Т-47D + NHF + BM-P) there ws detected significnt increse of quntity of р21+- (р <.5), cytoplsmic locliztion of p21, nd nucler locliztion of Slug. Expression of vimentin did not lter in ny vrint of co-cultivtion. Conclusion: The new integrtion cell system for investigtion of the mechnisms of interction etween tumor nd the tumor microenvironment in vitro ws developed. The significnt chnges in prolifertive ctivity of dependently on its ЕМT-sttus were detected fter their interction with firolsts nd endothelil in noncontct co-cultivtion system. BM of BC ptients hd different modifying influence on dependent on clinicl BC course. The ctivtion of ЕМT progrm ws reveled in upon noncontct co-cultivtion with BM of BC ptients with progression of the disese. Key Words: rest cncer, epithelil-mesenchyml trnsition, microenvironment, one mrrow, co-cultivtion. In most cses the cncer-relted mortlity is cused y hemtogenous spred of cncer into distnt orgns nd their susequent growth to overt metstses. After surgicl removl of the primry tumor, miniml residul disese (MRD) is defined s the presence of tumor () tht re not detectle y the current routine dignostic procedures used for tumor stging in cncer ptients, ut ecome pprent fter period of time [1]. The crosstlk etween tumor nd of its microenvironment crucilly determines the fte of tumor progression. The strom is very heterogeneous milieu including vrious cell types nd dhesion molecules, oth contriuting to the functionl ctivity nd structurl support of the tumor microenvironment (TME) [2]. S. Pget lid the foundtions of the TME reserch re y formulting the seed nd soil theory. The tumor is directed into one or severl possile moleculr evolution pthwys y signls originting Sumitted: My 8, 214. *Correspondence: E-mil: eznli@mil.ru Arevitions used: BC rest cncer; BM one mrrow; ECM extrcellulr mtrix; EMT epithelil-mesenchyml trnsition; MC mononucler ; MET mesenchyml-epithelil trnsition; MRD miniml residul disese; TAM tumor-ssocited mcrophge; tumor ; TME tumor microenvironment; TNF tumor necrosis fctor. in ntive nd/or modified microenvironmentl fctors. Mny of these pthwys my led to metstsis. The TME hs some chrcteristics: 1) the moleculr composition of the TME is estlished y nd y resident/ infil trting non-tumor ; 2) intercellulr interctions etween nd components of their microenvironment re crucil in determining cncer progression; 3) tumor microenvironment interctions re idirectionl; 4) certin tumor microenvironment interctions my initite tumor progression ; 5) microenvironmentl fctors ply opposing roles in tumor progression y either promoting or lterntively ntgonizing this process [3]. The TME ws recognized s the product of developing crosstlk etween different types: endothelil, crcinom-ssocited firolsts (CAFs), dipocytes, mesenchyml, mesenchyml stem (MSCs; one mrrow derived (BM-MSCs), or crcinom-ssocited (CA-MSCs)), nd from the immune nd inflmmtory systems (tumor-ssocited mcrophges (TAM), regultory T, etc.). The stroml interct not only with ut lso with ech other. induce chnges in firolsts, which contct with them [4], trnsforming them into myofirolsts, which ctivte progrm of epithelil-mesenchyml trnsition (EMT) in resulting in the domintion of mesenchyml fetures

2 Experimentl Oncology 36, 72 78, 214 (June) 73 nd development of highly metsttic phenotype [5]. All these processes tke plce with involvement of solule (humorl) fctors of microenvironment (cytokines, growth fctors, etc.), which, in turn, ctivte other links of interction, involved in mlignnt trnsition, in prticulr, trnscriptionl fctors. In the result of this, epithelil lost the cpcity to form dense intercellulr contcts, their dhesive properties decrese while cpcity for invsion nd migrtion develop. However, when such chieve their destintion point, the inverse process tkes plce mesenchyml-epithelil trnsition (MET), during which they gin cquire epithelil phenotype with incresed dhesion tht gives opportunity to them to fsten onto the sustrte nd to give growth to se condry metsttic focus [6]. Thus, mjor TAM-derived inflmmtory cytokine shown to e highly expressed in rest crcinoms is tumor necrosis fctor lph (TNF-α) which is multifctoril cytokine. TNF-α ctivity vries under different physiologicl conditions nd in cell-typedependent mnner contriutes to sense of miguity regrding its ntitumor effects. Indeed, the permnent expression of TNF-α in rest tumors ctully supports tumor growth nd plys role in the metsttic ehvior of rest crcinoms [7]. Furthermore, in BM of rest cncer (BC) ptients with dvnced cncer significntly higher TNF-α concentrtion ws detected [8]. Therefore, the further study of intercellulr interction nd cell trnsdifferentition my revel new trgets in ntimetsttic therpy. It is well known tht certin orgns nd tissues re typicl s dominnt sites of metstsis for mny types of tumor. For exmple, one mrrow (BM) nd ones often ecome such distnt sites in BC. Tody is well known the importnt role of stroml, in prticulr firolsts, s elements of TME, in control of their phenotype nd iologicl ehvior [9]. However, the role of BM s components of TME is poorly understood. Therefore, th e determintion of influence of BM, especilly in comprtive spect with firolst elements, on the phenotypic conversion nd prolifertive ctivity of of BC t their co-cultivtion, is of specil interest. In routine prctice for in vitro reserch of compounds of different nture (ntitumor drugs, cytotoxic fctors) isolted cell cultures (primry cell cultures nd stle cell lines) re used. Experimentl systems in vitro with use of humn cell lines represent importnt instrument for estimtion of the level nd mechnisms of toxicity of ntitumor drugs with the im of precise definition of their possile ntitumor ction in vivo. The min disdvntge of such in vitro system is n sence of numerous components of interction, which exist in vivo. The principlly new pproch for determintion of intercellulr interction in vitro hs een developed y A.P. Li nd co-uthors [1] who creted system of integrted discrete cultures of norml of different histogenesis nd BC for the study of simultneous comined toxic ction of drugs on the -trgets nd on the norml of orgnism (liver, kidneys, lungs, vsculr endothelium, etc.). The sme scheme with some modifiction hs een developed y us for testing the toxicity of chemicl compounds, in prticulr, hevy metl slts, in vitro, so-clled multi-orgn system of toxicity (MOST) [11]. For investigtion of cellulr interction in progression of disese, we hve developed in this work new cellulr system with use of primry nd stle cell lines of different genesis: humn nd rt BC, norml humn firolsts (NHF) nd norml rt firolsts (NRF), norml porcine endothelil (PAE), from BM spirtes from BC ptients with different disese stge. The im of the work ws to study in vitro the crosstlk etween nd stroml cell components using noncontct co-cultivtion in vitro system. MATERIALS AND METHODS Clinicl mterils. In co-cultivtion in vitro system, mononucler (MC) isolted from BM of BC ptients with II nd III stges of the disese (n = 6), were used. BC ptients were cured in Rivne Regionl Oncologicl Hospitl (Rivne, Ukrine). The ptients were informed out the survey nd provided written consent for prticiption in the reserch. The study ws crried out with pprovl of the locl Ethics Committee (R.E. Kvetsky Institute of Experimentl Pthology, Oncology nd Rdioiology, NAS of Ukrine). After conducted tretment (rdicl surgicl intervention nd djuvnt chemotherpy) BC ptients hve een distriuted into 2 groups: Progression (n = 3) nd Remission (n = 3), t the sis of their clinicl stte, presence of in BM nd high level of tumor-ssocited cytokines in plsm of BM nd peripherl lood [12, 13]. The BM spirtes (3 4 ml) were tken from sternum with n incision in the skin to void contmintion with epithelil. BM smples were otined from BC ptients prior to therpy. MC were isolted y Ficoll-Hypque density grdient LSM 177 (PAA, Austri) centrifugtion t 1,5 rpm for 2 min. MC were wshed three times with RPMI-164 nd were cryopreserved ( per vil). Cell lines. In our co-cultivtion study the following cell lines were used: NHF, NRF of Wistr rts, norml porcine endothelil (PAE), humn BC cell lines T-47D nd MCF-7 nd their sulines modified y longterm ction of interferon (IFN) (MCF-7 + IFN nd T-47D + IFN), rt mmmry crcinom [14, 15], which re chrcterized y domintion in popultion of low tumorigenic with epithelil phenotype s well s highly tumorigenic suline -, which is chrcterized y the presence of of only me senchyml phenotype. All cell lines hve een otined from Bnk of Cell Lines from Humn nd Animl Tissues of R.E. Kvetsky Institute of Experimentl Pthology, Oncology nd Rdioiology of Ntionl Acdemy of Sciences of Ukrine (Kyiv, Ukrine). The modeling system of noncontct cell co-cultivtion. The of different type which were cultured seprtely, serve s control (Fig. 1, ). For co-cultivtion experiment, the wells were comined with ech other vi specilly mde chnnels in the wll of wells in such wy

3 74 Experimentl Oncology 36, 72 78, 214 (June) tht interction of through their nutrient medium could occur (Fig. 1, ). NHF NHF BM BM Stroml Stroml Fig. 1. Scheme of noncontct co-cultivtion in vitro: control wells isolted; wells with noncontct co-cultivtion Cells were cultivted in plstic glsswre ( ТРР, Itly) in DMEM medium ( PAA, Austri), which contined 2 mm of L-glutmine nd NHCO 3 with 1% of FBS ( PAA, Austri) in humidified tmosphere t 37 С nd 5% CO 2. Quntity of fter their co-cultivtion hs een determined y stining with crystl violet ( Sigm, USA) with further registrtion of opticl density of well content using multiwell spectrometer (Lsystems Multiskn PLUS, Finlnd) [16]. Immunocytochemicl nlysis. The slides of cytospins were fixed in methnol + cetone (1:1) solution for 2 h t 2 С, nd then incuted with 1% BSA solution for 2 min. For immunocytochemicl nlysis, the following monoclonl ntiodies hve een pplied: nti-e-cdherin (Thermo Scientific, UK), nti-cd325 ( N-Cdherin) (BioLegend, USA), nti-vimentin (Dignostic BioSystems, USA), nti-cd44 (Dignostic BioSystems, USA), Slug (GeneTex, USA), р21/wf1 (NeoMrkers, USA), Ki-67 (Thermo Scientific, UK). Primry ntiodies were used ccording to the instructions of mnufcturers. For visuliztion, Poly Vue system (Thermo Scientific, UK) ws used. The slides were lso stined with Myer s hemtoxylin ( Sigm, USA) nd exmined using microscope AxioVert ( Сrl Zeiss, Germny) with 32 mgnifiction. Dt were nlyzed y clcultion of positive (+) using clssicl H-score method: S = 1В A + 2В B + 3В C, where S H-score index, which vlue lies within the limits from (no expression) to 3 (intensive expression in % of ); А percentge of poorly stined ; В percentge of modertely stined ; C percen tge of strongly stined. Sttistics. Sttisticl processing of otined results hs een crried out using mthemticl progrm STATISTIСA 6.. Significnce of differences etween men vlues hs een conducted with use of Student s t-criterion. RESULTS AND DISCUSSION First of ll, the expression of ЕМT mrkers (E- nd N-cdherin, vimentin nd Slug) ws nlyzed in which were cultured seprtely s control nd lter were used in noncontct co-cultivtion system. It ws found tht the mesenchyml ntigen expression profile ws the most Tle. EMT mrker expression in humn BC cell lines typicl for of T-47D nd - lines. MCF-7 nd cell lines re chrcterized y the prevlence of with epithelil phenotype with the presence of with mesenchyml fetures (Tle). Then, nd stroml elements (firolsts, endothelil ) were co-cultivted in vitro using the system descried ove. In the cse of co-cultivtion of nd firolsts, the prolifertion of firolsts ws close to tht of control ut the quntity of vried dependently on domintion of epithelil or mesenchyml phenotype of these. In prticulr, the quntity of with domintion of mesenchyml fetures (Т-47D, -Т5 ) in noncontct co-cultivtion with firolsts ws more prominently incresed compred to control (р <.5), thn quntity of with prevlence of epithelil phenotype (MCF-7, ) (Fig. 2). In the cse of co-cultivtion of firolsts (RNF) with rt with me senchyml or epithelil fetures (vrint of co-cultivtion + RNF + -) significnt inhiition of - with me senchyml phenotype in presence of epithelil ws detected (see Fig. 2). So, the with more differentited epithelil phenotype my significntly repress growth potentil of with mesenchyml phenotype [17] NRF NHF1 T-47D NHF2 MCF-7 NRF Fig. 2. Quntity of vile NHF nd humn () nd NRF nd rt () upon their comined noncontct co-cultivtion. OY xis represents the percent of vile compred to respective control cultured in seprte wells (tken s % of vile ) It is well known tht tumor nd stroml in microenvironment cn interct nd crosstlk etween them might e idirectionl. Tumor ngiogenesis is prerequisite for tumor progression nd metstsis. It is complex process Antigens E-cdherin N-cdherin Slug T-47D MCF-7 T-47D MCF-7 T-47D MCF-7 E M E M E M Scores (H-score system) 98±11 126±7 27±21 22±9 39±4 81±11 53±3 72±8 221±28 174± ±21 54±1 156±26 224±32 269±11 NRF

4 Experimentl Oncology 36, 72 78, 214 (June) 75 tht requires coopertive reciprocl interction of tumor nd endothelil nd involves solule nd cellulr components. This requires coordinted expression of prongiogenic fctors nd suppression of ntingiogenic fctors, which leds to endothelil cell prolifertion, migrtion nd vessel formtion. M. Buess et l. [18] suggest tht the interction of endothelil with tht express the CD44 +/ CD24 signture (indicting low prolifertive potentil) might explin ssocition of the CD44 + / CD24 signture with highly prolifertive tumors tht hve n unfvorle prognosis. The differences in prolifertive ctivities of humn BC (control nd IFN-modified ) in cocultivtion with cellulr TME components (firolsts nd endothelil ) were identified. For this study, we used the following design of noncontct cultivtion cell system: set 1: 1) MCF-7/MCF-7 (IFN modified) + NHF; 2) MCF-7/MCF-7 (IFN modified) + PAE; set 2: 1) T-47D/T-47D (IFN modified) + NHF; 2) T-47D/T-47D (IFN modified) + PAE. So, we otined the following results: using MCF-7 cell line nd MCF-7 + IFN suline, we oserved n inhiition the BC prolifertion independently on vrint of stroml (NHF or PAE). This inhiition ws more significnt in the cse with MCF-7 + IFN (2 25%) compred with control (Fig. 3) MCF-7 PAE MCF-7 NHF MCF-7 + IFN PAE MCF-7 + ІFN NHF Fig. 3. Prolifertive ctivities of MCF-7 () nd IFN-modified MCF-7 () in noncontct co-cultivtion system with NHF nd PAE. OY xis represents the percent of vile compred to respective control cultured in seprte wells (tken s % of vile ) Interestingly, prolifertive ctivity of NHF nd PAE ws chnged, too: quntities of stroml elements were higher (out 1% in cse co-cultivtion with PAE nd 3 2% in cse co-cultivtion with NHF). To explin this fct we will investigte the phenotype of these. It is known tht tumor cn induce phenotypic chnges ssocited with trnscriptionl reprogrmming of endothelil, which cn e detected y expression profiling: production of growth fctors y tht induce endothelil to express specific lignds nd their cognte receptors coordintely [19]. In nother set of our study, the prolifertive ctivities of BC nd stroml were different indicting significnt interply of. In prticulr, cultivtion of T-47D with NHF or PAE s well s cultivtion of IFN-modified T-47D with NHF or PAE resulted in incresed prolifertion of oth cell types (Fig. 4) T-47D PAE T-47D NHF T-47D + IFN PAE T-47D + ІFN NHF Fig. 4. Prolifertive ctivities of T-47D () nd IFN-modified T-47D () in noncontct co-cultivtion system with NHF nd PAE. OY xis represents the percent of vile compred to respective control cultured in seprte wells (tken s % of vile ) BM is considered to e depot for nd is direct prticipnt of MRD [2 22]. So, the influence of BM components to s n dditionl fctor ws studied. Cells from spirtes of BM of BC ptients were used in new integrted system in vitro. The significnt chnges of phenotypic fetures of oth tumor nd norml in noncontct co-cultivtion system with ddition of BM were detected (Fig. 5): vrint of co-cultivtion Т-47D + NHF + BM ( Progression ) nd Т-47D + NHF + BM ( Remission ). To investigte chnges t the cellulr nd moleculr level we decided to nlyze the expression of some protein mrkers ssocited with EMT. The investigtion of expression of cell cycle regultor p21 hs shown tht the quntity of p21-positive T-47D s well s intrcellulr locliztion of p21 remined unchnged in different vrints of co-cultivtion (see Fig. 5, ). The sucellulr locliztion of p21 is very

5 76 Experimentl Oncology 36, 72 78, 214 (June) importnt ecuse this protein performs opposite functions in nucleus nd cytoplsm. In prticulr, if p21 loclizes in nucleus, it cts s regultor of cell cycle through inctivtion of trnscriptionl fctors Е2F1, c-myc, STAT3 [23], s well s inhiitor of repliction due to oppression of suunit of DNA-polymerse of δ-protein PCNА. When p21 loclizes in cytoplsm, it performs formtion of stress-firin nd focl contcts tht ssists the migrtion of. Moreover, p21 in cytoplsm demonstrtes ntipoptotic functions through decrese of propoptotic kinses ctivity (ASK-1, JNK, p38) nd inhiition of proxpse-2. Expression of р21, scores Expression of proteins, scores cytoplsmic Locliztion of p21 Т-47D isolted Т-47D + NHF + BM ( Progression ) Т-47D + NHF + BM ( Remission ) nucleus ** * *** CD44 Ki-67 Vimentin E-cdherin Protein mrkers Fig. 5. Expression of р21 (), nd CD44, Ki-67, vimentin, Е-cdherin () in T-47D in noncontct co-cultivtion system in vitro with NHF nd BM. *p.5; **p.5; ***p; evlution y Н-Score, scores Next, we investigted CD44 expression. CD44 is dhesion molecule, which plys importnt role in interction tumor microenvironment nd is involved in processes of migrtion nd invsion of. Moreover, CD44 is used s mrker of progression nd metstsis t different types of cncer, in prticulr, BC. s with phenotype CD44 + CD24 re considered s cncer stem (CSC). In our study, the quntity of CD44-positive (CD44 + ) Т-47D were incresed y 26% t their co-cultivtion with BM (vrint Т-47D + NHF + BM ( Progression )) compred with control of in isolted wells. Interestingly, the quntity of CD ws significntly decresed in vrint of co-cultivtion Т-47D + NHF + BM ( Remission ) compred to isolted control (р <.5) nd compred to these in co-cultivtion vrint with BM of ptients from Progression group (р <.5) (see Fig. 5, ). The next step ws to study of E-cdherin expression in T-47D in different vrints of co-cultivtion: the quntity of E-cdherin + T-47D in co-cultivtion vrint Т-47D + NHF + BM ( Progression ) ws decresed y 1.8 times compred to isolted control nd ws not chnged compred to these in co-cultivtion vrint Т-47D + NHF + BM ( Remission ) (see Fig. 5, ). E-cdherin is mrker of epithelil phenotype. Loss of the E-cdherin molecule is thought to enle metstsis y disrupting intercellulr contcts n erly step in metsttic dissemintion nd prominently ssocited with tumor invsiveness nd poor prognosis [24]. Vimentin is protein of cytoskeleton normlly expressed in of mesenchyml origin [25]. It is used widely s mrker of the EMT nd cn induces chnges in cell shpe, motility, nd dhesion during of this process [26]. Its expression ws investigted in T-47D which were included in different vrints of co-cultivtion. Thus, this protein ws not detected in control T-47D (isolted) s well s in T-47D of vrint co-cultivtion Т-47D + NHF + BM ( Remission ), ut the expression of vimentin ws oserved in T-47D in co-cultivtion with NHF nd BM ( Progression ) (see Fig. 5, ). Such chnges of phenotypic profile of T-47D re the results of their co-cultivtion with components of microenvironment (NHF nd BM of BC ptients with different BC course). These indicte the significnt influence of BM nd humorl fctors, which they produce, on the BC which depended on the stte of tumor process in BC ptients (progression or remission of the disese). The phenotypic profile of in co-cultivtion vrint with BM of BC ptients of Remission group did not differ from control. But the ctivtion of ЕМT progrm were oserved in in co-cultivtion vrint with BM of BC ptients of Progression group, which ws ssocited with otining of more ggressive fetures y nd possile increse of their metsttic potentil. The phenotypic profile of NHF fter their co-cultivtion with T-47D nd BM of BC ptients with different courses of tumor process ws chnged too. The expression of vimentin, p21 nd Slug ws investigted. So, chnges of expression of vimentin were not detected in NHF in ny vrints of co-cultivtion. Chnges of expression of trnscription fctor of mesenchyml Slug were detected. In co-cultivtion NHF with nd BM of BC ptients Slug ws reloclized to the nucleus of NHF (p <.2). Interestingly, the presence of p21 protein only in cytoplsm of NHF ws oserved (Fig. 6). The quntity of p21+ NHF ws significntly incresed upon co-cultivtion with BM of BC ptients ( Progression group) compred with control (р <.5). These results in some wy explin the wellknown dt out differentition of firolsts into myofirolsts in co-cultivtion with [27]. Stroml elements, which include lso firolsts, my e inductors of trnscription fctors in tht support the domintion of mesenchyml fetures in. Such modultions of intrcellulr processes occur through the secretion y of series of fctors (cytokines, fctors of growth, etc.). One of such fc-

6 Experimentl Oncology 36, 72 78, 214 (June) 77 tors my e epi thelil-stroml interction 1 (EPST1). EPST1 hs een identified s interferon response gene exctly in co-cultivtion of BC with stroml firolsts [28, 29]. Its expression ws incresed in postsurgicl tumor mteril of BC ptients in contrst to control mmmry glnd tissues. The highest intensity of expression of EPST1 ws determined exctly in regions of tumor, which closely contcted with strom [29]. 35 NHF isolted 3 NHF + Т-47D + BM ( Progression ) NHF + Т-47D + BM ( Remission ) 25 Expression of proteins, scores 2 15 * *** 5 **** ** **** p2 SLUG SLUG Vimentin cytoplsmic cytoplsmic nucleus Protein mrkers Fig. 6. Expression of р21 (), Slug nd vimentin in NHF upon noncontct co-cultivtion with nd BM from BC ptients. *p <.5; **p <.5; ***p <.2; ****p <.2; evlution y Н-Score, scores In conclusion, the new integrtion cell system for investigtion of the mechnisms of interction etween nd TME in vitro ws developed. The significnt chnges in prolifertive ctivity of dependently on its ЕМT-sttus were detected fter their interction with firolsts nd endothelil in noncontct co-cultivtion system. BM of BC ptients hd different modifying influence on dependent on clinicl BC course. The ctivtion of ЕМT progrm ws reveled in upon noncontct co-cultivtion with BM of BC ptients with progression of the disese. ACKNOWLEDGEMENTS This study ws supported with the grnt NAS of Ukrine for Young Scientists Development of new integrtion cell system for investigtion of the mechnisms of interction with microenvironment in vitro, from REFERENCES 1. Cstco Z, Trcy K, McAllister SS. The tumor mcroenvironment nd systemic regultion of rest cncer progression. Int J Dev Biol 211; 55: Schivoni G, Griele L, Mttei F. The tumor microenvironment: pitch for multiple plyers Front Oncol 213; 3: Witz IP. The tumor microenvironment: the mking of prdigm cncer microenvironment. Cncer Microenviron 29; 2: Trn-Thnh D, Done SJ. 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