Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival

RESEARCH ARTICLE Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival Diana Braunholz1,2*, Mohammad Saki1,2, Franziska Niehr1,2, Merve Öztürk1, Berta Borràs Puértolas1, Robert Konschak1,2, Volker Budach1, Ingeborg Tinhofer1,2 a11111 1 Translational Radiooncology and Radiobiology Research Laboratory, Department of Radiooncology and Radiotherapy, Charité University Hospital Berlin, Berlin, Germany, 2 German Cancer Consortium (DKTK), Deutsches Krebsforschungszentrum (DKFZ), Partner Site Berlin, Charité University Hospital Berlin, Berlin, Germany * Abstract OPEN ACCESS Citation: Braunholz D, Saki M, Niehr F, Öztürk M, Borràs Puértolas B, Konschak R, et al. (2016) Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival. PLoS ONE 11(9): e0163149. doi:10.1371/journal.pone.0163149 Editor: Min-Hsien Wu, Chang Gung University, TAIWAN Received: March 14, 2016 Accepted: September 2, 2016 Published: September 19, 2016 Copyright: © 2016 Braunholz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist. In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UTSCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti-metastatic treatment approach in HNSCC. Abbreviations: AREG, amphiregulin; CSC, cancer stem cells; CTCs, circulating tumour cells; CTM, circulating microemboli; CTX, cetuximab; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FS, forced suspension; HNSCC, PLOS ONE | DOI:10.1371/journal.pone.0163149 September 19, 2016 1 / 14 In Vitro Analysis of EGFR Signalling in Anchorage Independent Survival head and neck squamous cell carcinoma; M, monolayer; MTT, Methylthiazolyldiphenyl-tetrazolium bromide; NSCLC, non-small cell lung cancer; pEGFR, phospho-EGFR; PI, Propidium iodid; polyHEMA, Poly(2-hydroxyethyl methacrylate); SCLC, small cell lung cancer; SD, standard deviation. Introduction Each day millions of tumour cells are shed into the blood circulation from solid tumours [1]. Of these cells, only a small subpopulation is able to survive and demonstrates tumour-inducing potential enabling metastastic progression [2,3]. Circulating tumour cells (CTCs) have been detected in peripheral blood of patients in most epithelial tumour types and were significantly associated with poor prognosis [4–9]. Previous findings revealed the existence of socalled CTC clusters or circulating microembolis (CTM) which display an increased metastatic potential compared to solitary CTCs [10,11]. In agreement with this, in vivo spheroids were shown to be exclusively detectable in blood from patients with metastatic disease in various histological entities indicative of their role in tumour progression and metastasis [12]. CTC clusters can be built from CTCs alone or are mixed with accessory cells including leukocytes, platelets, endothelial cells or fibroblasts [13–15]. In contrast to solitary CTCs, these CTC aggregates (e.g.  3 CTCs in advanced NSLCLC) [16] were shown to have an advantage in the blood circulation in terms of protection from an immune attack and anoikis (apoptosis resulting from loss of cell–cell and cell–matrix contact) [14,17]. Identification of the molecular mechanisms underlying the CTC cluster formation ability and their maintenance in the blood circulation may lead to a better understanding of the mechanisms involved in the metastatic potential of CTCs and might identify novel therapeutic targets for anti-metastatic treatment. In the seminal study of Jost and coworkers, EGFR activation was identified as key factor for anchorage-independent cell survival of primary and immortalized human keratinocytes [18]. Subsequent studies demonstrated this function of EGFR in different epithelial tumour models as well [19–21]. EGFR is overexpressed in many tumours of epithelial origin including HNSCC showing upregulated expression in about 90% of patients [22]. Increased levels of EGFR expression and activation have been associated with poor prognosis, distant metastasis, and therapy resistance [23]. We have previously shown in a breast xenograft model that EGFR as well as mesenchymal markers are upregulated in the CTC fraction [24]. Additionally, in HNSCC patients with locally advanced disease, we have detected EGFR in the total fraction of CTCs and its phosphorylated form in more than 50% of CTCs [25]. However, the causative role of EGFR and its downstream signalling pathway for anchorage-independent cell survival of CTCs in HNSCC remains unresolved. Previous studies established the forced suspension culture as a near-physiological in-vitro mo (...truncated)