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Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond

Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond Epithelial mesenchymal crosstalk (EMC) describes the interaction of the tumor stroma and associated fibroblasts with epithelial cancer cells. In this study we analysed the effects of EMC on head and neck cancer cells. In tumor cell lines EMC was induced using media conditioned from a mix-culture of cancer cells and fibroblasts. Cell proliferation and chemotherapy response were assessed using direct cell counting. Flow cytometry, immunohistochemistry of markers of epithelial-mesenchymal transition (EMT) and subsequent TissueFaxs™ acquisition and quantification and western blot analysis were performed. Holotomographic microscopy imaging was used to visualize the effects of EMC on Cisplatin response of SCC-25 cells. EMC induced a hybrid epithelial-mesenchymal phenotype in SCC-25 cells with co-expression of vimentin and cytokeratin. This hybrid phenotype was associated with chemotherapy resistance and increased proliferation of the cells. The EMC conditioned medium led to an activation of the IL-6/STAT3 pathway with subsequent phosphorylation of STAT3. EMC induced a hybrid epithelial- mesenchymal phenotype in HNSCC cells accompanied by increased therapy resistance and cell proliferation. The IL-6/ STAT3 pathway might be one of the major pathways involved in these EMC-related effects. . . . . . Keywords IL-6 EMT JAK/STAT3/Snail signalling radiochemotherapy therapy resistance conditioned medium Introduction related effects include increased cancer cell mobility [2], in- vasiveness [2, 3], acquisition of a mesenchymal phenotype Tumor cells constantly interact with their surrounding tumor (EMT) [4], generation of cells with stem-like properties and microenvironment. Apart from tumor cells the tumor micro- radiochemotherapy resistance [4, 5]. These effects might be environment consists of blood vessels, extracellular matrix, associated with epithelial to mesenchymal transition (EMT), other non-malignant cells like fibroblasts, immune cells, but also occur without EMT meaning that EMT is just one of pericytes or adipocytes and signalling molecules. Especial fi- the effects caused by EMC [5]. In EMT, epithelial cells ac- broblasts secrete growth factors and chemokines that support quire mesenchymal characteristics under the influence of the growth and survival of tumor cells [1]. surrounding microenvironment. EMT is closely linked to em- This interaction of the tumor stroma and associated fibro- bryonic development, wound healing, tissue fibrosis in chron- blasts with tumor cells is also referred to as epithelial- ic inflammation and cancer progression [6]. During EMT, mesenchymal crosstalk (EMC). EMC relies on paracrine sig- epithelial cells lose their junctions and polarity, re-organize naling, cell-cell interactions and cell-matrix interactions. EMC their cytoskeleton, undergo a change in the signalling programmes that define cell shape and re-programme gene expression; this increases the motility of individual cells and enables the development of an invasive phenotype [7–9]. * T. B. Steinbichler Molecular markers of EMT are decreased expression of epi- teresa.steinbichler@i-med.ac.at thelial cadherin (E-cadherin) and cytokeratines and an in- creased expression of vimentin and neural cadherin (N- Department of Otorhinolaryngology, Head and Neck Surgery, cadherin) [10, 11]. EMT is further associated with the induc- Medical University of Innsbruck, Anichstr.35, tion of cancer stem cells (CSC) which efficiently are able to A-6020 Innsbruck, Austria generate new tumors or form metastasis. Through EMT dif- Department of Radiation Oncology, Medical University of ferentiated cancer cells can reversibly transit into CSC and Innsbruck, Anichstr.35, A-6020 Innsbruck, Austria 68 Steinbichler T.B. et al. vice versa enabling adaption to the varying requirements of monoclonal α-SMA antibody (Cat. Nr. A5228, Sigma, tumor progression and the metastatic cascade [12, 13]. Darmstadt, Germany). Secondary anti-mouse and anti-rabbit One of the major signalling factors in EMC is interleukin-6 Alexa Fluor™ 488 or 594 conjugated antibodies for detection (IL-6) [14–16]. IL-6 is a pleiotropic cytokine, widely appreci- of the immunoreaction were purchased from Molecular ated as a major regulator of the acute phase response, yet Probes (Life Technologies, Darmstadt, Germany). All anti- harbouring numerous functions outside the immune system, bodies were diluted as suggested by the manufacturers. The including lipid metabolism, insulin resistance, mitochondrial immunofluorescent-stained slides were cell nuclei counter- function, and neuroendocrine regulation [16, 17]. IL-6 signal- stained by DAPI (Molecular Probes) and covered in ing is mediated by a heterodimeric receptor complex com- Vectashield Vibrance (Vector Laboratories, Burlingame, CA, prised of the ligand-binding subunit and the signal- USA). Immunohistochemical reactions were acquired in transducing subunit [14]. IL-6 is one of the main chemokines TissueFaxs system and quantified using TissueQuest software present in serum samples of head and neck cancer patients and as published before [26]. elevated IL-6 levels independently predict tumor recurrence, poor survival and tumor metastasis [18]. The tumor- Cell Lines promoting activities of IL-6 are manifold and include the eva- sion of growth suppression by regulating the TP53 gene [19], Human gingival fibroblasts (HGF) and Detroit 562 cells were mediating resistance against cell death [20, 21], increasing purchased from Cell Line Service, Eppelheim, Germany [10, stemness of tumor cells [22, 23], and mediating tumor inva- 27]. SCC-25 cells were purchased from German Collection of sion and metastasis through EMT via the JAK/STAT3/Snail Microorganisms (DSMZ, Braunschweig, Germany). All cells signaling pathway [24, 25]. were cultured in DMEM/F12 (PAA, Pasching, Austria) sup- In this study we analysed the effect of EMC, especially plemented with 10% FBS (PAA), 2 mM l-glutamine, mediated through IL-6 in head and neck cancer cells. 100 units/ml penicillin, and 100 μg/ml streptomycin [10]. Production of Conditioned Medium Methods For the production of EMC-conditioned medium (EMC-CM), 4 4 Patient Samples, Immunohistochemistry 4×10 SCC-25 or Detroit 562 cells/ml and 1 × 10 HGF cells/ ml were plated in 250 ml cell culture flasks and cultured for The procedures followed were in accordance with the ethical 72 h in 15 ml foetal bovine serum-containing medium (1:1 standards of the committee on human experimentation of the mix of DMEM/F12 (PAA) and DMEM-low glucose (PAA) institution and in accord with the Helsinki Declaration of 1975 supplemented with 10% foetal bovine serum (FBS) (PAA), as revised in 1983. Permission was obtained from the local 2 mM l-glutamine, 100 units/ml penicillin, and 100 μg/ml ethics committee to collect pretreatment biopsy samples for streptomycin) [4, 5]. Then the cells were washed twice with molecular biological investigation, paraffin embedding, sec- Dulbecco’s Phosphate-Buffered Saline (DPBS) tioning, and immunohistochemical analysis (Reference (Biowhittaker®, Oud-Heverlee, Belgium) and the serum- Number: UN4428 303/4.14, 26 July 2011). Written informed containing medium was replaced by 15 ml albumin- consent was obtained from all patients. Immunohistochemical containing medium (7,5 ml DMEM/F12 (PAA) and 7,5 ml analysis of pan-cytokeratin, vimentin and α-SMA was per- DMEM-low glucose (PAA) supplemented with bovine serum formed in randomly selected specimens of incident locally albumin (BSA, PAA) (0.4 g albumin/100 ml medium) replac- advanced HNSCC patients treated between March 2010 and ing the protein content of 10% FBS, 2 mM l-glutamine, October 2017 at the Department of Otorhinolaryngology— 100 units/ml penicillin, and 100 μg/ml streptomycin). Head and Neck Surgery, Medical University of Innsbruck. Albumin-containing medium was left 48 h on the mix- Pretreatment tumor samples were obtained during diagnostic culture allowing interacting epithelial cells and fibroblasts to panendoscopy. Patient samples were paraffin embedded, sec- secrete EMC-related factors into the medium [4, 5]. tioned, and immuno-stained as published before [26]. Afterwards, EMC-CM was collected and cells were counted. EMC-CM was portioned according to cell numbers as de- Immunohistochemistry scribed by Hassona and colleagues [28]. EMC-CM cultured cells were used for flow cytometry, or embedded in agarose Indirect immunofluorescence staining was performed using and paraffin and subsequently used for indirect fluorescent pre-diluted mouse monoclonal pan-cytokeratin antibody (cat. immunohistochemistry as described above. Medium condi- nr. 760–2595, Roche Ventana, Mannheim, Germany) and tioned from SCC-25, Detroit 562 cells or HGF fibroblasts 1:100 diluted rabbit polyclonal anti-vimentin, clone SP20 an- was produced in a similar way but 5 × 10 /ml cells were tibody (Linaris, Dossenheim, Germany), 1:200 diluted mouse plated in 250 ml cell culture flasks and mono-cultured in Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond 69 serum containing medium for three days and afterwards in suspension was vortexed and incubated three-times for albumin-containing medium as described above [2]. The 15 min on ice, homogenized in 22G needles and centrifuged CM was sterile-filtered and stored at −80 °C. at 15000 g, 15 min, 4 °C. The cleared supernatant was sub- jected to protein concentration measurement using the Pierce Flow Cytometry of Mixed Cultured Cells 660 nm protein assay (Pierce, Rochford, IL, USA) according to the instructions of the manufacturer. 10 μg protein from all EMC culture was mixed as described above (4 × 10 SCC-25 samples was subsequently processed for western blot, using cells/ml and 1 × 10 HGF cells/ml) and processed immediate- Invitrogen NuPage gels, electrophoresis and blotting system. ly for flow cytometry or was cultured for 5 days (72 h with Western blot detection was done as published before [10], serum, 48 h serum-free). EMC-CM was collected, filtered and using primary antibodies: rabbit monoclonal anti-STAT the cultured cells were processed for flow cytometry using (1:1000) or phospho–STAT (1:2000) and positive controls PerFix-nc kit of Beckman Coulter (Marseille, France). Cells from Cell Signaling Technologies (Danvers, MA, USA), were fixed in formaldehyde containing solution of the kit, mouse monoclonal anti-Slug at 125 ng/ml (Cat. Nr. 564614, permeabilysed by the kit solution “2” and stained with conju- BD Pharmingen, Szabo Scandic, Vienna Austria), rabbit poly- gated antibodies as follows: 7 μl cytokeratin 18-Alexa Fluor clonal anti-Snail1:1000 (Cat. Nr. PA5–11923, Pierce, 488 / 10 cells (Invitrogen, Darmstadt, Germany), vimentin Darmstadt, Germany), mouse monoclonal anti-GAPDH, Phycoerythrin (PE) 1: 10 dilution (Exbio, Prague, 1:100(Santa Cruz Biotechnology, Szabo Scandic, Vienna Czech Republic), SMA–AF 488 1:300 dilution Austria), anti mouse monoclonal β-actin 1: 2 × 10 (eBioscience, Darmstadt, Germany). Isotype matching immu- (Proteintech, Manchester, UK). For signal detection horserad- noglobulins with the same conjugates were purchased from ish peroxidase coupled matched secondary antibodies (1:1000 Exbio, eBioscience and Invitrogen. PE and AF antibodies and 1:2000 dilutions, depending on the instruction of the man- were used combined in the flow cytometry. For preparation ufacturer) and chemiluminescent substrate of Thermo Fisher of the cell suspension samples the instructions of PerFix nc kit Scientific (Darmstadt, Germany,) were used in conditions sug- kit were followed. The fixed and immunostained cells were gested by the manufacturer. The chemilumescence signal was investigated in CytoFLEX flow cytometer of Beckman imaged by an Azure C500 documentation system (Biomedica, Coulter (Brea, CA, USA) following the instructions of the Vienna, Austria). manufacturer. Paraffin Embedding of Cultured Cells Stimulation of SCC-25/ Detroit 562 Cells with CM and IL-6 Routinely cultured cell lines and mixed cultures were collect- ed by centrifugation and embedded as cell pellets in agarose as SCC-25/Detroit 562 cells were treated with 7 ml EMC-CM published before [26, 30] and modified as follows: Cells were per 50 ml cell culture flask for 72 h. The medium was changed harvested by centrifugation at 290 × g for 10 min at 4 °C, and daily. To assess the effects of IL-6 SCC-25/ Detroit 562 cells the resulting pellet was fixed in 10 mL neutral buffered 4% were cultivated in albumin-containing medium supplemented formaldehyde solution (SAV Liquid Production Ltd., with IL-6 at 50 ng/ml (RnD Systems, Biomedica, Vienna, Flintsbach am Inn, Germany). After fixation the cells were Austria). The concentration of IL-6 was based on pre- centrifuged at 400 × g for 10 min at room temperature. The experiments and on a publication of Sullivan and colleagues cell pellet was resuspended in 300 μL PBS, transferred to [29]. Exposure conditions were the same as in the first exper- Eppendorf tube (1.5 mL), and kept on ice. Low melting point imental group, i.e., IL-6 supplemented medium was used over agarose (gelling temperature point 34–37 °C) was prepared in a period of 72 h. At the end of the stimulation period, cells PBS as 3% solution in labor glassware by microwave were used for immunohistochemistry, protein isolation and warming and it was equilibrated in a thermoblock to 65 °C MTT assays respectively. for at least 30 min. The 300 μL PBS-cell suspension was also equilibrated to 65 °C for not more than 10 min. Then, 600 μL Protein Isolation and Western Blotting melted equilibrated agarose was pipetted to the cell suspen- sion, followed by spinning at 2000 × g for 5 min at room Following treatments SCC-25 and Detroit 562 cells were temperature. After that, the tube was placed on ice, the cell washed twice with cold DPBS and scraped in 500 μl RIPA- pellet was trimmed, and it was placed in embedding cassette. buffer (50 mM Tris HCl/pH:7.4, 1 mM EDTA, 0.5 mM The cell pellet in the cassette was stored in PBS containing EGTA, 1% Triton X-100, 0.25% sodium deoxycholate, 0.05–0.1% sodium azide until embedded in paraffin as pub- 0.1% sodium dodecylsulfate, 150 mM NaCl, 10 mM NaF, lished in detail before [26]. 1 mM PMSF; 10 μl proteinase inhibitors of Invitrogen “Halt Similar to the tissue sections, from the cell pellets 5 μm Inhibitors mix/ml RIPA buffer)/culture dish. The cell thick sections were cut. The cell sections did not contain any 70 Steinbichler T.B. et al. overlaps as the cells were distributed. The cell sections were To study this phenomena more closely SCC-25 cells and stained immunohistochemically identical to the tissue sec- HGF fibroblasts were cultured separately and were mixed just tions. The percentage of positive cells for the required reaction before flow cytometry. This sample was used to set separate was identified after scanning the sections in the TissueFaxs gates for epithelial and the mesenchymal cells in the system and evaluating with Tissuequest software [26]. CytoFLEX™ flow cytometer. Most cells settled in the epithe- lial gate, meaning they expressed cytokeratin-18 (94.35%) and in the mesenchymal gate (3.35%), meaning they Holotomographic Microscopy expressed vimentin. Cells with an EMC phenotype expressing both cytokeratin-18 and vimentin were rare (0.48%) (Fig. 2). 10 SCC-25 cells/ml were plated in cell culture dishes (1.5ml/ After five days of mix-cultivation of SCC-25 cells and dish) (IbiDi Ltd., Planegg, Germany) in DMEM/12 supple- HGF cells the distribution of these three cell phenotypes mented with 10% FBS for 24 h. Afterwards the cells were changed completely. The most abundant cell type was the washed with PBS and cultured in albumine medium or EMC-cytokeratin-18 and vimentin double positive cell EMC-CM containing IC (6.2 μM) Cisplatin (Ebewe, (55.91%). 34.99% of the cells expressed only cytokeratin-18 Unterach am Attersee, Austria, Ref. 4)for 3days. and 1.32% expressed only vimentin (Fig. 2). Morphological properties of albumin-medium/cisplatin and Furthermore if the embedded cells were stained with EMC-CM/cisplatin cultured cells were assessed by live cell vimentin and SMA-besides the double-negative epithelial imaging using the Nanolive 3D Cell Explorer cells and the only vimentin-positive fibroblasts-a significant holotomographic microscope (Ecublens, Switzerland) without vimentin/SMA double positive cell population was observed, any additional materials or components. which might represent CAFs (Fig. 2). To further demonstrate that this double-positive ‘hybrid’ epithelial-mesenchymal cell population is not just a laboratory phenomena but could be Results also found in vivo in head and neck cancer patients, tissue biopsies were analysed for these ‘hybrid’ epithelial- EMC and its Cells mesenchymal cells. Immunohistochemistry revealed tumor cell nests stained with pan-cytokeratin, that are surrounded Mixed culture of SCC-25 and HGF cells, functioned as model by fibroblasts stained with vimentin and build functional units in head and neck cancer. Nevertheless, in several HNSCC for EMC (Fig. 1 and 2). During direct mix culture of SCC-25 cells and HGF fibroblasts for production of EMC-CM, the samples, this structure was replaced by a mixture of epithelial main component was a high cytokeratin and high vimentin and mesenchymal cells containing a significant amount of expressing cell population (Fig. 1 and 2), which is considered double positive ‘hybrid’ EMC cells (Fig. 3). as mesenchymal trans-differentiated epithelial cell type (EMC-cell) [6]. A further identified cell population showed high vimentin EMC Increased Cell Proliferation and Cisplatin Resistance and SMA levels and might represent cancer-associated fibro- blasts (CAFs) (Fig. 1 and 2). SMA is a marker widely used for SCC-25 and Detroit 562 cells were treated with EMC-CM or the identification of CAFs, even if it is also expressed in vas- cular smooth muscle cells, pericytes, and myoepithelial cells with media conditioned from a mono-culture of SCC-25/ Detroit 562 cells. [31]. Fig. 1 EMC model of HNSCC in cell culture. The mixed culture of smooth muscle alpha actin (SMA, green) and vimentin (red) antibodies SCC-25 cells and HGF fibroblasts functioned as model for EMC. After (b). The most abundant component of EMC in cell culture is the EMT cell culture and production of conditioned medium (EMC-CM) the cells cell, showing positive reaction for both pan-cytokeratin and vimentin were embedded in agarose and in paraffin, sectioned and immunostained (coloured in yellow or orange), but SMA myofibroblasts (B,green) are using anti-pan-cytokeratin (green) and vimentin (red) antibodies (a)or also detected in this complex. Bars: 20 μm(n =5) Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond Fig. 2 Flow cytometry and TissueFaxs™/ TissueQuest™ analysis of of EMC showed comparable results to B, providing an abundant pan- the EMC model of HNSCC in cell culture. A) SCC-25 oral and HGF cytokeratin (detected with Alexa Fluor 488; x-axis on C photographed in fibroblasts were cultured separately and were mixed before flow cytom- 44Fl channel)–vimentin (detected with Alexa Fluor 594; y-axis on C etry. Cells were fixed and stained using the PerFix-nc kit of Beckman photographed in A594 channel) double positive cell population (light Coulter and cytokeratin-18-Alexa Fluor 488, and vimentin- blue on panel C). If the embedded cells were stained with vimentin Phycoerythrin direct conjugated antibodies. This sample was used to set (detected with Alexa Fluor 594; y-axis on D photographed in A594 chan- the epithelial (blue) and fibroblast (green) gates in the CytoFLEX™ flow nel) and SMA (detected with Alexa Fluor 488; x-axis on D photographed cytometer (A). B) If SCC-25 cells and HGF fibroblasts were cultured for in 44Fl channel); besides the double–negative epithelial cell population production of EMC-CM, the most abundant component of this mixed and only vimentin–positive fibroblasts (black gate on panel D) a signif- EMC-culture was the cytokeratin-18–vimentin double positive cell type, icant vimentin–SMA–double positive cell population (orange gate on which represents the EMT cell (labelled as magenta in panel B). C-D) panel D) was detectable, which is identified as cancer-associated Agarose and paraffin embedding and subsequent TissueFaxs™ aquisition fibroblast (n = 5) and TissueQuest™ evaluation of the mix cultured SCC-25 and HGF cells All of these treatment arms significantly increased cell derived factor-1 (SDF-1) and tumor necrosis factor-α growth measured by MTT test (Fig. 4). (TNF-α) were highly expressed. In SCC-25 conditioned me- A cytokine array from EMC-CM was performed and the dia angiopoietin (ANG), CC-chemokine ligand 23 (CCL23), most abundant cytokine in EMC-CM was IL-6. Treatment BLCandEotaxin1werehighlyexpressedandinHGF- with albumin media containing IL-6 at a concentration of fibroblast conditioned media stem cell factor (SCF) was the 50 ng/ml significantly increased cell growth, too, but in most abundant cytokine. SCC-25 cells, to a lower extent than EMC-CM (Fig. 4). As published by us before, treatment with EMC-CM in- Further cytokines highly expressed in EMC-CM were brain- creased the IC of Cisplatin in SCC-25 and Detroit 562 cells derived neurotrophic factor (BNDF), interleukin-2 (IL-2), B [4]. The IC of Cisplatin of SCC-25 cells increased from lymphocyte chemoattractant (BLC) and Eotaxin 2. 6.2 μMto 13.1 μM(p < 0.001) in MTT assays after treatment In contrast, in indirect co-culture conditioned media fibro- with EMC-CM. The IC of Cisplatin of Detroit 562 cells was blast growth factor (FGF)-6 and −7, interleukin 16, stromal increased following treatment with EMC-CM from 13.1 μM Steinbichler T.B. et al. Fig. 3 EMC in HNSCC tissue of oral cavity. a) Immunohistochemical and mesenchymal cells (B) as pan – cytokeratin – positive epithelial reaction of pan-cytokeratin (green) and vimentin (red) in HNSCC tissue. tumour cells (green) and vimentin-positive fibroblasts (red), but also The tumour cell nests (green) are surrounded by fibroblasts (red) and yellow–orange double positive cells, which are mesenchymal seemed to build functional units in head and neck cancer. b) In several transdifferentiated epithelial cells (EMT). Bar represents 50 μm in panel HNSCC samples, this structure was replaced by a mixture of epithelial Aand 20 μminpanel B(n=5) to 26.8 μM(p <0.01) [4]. These results were also confirmed in vitro [32]. Colony forming ability after treatment with 5 or by clonogenic assays, which are considered the gold standard 10 μM Cisplatin was significantly higher in HNSCC cells for the assessment of chemotherapy or radiotherapy resistance treated with EMC-CM than in controls (p <0.05) [4]. These Fig. 4 EMC increased cell growth in HNSCC cells. SCC-25 (a)and cytokine/chemokine array the most abundant cytokine especially in Detroit 562 (b) cells were treated with conditioned medium of single EMC-CM was interleukin-6 (IL-6), which also supported increased cell tumor cells, single fibroblasts and mix culture of tumor cells and fibro- growth in both SCC-25 and Detroit 562 cells if used for treatment of the blasts (EMC). All conditioned media provided a significantly increased cells at 50 ng/ml. In both panels A and B, the used statistical test was One cell proliferation compared to the controls measured by cell couting, but Way Analysis of Variance (ANOVA) with multiple comparisons of all in SCC-25 cells the highest effect was observed after treatment with treatments to controls using Graphpad Prism 7.00 (n =24) EMC-conditioned medium (EMC-CM; SCC-25-Fibs Mix CM). Using Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond 73 effects of EMC-CM on cisplatin resistance were visualized treatment arms. In contrast, phosphorylation of STAT-3 on with holotomographic microscopy images of IC Cisplatin- Tyrosine 705 was only induced by EMC-CM treatment, treated SCC-25 cells using the NanoLIVE system. If SCC-25 which also achieved a significant induction of Slug protein cells were treated with cisplatin in albumin containing medi- synthesis. um for 72 h the cells died and released their adhesion from the On a second experimental setting the role of IL-6 in the cell culture dish. In contrast, SCC-25 cells treated with the observed effects from EMC-CM was analysed with western IC of cisplatin and EMC-CM, were still alive and metabol- blots. The effect of EMC-CM on STAT3 phosphorylation was ically active. Furthermore, the induction of a large, multinu- compared with the effect of medium conditioned from mono- clear morphology was observed with several mitochondria cultures of SCC-25 or HGF and with albumin medium con- and lipid droplets (Fig. 5). taining 50 ng/ml IL-6. STAT3 phosphorylation was only achieved by EMC-CM or by IL-6. EMC Effects Are Mediated by IL-6-STAT3 Signalling To further elucidate the mechanistic backgrounds of EMC- Discussion CM the IL-6-STAT3 signalling cascade was hypothesized to be involved in the functions of EMC-CM, and it was investi- EMT in tumor progression is nowadays considered as a re- gated in more detail with western blot assays. versible process, which is reversed by its contrary process, SCC-25 cells were either treated with EMC-CM or with mesenchymal to epithelial transition (MET). This phenotypic media conditioned from a mono-culture of SCC-25 cells or plasticity allows tumor cells to adapt to the different require- HGF cells. SCC-25 cells constitutively synthesized STAT3 ments of cancer progression [12]. EMT is closely linked to the and Snail so these signalling factors could be found in all induction of cancer stemness in metastatic disease allowing Fig. 5 EMC rescues cultured HNSCC cells from chemotherapy induced cell death. As published by us before (4) EMC-conditioned media (EMC-CM) doubled the IC value of cisplatin in SCC-25 cells. If SCC-25 cells were treated with cisplatin at IC in albumin con- taining medium for 72 h the cells died and released their adhesion from culture dish (A, B), whereas, the SCC-25 treated with cisplatin at IC in EMC CM were alive, metabolically active and large. Furthermore the initiation of multinuclear morphology was observed with several mitochon- dria and lipid droplets (C, D). A, B, C, D: holotomographic mi- croscopy images of SCC-25 cells using the NanoLIVE system. Scale bars 20 μm Steinbichler T.B. et al. CSC to migrate to different organs. However MET, which epithelial – mesenchymal crosstalk (EMC). The treatment increases proliferative capabilities of cancer cells in return is with EMC-CM induced a hybrid phenotype in naïve SCC- necessary, after CSC have reached their metastatic niche, for 25 cells and also the protein synthesis of EMT-related tran- metastatic colonization of the host organ [6, 12, 33]. This scription factors as Snail or Slug (Fig. 6). Furthermore, this dynamic model of EMT and MET is further supported by hybrid phenotype could be found in the invasive front of tissue the finding of hybrid epithelial-mesenchymal cells which are biopsies from patients with head and neck cancer (Fig. 3). favourable for the establishment of metastasis. These hybrid These findings might be clinical relevant as we were able to phenotypes could be found in circulating tumor cells from demonstrate before that EMC-CM led to chemotherapy and patients with non-small cell lung cancer [34], prostate cancer radiotherapy resistance in HNSCC cells, indicating that this [35] and breast cancer [36–38]. This phenotypic plasticity was hybrid epithelial-mesenchymal phenotype might be involved described as stemness related feature, as these hybrids cells in therapy resistance, which is also described as a key feature were demonstrated to express high levels of CSC markers, too of CSC [4, 5, 40]. [35, 39]. Visualisation of these EMC-induced hybrid cells with In this study a cell culture model of EMTwas established in holotomographic microscopy after exposure to 6.2 μMcis- the mixed culture of epithelial tumor cells and fibroblasts as platin showed that these cells survived cisplatin treatment Fig. 6 EMC-CM transmits induction of EMT through IL-6 - STAT3 actin was used as loading control. 1: cells cultured in albumin- signalling. SCC-25 cells were treated with conditioned media of single containing medium, 2: cells treated with CM of single culture of SCC- culture of SCC-25 cells (A, 1), single culture of HGF fibroblasts (A,2), 25 cells, 3: cells treated with CM of single culture of HGF fibroblasts, 4: mixed EMC culture (A, 3), or were indirect co-cultured with HGF fibro- cells treated with CM of mixed EMC culture, 5: cells treated with blasts (A,4). A) Western blot analysis of treated SCC-25 for STAT3, 50 ng/ml IL-6, 6: commercial available positive control of induced phospho-STAT3, Snail and Slug. GAPDH was used as loading control. STAT3 phosphorylation. STAT3 phosphorylation was achieved by CM The SCC-25 cells constitutively synthesized STAT3 and Snail. STAT-3 of mixed EMC culture or by IL-6. C-D) Normalized optical density was only phosphorylated by mixed EMC-CM treatment, which also quantification of western blots presented in B, C: STAT3,D: phospho- achieved a significant induction of Slug protein synthesis. B) Western STAT3 (n =3) blot analysis of treated SCC-25 cells for STAT3, phospho-STAT3. β- Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond 75 Separation of cell survival, growth, migration, and mesenchymal and a large, multinuclear morphology was induced with sev- transdifferentiation effects of fibroblast secretome on tumor cells of eral mitochondria and lipid droplets. Native SCC-25 in con- head and neck squamous cell carcinoma. Tumour biology : the trast died and released their adhesion from the cell culture dish journal of the International Society for Oncodevelopmental (Fig. 5). Similarly to previous studies these EMC-induced Biology and Medicine 39(11):1010428317705507 3. Chan R, Sethi P, Jyoti A, McGarry R, Upreti M (2016) hybrid cancer cells showed a higher proliferation and conse- Investigating the Radioresistant properties of lung Cancer stem quently tumor-initiating ability (Fig. 4)[41, 42]. cells in the context of the tumor microenvironment. Radiat Res As a possible mediator of these effects we suggested IL-6, 185(2):169–181 as it is the most abundant cytokine in EMC-CM. IL-6 has been 4. Steinbichler TB, Metzler V, Pritz C, Riechelmann H, Dudas J (2016) Tumor-associated fibroblast-conditioned medium induces shown to regulate cancer cell stemness in several studies [43, CDDP resistance in HNSCC cells. Oncotarget. 7(3):2508–2518 44]. 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Exp Cell Res 319(6): type is associated with increased self-renewal, therapy resis- 800–809 12. Steinbichler TB, Dudas J, Riechelmann H, Skvortsova II (2017) tance and migration and can also be found in the invasive front The role of exosomes in cancer metastasis. Semin Cancer Biol 44: of tissue biopsies from head and neck cancer patients. These 170–181 observed effects might be mediated by IL-6-STAT3 13. Steinbichler TB, Dudas J, Skvortsov S, Ganswindt U, Riechelmann signalling. H, Skvortsova II (2018) Therapy resistance mediated by cancer stem cells. Semin Cancer Biol 53:156–167 14. Ebbing EA, van der Zalm AP, Steins A, Creemers A, Hermsen S, Funding Information Open access funding provided by University of Rentenaar R, Klein M, Waasdorp C, Hooijer GKJ, Meijer SL, Innsbruck and Medical University of Innsbruck. 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Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond

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Springer Journals
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Copyright © 2019 by The Author(s)
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Biomedicine; Cancer Research; Oncology; Immunology; Cell Biology; Biochemistry, general; Biomedicine, general
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1875-2292
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1875-2284
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10.1007/s12307-019-00228-y
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Abstract

Epithelial mesenchymal crosstalk (EMC) describes the interaction of the tumor stroma and associated fibroblasts with epithelial cancer cells. In this study we analysed the effects of EMC on head and neck cancer cells. In tumor cell lines EMC was induced using media conditioned from a mix-culture of cancer cells and fibroblasts. Cell proliferation and chemotherapy response were assessed using direct cell counting. Flow cytometry, immunohistochemistry of markers of epithelial-mesenchymal transition (EMT) and subsequent TissueFaxs™ acquisition and quantification and western blot analysis were performed. Holotomographic microscopy imaging was used to visualize the effects of EMC on Cisplatin response of SCC-25 cells. EMC induced a hybrid epithelial-mesenchymal phenotype in SCC-25 cells with co-expression of vimentin and cytokeratin. This hybrid phenotype was associated with chemotherapy resistance and increased proliferation of the cells. The EMC conditioned medium led to an activation of the IL-6/STAT3 pathway with subsequent phosphorylation of STAT3. EMC induced a hybrid epithelial- mesenchymal phenotype in HNSCC cells accompanied by increased therapy resistance and cell proliferation. The IL-6/ STAT3 pathway might be one of the major pathways involved in these EMC-related effects. . . . . . Keywords IL-6 EMT JAK/STAT3/Snail signalling radiochemotherapy therapy resistance conditioned medium Introduction related effects include increased cancer cell mobility [2], in- vasiveness [2, 3], acquisition of a mesenchymal phenotype Tumor cells constantly interact with their surrounding tumor (EMT) [4], generation of cells with stem-like properties and microenvironment. Apart from tumor cells the tumor micro- radiochemotherapy resistance [4, 5]. These effects might be environment consists of blood vessels, extracellular matrix, associated with epithelial to mesenchymal transition (EMT), other non-malignant cells like fibroblasts, immune cells, but also occur without EMT meaning that EMT is just one of pericytes or adipocytes and signalling molecules. Especial fi- the effects caused by EMC [5]. In EMT, epithelial cells ac- broblasts secrete growth factors and chemokines that support quire mesenchymal characteristics under the influence of the growth and survival of tumor cells [1]. surrounding microenvironment. EMT is closely linked to em- This interaction of the tumor stroma and associated fibro- bryonic development, wound healing, tissue fibrosis in chron- blasts with tumor cells is also referred to as epithelial- ic inflammation and cancer progression [6]. During EMT, mesenchymal crosstalk (EMC). EMC relies on paracrine sig- epithelial cells lose their junctions and polarity, re-organize naling, cell-cell interactions and cell-matrix interactions. EMC their cytoskeleton, undergo a change in the signalling programmes that define cell shape and re-programme gene expression; this increases the motility of individual cells and enables the development of an invasive phenotype [7–9]. * T. B. Steinbichler Molecular markers of EMT are decreased expression of epi- teresa.steinbichler@i-med.ac.at thelial cadherin (E-cadherin) and cytokeratines and an in- creased expression of vimentin and neural cadherin (N- Department of Otorhinolaryngology, Head and Neck Surgery, cadherin) [10, 11]. EMT is further associated with the induc- Medical University of Innsbruck, Anichstr.35, tion of cancer stem cells (CSC) which efficiently are able to A-6020 Innsbruck, Austria generate new tumors or form metastasis. Through EMT dif- Department of Radiation Oncology, Medical University of ferentiated cancer cells can reversibly transit into CSC and Innsbruck, Anichstr.35, A-6020 Innsbruck, Austria 68 Steinbichler T.B. et al. vice versa enabling adaption to the varying requirements of monoclonal α-SMA antibody (Cat. Nr. A5228, Sigma, tumor progression and the metastatic cascade [12, 13]. Darmstadt, Germany). Secondary anti-mouse and anti-rabbit One of the major signalling factors in EMC is interleukin-6 Alexa Fluor™ 488 or 594 conjugated antibodies for detection (IL-6) [14–16]. IL-6 is a pleiotropic cytokine, widely appreci- of the immunoreaction were purchased from Molecular ated as a major regulator of the acute phase response, yet Probes (Life Technologies, Darmstadt, Germany). All anti- harbouring numerous functions outside the immune system, bodies were diluted as suggested by the manufacturers. The including lipid metabolism, insulin resistance, mitochondrial immunofluorescent-stained slides were cell nuclei counter- function, and neuroendocrine regulation [16, 17]. IL-6 signal- stained by DAPI (Molecular Probes) and covered in ing is mediated by a heterodimeric receptor complex com- Vectashield Vibrance (Vector Laboratories, Burlingame, CA, prised of the ligand-binding subunit and the signal- USA). Immunohistochemical reactions were acquired in transducing subunit [14]. IL-6 is one of the main chemokines TissueFaxs system and quantified using TissueQuest software present in serum samples of head and neck cancer patients and as published before [26]. elevated IL-6 levels independently predict tumor recurrence, poor survival and tumor metastasis [18]. The tumor- Cell Lines promoting activities of IL-6 are manifold and include the eva- sion of growth suppression by regulating the TP53 gene [19], Human gingival fibroblasts (HGF) and Detroit 562 cells were mediating resistance against cell death [20, 21], increasing purchased from Cell Line Service, Eppelheim, Germany [10, stemness of tumor cells [22, 23], and mediating tumor inva- 27]. SCC-25 cells were purchased from German Collection of sion and metastasis through EMT via the JAK/STAT3/Snail Microorganisms (DSMZ, Braunschweig, Germany). All cells signaling pathway [24, 25]. were cultured in DMEM/F12 (PAA, Pasching, Austria) sup- In this study we analysed the effect of EMC, especially plemented with 10% FBS (PAA), 2 mM l-glutamine, mediated through IL-6 in head and neck cancer cells. 100 units/ml penicillin, and 100 μg/ml streptomycin [10]. Production of Conditioned Medium Methods For the production of EMC-conditioned medium (EMC-CM), 4 4 Patient Samples, Immunohistochemistry 4×10 SCC-25 or Detroit 562 cells/ml and 1 × 10 HGF cells/ ml were plated in 250 ml cell culture flasks and cultured for The procedures followed were in accordance with the ethical 72 h in 15 ml foetal bovine serum-containing medium (1:1 standards of the committee on human experimentation of the mix of DMEM/F12 (PAA) and DMEM-low glucose (PAA) institution and in accord with the Helsinki Declaration of 1975 supplemented with 10% foetal bovine serum (FBS) (PAA), as revised in 1983. Permission was obtained from the local 2 mM l-glutamine, 100 units/ml penicillin, and 100 μg/ml ethics committee to collect pretreatment biopsy samples for streptomycin) [4, 5]. Then the cells were washed twice with molecular biological investigation, paraffin embedding, sec- Dulbecco’s Phosphate-Buffered Saline (DPBS) tioning, and immunohistochemical analysis (Reference (Biowhittaker®, Oud-Heverlee, Belgium) and the serum- Number: UN4428 303/4.14, 26 July 2011). Written informed containing medium was replaced by 15 ml albumin- consent was obtained from all patients. Immunohistochemical containing medium (7,5 ml DMEM/F12 (PAA) and 7,5 ml analysis of pan-cytokeratin, vimentin and α-SMA was per- DMEM-low glucose (PAA) supplemented with bovine serum formed in randomly selected specimens of incident locally albumin (BSA, PAA) (0.4 g albumin/100 ml medium) replac- advanced HNSCC patients treated between March 2010 and ing the protein content of 10% FBS, 2 mM l-glutamine, October 2017 at the Department of Otorhinolaryngology— 100 units/ml penicillin, and 100 μg/ml streptomycin). Head and Neck Surgery, Medical University of Innsbruck. Albumin-containing medium was left 48 h on the mix- Pretreatment tumor samples were obtained during diagnostic culture allowing interacting epithelial cells and fibroblasts to panendoscopy. Patient samples were paraffin embedded, sec- secrete EMC-related factors into the medium [4, 5]. tioned, and immuno-stained as published before [26]. Afterwards, EMC-CM was collected and cells were counted. EMC-CM was portioned according to cell numbers as de- Immunohistochemistry scribed by Hassona and colleagues [28]. EMC-CM cultured cells were used for flow cytometry, or embedded in agarose Indirect immunofluorescence staining was performed using and paraffin and subsequently used for indirect fluorescent pre-diluted mouse monoclonal pan-cytokeratin antibody (cat. immunohistochemistry as described above. Medium condi- nr. 760–2595, Roche Ventana, Mannheim, Germany) and tioned from SCC-25, Detroit 562 cells or HGF fibroblasts 1:100 diluted rabbit polyclonal anti-vimentin, clone SP20 an- was produced in a similar way but 5 × 10 /ml cells were tibody (Linaris, Dossenheim, Germany), 1:200 diluted mouse plated in 250 ml cell culture flasks and mono-cultured in Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond 69 serum containing medium for three days and afterwards in suspension was vortexed and incubated three-times for albumin-containing medium as described above [2]. The 15 min on ice, homogenized in 22G needles and centrifuged CM was sterile-filtered and stored at −80 °C. at 15000 g, 15 min, 4 °C. The cleared supernatant was sub- jected to protein concentration measurement using the Pierce Flow Cytometry of Mixed Cultured Cells 660 nm protein assay (Pierce, Rochford, IL, USA) according to the instructions of the manufacturer. 10 μg protein from all EMC culture was mixed as described above (4 × 10 SCC-25 samples was subsequently processed for western blot, using cells/ml and 1 × 10 HGF cells/ml) and processed immediate- Invitrogen NuPage gels, electrophoresis and blotting system. ly for flow cytometry or was cultured for 5 days (72 h with Western blot detection was done as published before [10], serum, 48 h serum-free). EMC-CM was collected, filtered and using primary antibodies: rabbit monoclonal anti-STAT the cultured cells were processed for flow cytometry using (1:1000) or phospho–STAT (1:2000) and positive controls PerFix-nc kit of Beckman Coulter (Marseille, France). Cells from Cell Signaling Technologies (Danvers, MA, USA), were fixed in formaldehyde containing solution of the kit, mouse monoclonal anti-Slug at 125 ng/ml (Cat. Nr. 564614, permeabilysed by the kit solution “2” and stained with conju- BD Pharmingen, Szabo Scandic, Vienna Austria), rabbit poly- gated antibodies as follows: 7 μl cytokeratin 18-Alexa Fluor clonal anti-Snail1:1000 (Cat. Nr. PA5–11923, Pierce, 488 / 10 cells (Invitrogen, Darmstadt, Germany), vimentin Darmstadt, Germany), mouse monoclonal anti-GAPDH, Phycoerythrin (PE) 1: 10 dilution (Exbio, Prague, 1:100(Santa Cruz Biotechnology, Szabo Scandic, Vienna Czech Republic), SMA–AF 488 1:300 dilution Austria), anti mouse monoclonal β-actin 1: 2 × 10 (eBioscience, Darmstadt, Germany). Isotype matching immu- (Proteintech, Manchester, UK). For signal detection horserad- noglobulins with the same conjugates were purchased from ish peroxidase coupled matched secondary antibodies (1:1000 Exbio, eBioscience and Invitrogen. PE and AF antibodies and 1:2000 dilutions, depending on the instruction of the man- were used combined in the flow cytometry. For preparation ufacturer) and chemiluminescent substrate of Thermo Fisher of the cell suspension samples the instructions of PerFix nc kit Scientific (Darmstadt, Germany,) were used in conditions sug- kit were followed. The fixed and immunostained cells were gested by the manufacturer. The chemilumescence signal was investigated in CytoFLEX flow cytometer of Beckman imaged by an Azure C500 documentation system (Biomedica, Coulter (Brea, CA, USA) following the instructions of the Vienna, Austria). manufacturer. Paraffin Embedding of Cultured Cells Stimulation of SCC-25/ Detroit 562 Cells with CM and IL-6 Routinely cultured cell lines and mixed cultures were collect- ed by centrifugation and embedded as cell pellets in agarose as SCC-25/Detroit 562 cells were treated with 7 ml EMC-CM published before [26, 30] and modified as follows: Cells were per 50 ml cell culture flask for 72 h. The medium was changed harvested by centrifugation at 290 × g for 10 min at 4 °C, and daily. To assess the effects of IL-6 SCC-25/ Detroit 562 cells the resulting pellet was fixed in 10 mL neutral buffered 4% were cultivated in albumin-containing medium supplemented formaldehyde solution (SAV Liquid Production Ltd., with IL-6 at 50 ng/ml (RnD Systems, Biomedica, Vienna, Flintsbach am Inn, Germany). After fixation the cells were Austria). The concentration of IL-6 was based on pre- centrifuged at 400 × g for 10 min at room temperature. The experiments and on a publication of Sullivan and colleagues cell pellet was resuspended in 300 μL PBS, transferred to [29]. Exposure conditions were the same as in the first exper- Eppendorf tube (1.5 mL), and kept on ice. Low melting point imental group, i.e., IL-6 supplemented medium was used over agarose (gelling temperature point 34–37 °C) was prepared in a period of 72 h. At the end of the stimulation period, cells PBS as 3% solution in labor glassware by microwave were used for immunohistochemistry, protein isolation and warming and it was equilibrated in a thermoblock to 65 °C MTT assays respectively. for at least 30 min. The 300 μL PBS-cell suspension was also equilibrated to 65 °C for not more than 10 min. Then, 600 μL Protein Isolation and Western Blotting melted equilibrated agarose was pipetted to the cell suspen- sion, followed by spinning at 2000 × g for 5 min at room Following treatments SCC-25 and Detroit 562 cells were temperature. After that, the tube was placed on ice, the cell washed twice with cold DPBS and scraped in 500 μl RIPA- pellet was trimmed, and it was placed in embedding cassette. buffer (50 mM Tris HCl/pH:7.4, 1 mM EDTA, 0.5 mM The cell pellet in the cassette was stored in PBS containing EGTA, 1% Triton X-100, 0.25% sodium deoxycholate, 0.05–0.1% sodium azide until embedded in paraffin as pub- 0.1% sodium dodecylsulfate, 150 mM NaCl, 10 mM NaF, lished in detail before [26]. 1 mM PMSF; 10 μl proteinase inhibitors of Invitrogen “Halt Similar to the tissue sections, from the cell pellets 5 μm Inhibitors mix/ml RIPA buffer)/culture dish. The cell thick sections were cut. The cell sections did not contain any 70 Steinbichler T.B. et al. overlaps as the cells were distributed. The cell sections were To study this phenomena more closely SCC-25 cells and stained immunohistochemically identical to the tissue sec- HGF fibroblasts were cultured separately and were mixed just tions. The percentage of positive cells for the required reaction before flow cytometry. This sample was used to set separate was identified after scanning the sections in the TissueFaxs gates for epithelial and the mesenchymal cells in the system and evaluating with Tissuequest software [26]. CytoFLEX™ flow cytometer. Most cells settled in the epithe- lial gate, meaning they expressed cytokeratin-18 (94.35%) and in the mesenchymal gate (3.35%), meaning they Holotomographic Microscopy expressed vimentin. Cells with an EMC phenotype expressing both cytokeratin-18 and vimentin were rare (0.48%) (Fig. 2). 10 SCC-25 cells/ml were plated in cell culture dishes (1.5ml/ After five days of mix-cultivation of SCC-25 cells and dish) (IbiDi Ltd., Planegg, Germany) in DMEM/12 supple- HGF cells the distribution of these three cell phenotypes mented with 10% FBS for 24 h. Afterwards the cells were changed completely. The most abundant cell type was the washed with PBS and cultured in albumine medium or EMC-cytokeratin-18 and vimentin double positive cell EMC-CM containing IC (6.2 μM) Cisplatin (Ebewe, (55.91%). 34.99% of the cells expressed only cytokeratin-18 Unterach am Attersee, Austria, Ref. 4)for 3days. and 1.32% expressed only vimentin (Fig. 2). Morphological properties of albumin-medium/cisplatin and Furthermore if the embedded cells were stained with EMC-CM/cisplatin cultured cells were assessed by live cell vimentin and SMA-besides the double-negative epithelial imaging using the Nanolive 3D Cell Explorer cells and the only vimentin-positive fibroblasts-a significant holotomographic microscope (Ecublens, Switzerland) without vimentin/SMA double positive cell population was observed, any additional materials or components. which might represent CAFs (Fig. 2). To further demonstrate that this double-positive ‘hybrid’ epithelial-mesenchymal cell population is not just a laboratory phenomena but could be Results also found in vivo in head and neck cancer patients, tissue biopsies were analysed for these ‘hybrid’ epithelial- EMC and its Cells mesenchymal cells. Immunohistochemistry revealed tumor cell nests stained with pan-cytokeratin, that are surrounded Mixed culture of SCC-25 and HGF cells, functioned as model by fibroblasts stained with vimentin and build functional units in head and neck cancer. Nevertheless, in several HNSCC for EMC (Fig. 1 and 2). During direct mix culture of SCC-25 cells and HGF fibroblasts for production of EMC-CM, the samples, this structure was replaced by a mixture of epithelial main component was a high cytokeratin and high vimentin and mesenchymal cells containing a significant amount of expressing cell population (Fig. 1 and 2), which is considered double positive ‘hybrid’ EMC cells (Fig. 3). as mesenchymal trans-differentiated epithelial cell type (EMC-cell) [6]. A further identified cell population showed high vimentin EMC Increased Cell Proliferation and Cisplatin Resistance and SMA levels and might represent cancer-associated fibro- blasts (CAFs) (Fig. 1 and 2). SMA is a marker widely used for SCC-25 and Detroit 562 cells were treated with EMC-CM or the identification of CAFs, even if it is also expressed in vas- cular smooth muscle cells, pericytes, and myoepithelial cells with media conditioned from a mono-culture of SCC-25/ Detroit 562 cells. [31]. Fig. 1 EMC model of HNSCC in cell culture. The mixed culture of smooth muscle alpha actin (SMA, green) and vimentin (red) antibodies SCC-25 cells and HGF fibroblasts functioned as model for EMC. After (b). The most abundant component of EMC in cell culture is the EMT cell culture and production of conditioned medium (EMC-CM) the cells cell, showing positive reaction for both pan-cytokeratin and vimentin were embedded in agarose and in paraffin, sectioned and immunostained (coloured in yellow or orange), but SMA myofibroblasts (B,green) are using anti-pan-cytokeratin (green) and vimentin (red) antibodies (a)or also detected in this complex. Bars: 20 μm(n =5) Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond Fig. 2 Flow cytometry and TissueFaxs™/ TissueQuest™ analysis of of EMC showed comparable results to B, providing an abundant pan- the EMC model of HNSCC in cell culture. A) SCC-25 oral and HGF cytokeratin (detected with Alexa Fluor 488; x-axis on C photographed in fibroblasts were cultured separately and were mixed before flow cytom- 44Fl channel)–vimentin (detected with Alexa Fluor 594; y-axis on C etry. Cells were fixed and stained using the PerFix-nc kit of Beckman photographed in A594 channel) double positive cell population (light Coulter and cytokeratin-18-Alexa Fluor 488, and vimentin- blue on panel C). If the embedded cells were stained with vimentin Phycoerythrin direct conjugated antibodies. This sample was used to set (detected with Alexa Fluor 594; y-axis on D photographed in A594 chan- the epithelial (blue) and fibroblast (green) gates in the CytoFLEX™ flow nel) and SMA (detected with Alexa Fluor 488; x-axis on D photographed cytometer (A). B) If SCC-25 cells and HGF fibroblasts were cultured for in 44Fl channel); besides the double–negative epithelial cell population production of EMC-CM, the most abundant component of this mixed and only vimentin–positive fibroblasts (black gate on panel D) a signif- EMC-culture was the cytokeratin-18–vimentin double positive cell type, icant vimentin–SMA–double positive cell population (orange gate on which represents the EMT cell (labelled as magenta in panel B). C-D) panel D) was detectable, which is identified as cancer-associated Agarose and paraffin embedding and subsequent TissueFaxs™ aquisition fibroblast (n = 5) and TissueQuest™ evaluation of the mix cultured SCC-25 and HGF cells All of these treatment arms significantly increased cell derived factor-1 (SDF-1) and tumor necrosis factor-α growth measured by MTT test (Fig. 4). (TNF-α) were highly expressed. In SCC-25 conditioned me- A cytokine array from EMC-CM was performed and the dia angiopoietin (ANG), CC-chemokine ligand 23 (CCL23), most abundant cytokine in EMC-CM was IL-6. Treatment BLCandEotaxin1werehighlyexpressedandinHGF- with albumin media containing IL-6 at a concentration of fibroblast conditioned media stem cell factor (SCF) was the 50 ng/ml significantly increased cell growth, too, but in most abundant cytokine. SCC-25 cells, to a lower extent than EMC-CM (Fig. 4). As published by us before, treatment with EMC-CM in- Further cytokines highly expressed in EMC-CM were brain- creased the IC of Cisplatin in SCC-25 and Detroit 562 cells derived neurotrophic factor (BNDF), interleukin-2 (IL-2), B [4]. The IC of Cisplatin of SCC-25 cells increased from lymphocyte chemoattractant (BLC) and Eotaxin 2. 6.2 μMto 13.1 μM(p < 0.001) in MTT assays after treatment In contrast, in indirect co-culture conditioned media fibro- with EMC-CM. The IC of Cisplatin of Detroit 562 cells was blast growth factor (FGF)-6 and −7, interleukin 16, stromal increased following treatment with EMC-CM from 13.1 μM Steinbichler T.B. et al. Fig. 3 EMC in HNSCC tissue of oral cavity. a) Immunohistochemical and mesenchymal cells (B) as pan – cytokeratin – positive epithelial reaction of pan-cytokeratin (green) and vimentin (red) in HNSCC tissue. tumour cells (green) and vimentin-positive fibroblasts (red), but also The tumour cell nests (green) are surrounded by fibroblasts (red) and yellow–orange double positive cells, which are mesenchymal seemed to build functional units in head and neck cancer. b) In several transdifferentiated epithelial cells (EMT). Bar represents 50 μm in panel HNSCC samples, this structure was replaced by a mixture of epithelial Aand 20 μminpanel B(n=5) to 26.8 μM(p <0.01) [4]. These results were also confirmed in vitro [32]. Colony forming ability after treatment with 5 or by clonogenic assays, which are considered the gold standard 10 μM Cisplatin was significantly higher in HNSCC cells for the assessment of chemotherapy or radiotherapy resistance treated with EMC-CM than in controls (p <0.05) [4]. These Fig. 4 EMC increased cell growth in HNSCC cells. SCC-25 (a)and cytokine/chemokine array the most abundant cytokine especially in Detroit 562 (b) cells were treated with conditioned medium of single EMC-CM was interleukin-6 (IL-6), which also supported increased cell tumor cells, single fibroblasts and mix culture of tumor cells and fibro- growth in both SCC-25 and Detroit 562 cells if used for treatment of the blasts (EMC). All conditioned media provided a significantly increased cells at 50 ng/ml. In both panels A and B, the used statistical test was One cell proliferation compared to the controls measured by cell couting, but Way Analysis of Variance (ANOVA) with multiple comparisons of all in SCC-25 cells the highest effect was observed after treatment with treatments to controls using Graphpad Prism 7.00 (n =24) EMC-conditioned medium (EMC-CM; SCC-25-Fibs Mix CM). Using Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond 73 effects of EMC-CM on cisplatin resistance were visualized treatment arms. In contrast, phosphorylation of STAT-3 on with holotomographic microscopy images of IC Cisplatin- Tyrosine 705 was only induced by EMC-CM treatment, treated SCC-25 cells using the NanoLIVE system. If SCC-25 which also achieved a significant induction of Slug protein cells were treated with cisplatin in albumin containing medi- synthesis. um for 72 h the cells died and released their adhesion from the On a second experimental setting the role of IL-6 in the cell culture dish. In contrast, SCC-25 cells treated with the observed effects from EMC-CM was analysed with western IC of cisplatin and EMC-CM, were still alive and metabol- blots. The effect of EMC-CM on STAT3 phosphorylation was ically active. Furthermore, the induction of a large, multinu- compared with the effect of medium conditioned from mono- clear morphology was observed with several mitochondria cultures of SCC-25 or HGF and with albumin medium con- and lipid droplets (Fig. 5). taining 50 ng/ml IL-6. STAT3 phosphorylation was only achieved by EMC-CM or by IL-6. EMC Effects Are Mediated by IL-6-STAT3 Signalling To further elucidate the mechanistic backgrounds of EMC- Discussion CM the IL-6-STAT3 signalling cascade was hypothesized to be involved in the functions of EMC-CM, and it was investi- EMT in tumor progression is nowadays considered as a re- gated in more detail with western blot assays. versible process, which is reversed by its contrary process, SCC-25 cells were either treated with EMC-CM or with mesenchymal to epithelial transition (MET). This phenotypic media conditioned from a mono-culture of SCC-25 cells or plasticity allows tumor cells to adapt to the different require- HGF cells. SCC-25 cells constitutively synthesized STAT3 ments of cancer progression [12]. EMT is closely linked to the and Snail so these signalling factors could be found in all induction of cancer stemness in metastatic disease allowing Fig. 5 EMC rescues cultured HNSCC cells from chemotherapy induced cell death. As published by us before (4) EMC-conditioned media (EMC-CM) doubled the IC value of cisplatin in SCC-25 cells. If SCC-25 cells were treated with cisplatin at IC in albumin con- taining medium for 72 h the cells died and released their adhesion from culture dish (A, B), whereas, the SCC-25 treated with cisplatin at IC in EMC CM were alive, metabolically active and large. Furthermore the initiation of multinuclear morphology was observed with several mitochon- dria and lipid droplets (C, D). A, B, C, D: holotomographic mi- croscopy images of SCC-25 cells using the NanoLIVE system. Scale bars 20 μm Steinbichler T.B. et al. CSC to migrate to different organs. However MET, which epithelial – mesenchymal crosstalk (EMC). The treatment increases proliferative capabilities of cancer cells in return is with EMC-CM induced a hybrid phenotype in naïve SCC- necessary, after CSC have reached their metastatic niche, for 25 cells and also the protein synthesis of EMT-related tran- metastatic colonization of the host organ [6, 12, 33]. This scription factors as Snail or Slug (Fig. 6). Furthermore, this dynamic model of EMT and MET is further supported by hybrid phenotype could be found in the invasive front of tissue the finding of hybrid epithelial-mesenchymal cells which are biopsies from patients with head and neck cancer (Fig. 3). favourable for the establishment of metastasis. These hybrid These findings might be clinical relevant as we were able to phenotypes could be found in circulating tumor cells from demonstrate before that EMC-CM led to chemotherapy and patients with non-small cell lung cancer [34], prostate cancer radiotherapy resistance in HNSCC cells, indicating that this [35] and breast cancer [36–38]. This phenotypic plasticity was hybrid epithelial-mesenchymal phenotype might be involved described as stemness related feature, as these hybrids cells in therapy resistance, which is also described as a key feature were demonstrated to express high levels of CSC markers, too of CSC [4, 5, 40]. [35, 39]. Visualisation of these EMC-induced hybrid cells with In this study a cell culture model of EMTwas established in holotomographic microscopy after exposure to 6.2 μMcis- the mixed culture of epithelial tumor cells and fibroblasts as platin showed that these cells survived cisplatin treatment Fig. 6 EMC-CM transmits induction of EMT through IL-6 - STAT3 actin was used as loading control. 1: cells cultured in albumin- signalling. SCC-25 cells were treated with conditioned media of single containing medium, 2: cells treated with CM of single culture of SCC- culture of SCC-25 cells (A, 1), single culture of HGF fibroblasts (A,2), 25 cells, 3: cells treated with CM of single culture of HGF fibroblasts, 4: mixed EMC culture (A, 3), or were indirect co-cultured with HGF fibro- cells treated with CM of mixed EMC culture, 5: cells treated with blasts (A,4). A) Western blot analysis of treated SCC-25 for STAT3, 50 ng/ml IL-6, 6: commercial available positive control of induced phospho-STAT3, Snail and Slug. GAPDH was used as loading control. STAT3 phosphorylation. STAT3 phosphorylation was achieved by CM The SCC-25 cells constitutively synthesized STAT3 and Snail. STAT-3 of mixed EMC culture or by IL-6. C-D) Normalized optical density was only phosphorylated by mixed EMC-CM treatment, which also quantification of western blots presented in B, C: STAT3,D: phospho- achieved a significant induction of Slug protein synthesis. B) Western STAT3 (n =3) blot analysis of treated SCC-25 cells for STAT3, phospho-STAT3. β- Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond 75 Separation of cell survival, growth, migration, and mesenchymal and a large, multinuclear morphology was induced with sev- transdifferentiation effects of fibroblast secretome on tumor cells of eral mitochondria and lipid droplets. Native SCC-25 in con- head and neck squamous cell carcinoma. Tumour biology : the trast died and released their adhesion from the cell culture dish journal of the International Society for Oncodevelopmental (Fig. 5). Similarly to previous studies these EMC-induced Biology and Medicine 39(11):1010428317705507 3. Chan R, Sethi P, Jyoti A, McGarry R, Upreti M (2016) hybrid cancer cells showed a higher proliferation and conse- Investigating the Radioresistant properties of lung Cancer stem quently tumor-initiating ability (Fig. 4)[41, 42]. cells in the context of the tumor microenvironment. Radiat Res As a possible mediator of these effects we suggested IL-6, 185(2):169–181 as it is the most abundant cytokine in EMC-CM. IL-6 has been 4. Steinbichler TB, Metzler V, Pritz C, Riechelmann H, Dudas J (2016) Tumor-associated fibroblast-conditioned medium induces shown to regulate cancer cell stemness in several studies [43, CDDP resistance in HNSCC cells. Oncotarget. 7(3):2508–2518 44]. 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Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial- EMC-CM induced IL-6 dependent STAT3 phosphorylation of mesenchymal transitions in development and disease. Cell. SCC-25 cells suggesting IL-6 as mediator balancing this 139(5):871–890 EMC-related hybrid phenotype (Fig. 6). 9. Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7(2): 131–142 10. Dudas J, Bitsche M, Schartinger V, Falkeis C, Sprinzl GM, Conclusion Riechelmann H (2011) Fibroblasts produce brain-derived neuro- trophic factor and induce mesenchymal transition of oral tumor cells. Oral Oncol 47(2):98–103 EMC conditioned medium led to the induction of a hybrid 11. Dudas J, Fullar A, Romani A, Pritz C, Kovalszky I, Hans epithelial-mesenchymal cancer cell phenotype, characterised Schartinger V et al (2013) Curcumin targets fibroblast-tumor cell by co-expression of vimentin and cytokeratins. This pheno- interactions in oral squamous cell carcinoma. Exp Cell Res 319(6): type is associated with increased self-renewal, therapy resis- 800–809 12. Steinbichler TB, Dudas J, Riechelmann H, Skvortsova II (2017) tance and migration and can also be found in the invasive front The role of exosomes in cancer metastasis. Semin Cancer Biol 44: of tissue biopsies from head and neck cancer patients. These 170–181 observed effects might be mediated by IL-6-STAT3 13. Steinbichler TB, Dudas J, Skvortsov S, Ganswindt U, Riechelmann signalling. H, Skvortsova II (2018) Therapy resistance mediated by cancer stem cells. Semin Cancer Biol 53:156–167 14. Ebbing EA, van der Zalm AP, Steins A, Creemers A, Hermsen S, Funding Information Open access funding provided by University of Rentenaar R, Klein M, Waasdorp C, Hooijer GKJ, Meijer SL, Innsbruck and Medical University of Innsbruck. Krishnadath KK, Punt CJA, van Berge Henegouwen MI, Gisbertz SS, van Delden OM, Hulshof MCCM, Medema JP, van Laarhoven Compliance with Ethical Standards HWM, Bijlsma MF (2019) Stromal-derived interleukin 6 drives epithelial-to-mesenchymal transition and therapy resistance in Conflict of Interest The authors declare that they have no conflict of esophageal adenocarcinoma. Proc Natl Acad Sci U S A 116(6): interest. 2237–2242 15. Gao J, Zhao S, Halstensen TS (2016) Increased interleukin-6 ex- Open Access This article is distributed under the terms of the Creative pression is associated with poor prognosis and acquired cisplatin Commons Attribution 4.0 International License (http:// resistance in head and neck squamous cell carcinoma. Oncol Rep creativecommons.org/licenses/by/4.0/), which permits unrestricted use, 35(6):3265–3274 distribution, and reproduction in any medium, provided you give appro- 16. 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Published: Jul 11, 2019

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