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. 2016 Sep 19;11(9):e0163149.
doi: 10.1371/journal.pone.0163149. eCollection 2016.

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

Diana Braunholz  1   2 , Mohammad Saki  1   2 , Franziska Niehr  1   2 , Merve Öztürk  1 , Berta Borràs Puértolas  1 , Robert Konschak  1   2 , Volker Budach  1 , Ingeborg Tinhofer  1   2
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Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival

"V体育官网入口" Diana Braunholz et al. PLoS One. .

"V体育ios版" Abstract

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 VSports手机版. 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 UT-SCC-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. .

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Conflict of interest statement (VSports注册入口)

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Spheroid generation is cell line dependent.
(A) 2,500 cells were seeded into 96-well plates (poly-HEMA coated). After 96h, total number of spheroids per well were counted. (B) 2 ×104cells/well were plated into 12-well culture plates and at indicated times cells were counted. The data points shown in (A) and (B) represent the mean values ± standard deviation (SD) of three independent experiments. (C) Cells were lysed, and protein samples were subjected to western blot analysis with specific antibodies against EGFR. GAPDH was used as a loading control.
Fig 2
Fig 2. Autocrine EGFR signalling is maintained in spheroid culture of HNSCC cell lines.
Cells were cultured as monolayer (M) or spheroids (FS) for 24h, 48h or 72h in serum-free medium. The expression levels of pERK1/2 (42/44 kDa) and vinculin (internal loading control; 124 kDa) were analysed by immunoblotting. The values of the quantitative analysis are depicted as pERK1/2 protein levels relative to vinculin expression levels.
Fig 3
Fig 3. Effects of EGFR activation/blockade on spheroid formation.
Cells were seeded at a density of 300,000 cells/well into 6-well plates and cultured under non-adherent conditions in the absence or presence of EGFR ligands (AREG, EGF) or EGFR blocking agents (CTX, gefitinib). Pictures were taken 72h after cell seeding. (5x magnification)
Fig 4
Fig 4. Effects of EGFR activation/blockade on spheroid volume.
Cells were seeded at a density of 300,000 cells/well into poly-HEMA coated 6-well plates and treated with the indicated agents. After 72h, the diameters of at least 15 spheroids were measured and spheroid volumes calculated relative to the untreated spheroids. Bars represent mean values ± SD of four independent experiments. For the cell line SCC-9 the y-axis was adapted due to the EGF-induced strong increase in spheroid volumes.
Fig 5
Fig 5. Activation of EGFR protects HNSCC cells against anoikis.
Cells were seeded at a density of 300,000 cells/well in non-coated or poly-HEMA coated 6-well plates and were immediately treated with either EGFR ligands or blocking reagents. After 72h, cells from monolayer (M) and forced suspension (FS) cultures were harvested and the apoptotic fraction was determined by PI staining and subsequent flow cytometry. Bars represent the mean percentages of apoptotic cells ± SD of at least three independent experiments. Significant changes compared to control are marked with an asterisk.
Fig 6
Fig 6. CTX affects proliferation of spheroids in sensitive cell lines.
MTT assays were performed in 96-well plates with cells treated with EGFR stimulating or blocking reagents in monolayer (M) or forced suspension (FS) culture. Bars represent mean percentages ± SD of four independent experiments. Significant changes compared to control are marked with an asterisk.
Fig 7
Fig 7. EGFR signalling regulates clonogenic survival of HNSCC cells derived from forced suspension cultures.
Cells were cultured as monolayer (M) or in forced suspension (FS) in the absence or presence of EGF, AREG or CTX. 72h later, cells were harvested, disaggregated to single cells and subjected to clonogenic survival analysis. Bars represent the surviving fractions (SF) ± SD from three independent experiments. Significant changes compared to control are marked with an asterisk.
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