OR Oncology Reports 1021-335X 1791-2431 D.A. Spandidos 10.3892/or.2016.4581 or-35-04-2431 Articles Spheroid-based 3-dimensional culture models: Gene expression and functionality in head and neck cancer SCHMIDTMARIANNE1 SCHOLZCLAUS-JUERGEN2 POLEDNIKCHRISTINE1 ROLLERJEANETTE1 Department of Otorhinolaryngology, University of Würzburg, D-97080 Würzburg, Germany Interdisciplinary Center for Clinical Research, Microarray Core Unit, University of Würzburg, D-97078 Würzburg, Germany Correspondence to: Dr Marianne Schmidt, Department of Otorhinolaryngology, University of Wuerzburg, Josef-Schneider-Strasse 11, D-97080 Wuerzburg, Germany, E-mail: schmidt_m2@ukw.de 04 2016 21 01 2016 35 4 2431 2440 26 10 2015 05 12 2015 Copyright © 2016, Spandidos Publications 2016

In the present study a panel of 12 head and neck cancer (HNSCC) cell lines were tested for spheroid formation. Since the size and morphology of spheroids is dependent on both cell adhesion and proliferation in the 3-dimensional (3D) context, morphology of HNSCC spheroids was related to expression of E-cadherin and the proliferation marker Ki67. In HNSCC cell lines the formation of tight regular spheroids was dependent on distinct E-cadherin expression levels in monolayer cultures, usually resulting in upregulation following aggregation into 3D structures. Cell lines expressing only low levels of E-cadherin in monolayers produced only loose cell clusters, frequently decreasing E-cadherin expression further upon aggregation. In these cell lines no epidermal growth factor receptor (EGFR) upregulation occurred and proliferation generally decreased in spheroids/aggregates independent of E-cadherin expression. In a second approach a global gene expression analysis of the larynx carcinoma cell line HLaC78 monolayer and the corresponding spheroids was performed. A global upregulation of gene expression in HLaC78 spheroids was related to genes involved in cell adhesion, cell junctions and cytochrome P450-mediated metabolism of xenobiotics. Downregulation was associated with genes controlling cell cycle, DNA-replication and DNA mismatch repair. Analyzing the expression of selected genes of each functional group in monolayer and spheroid cultures of all 12 cell lines revealed evidence for common gene expression shifts in genes controlling cell junctions, cell adhesion, cell cycle and DNA replication as well as genes involved in the cytochrome P450-mediated metabolism of xenobiotics.

 cancer carcinoma head and neck 3D culture in vitro HNSCC E-cadherin microarray proliferation Introduction

According to an analysis of over 3,000 cases of primary head and neck tumours in Germany in 2009, the patient benefit has not significantly improved from 1995 to 2006, despite new treatment strategies. Particularly, the 5-year overall survival rate for carcinomas of hypopharyngeal origin is very low with 27.2% (1). Hypopharyngeal cancer is often detected in later stages, since early signs and symptoms rarely occur. Late stages frequently involve multiple node affection, leading to poor prognosis (http://emedicine.medscape.com/article/1375268).

In vivo cells in a tissue context interact with neighbouring cells and extracellular matrix components. However, in 2-dimensional (2D) monolayer cultures cells tend to loose tissue-specific properties (2). 2D culture models therefore seem unsuitable to mimic the behaviour of cells in a natural, 3-dimensional (3D) microenvironment, particularly for testing of growth or invasion inhibiting substances, just as proposed in 1988 (3). In many aspects spheroids resemble the solid, nonvascularized tumour tissue and spheroidal cells show higher morphological and functional differentiation than monolayer cells. Upon proliferation of cells inside spheroids a tumourlike microenvironment develops with a hypoxic/necrotic core (reviewed in ref. 4), surrounded by quiescent and proliferating zones. Cells inside spheroids secrete extracellular matrix and functional cell junctions arise between cells, providing an advanced model system for examination of growth and invasion of carcinomas (4).

In the present study we compared the morphology and size of 12 HNSCC cell lines with the expression of the proliferation marker Ki67 and the cell-cell adhesion marker E-cadherin. Furthermore, the larynx carcinoma cell line HLaC78 was used as a model for comparing overall gene expression in 2D monolayer cultures with 72 h old HLaC78 multicellular spheroids. The results provide evidence for extensive restructuring of gene expression in the 3D context.

 Materials and methods Cell lines and cell culture

The cell lines used for spheroid analysis are listed in Table I. The cell lines were kept in RPMI-1640, 10% FCS and standard antibiotics, except Hep2 (MEM), Hep2-Tax (MEM+20 μM paclitaxel), SCC4 [Dulbecco's modified Eagle's medium (DMEM/F12)] and HNO 210 (DMEM).

 Spheroid formation

Tumour spheroids were generated by seeding 5,000 cells/well of Hep2 and Hep2-Tax cells in ultra-low attachment (ULA) 96-well round-bottomed plates (Corning, Amsterdam, The Netherlands) (8) or alternatively in ULA T75 flasks. Images were captured after 72 h with a Leica DMI 4000 inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

 Expression micro array RNA extraction and RNA quality control

RNA of HLaC78 monolayer and spheroidal cell cultures was isolated with the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA quality was assessed with the RNA 6000 Nano kit using the Bioanalyzer 2100 instrument (Agilent, Böblingen, Germany). RNA integrity numbers (RINs) of our samples ranged between 9.4 and 9.9.

 Microarray analysis

For microarray hybridization, 100 ng total RNA were amplified and labelled using the IVT Express kit and hybridized to GeneChip PrimeView Human Gene Expression arrays (both from Affymetrix, Santa Clara, CA, USA) according to the manufacturer's instructions.

Raw microarray data was background corrected, normalized and summarized to probe set expression values using the robust microarray average (RMA) [algorithm (9,10)]. Data preprocessing and calculation of log2 fold-changes between spheroid and monolayer expression values was performed in the R environment (http://www.r-project.org) using the Bioconductor (http://www.bioconductor.org) package ʻaffyʼ. Pathway enrichment analyses were performed with gene set enrichment analysis (GSEA) (11). Microarray data were deposited in MIAME-compliant form at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) with the identifier GSE67495.

 Real-time PCR

Gene expression rates were determined by real-time TaqMan® PCR (Applied Biosystems, Foster City, CA, USA). RNA was isolated from cell lines and primary cells with the RNeasy kit according to the manufacturer's instructions. The High Capacity RNA-to-cDNA Master Mix (Applied Biosystems, Darmstadt, Germany) was used for cDNA reverse transcription. Real-time PCR was performed in triplicates on a Real-time PCR cycler (Applied Biosytems, Darmstadt, Germany) using the TaqMan® gene expression assays for E-cadherin (CDH1), N-cadherin (CDH2), Ki67 (MKI), gap junction protein β6 (GJB6), aldo-keto reductase family 1 member C1 (AKR1C1), cytochrome P450 1B1 (CYP1b1), flap endonuclease 1 (FEN1), mutL homolog 1 (MLH1), [replication factor C (activator 1) 3 (RFC3)], and epidermal growth factor receptor 1 (EGFR1).

Relative quantification was calculated according to the 2−ΔΔCt method (12). Expression values were normalized to the expression of GAPDH as an endogenous control which proved to be expressed most stably throughout the cell lines.

 Scanning electron microscopy

For examination of morphology of HLaC78 spheroidal surface SEM was performed. Spheroids were fixed overnight at 4°C with 2% glutaraldehyde in 0.1 M phosphate-buffer. Subsequently, spheroids were treated with 2% osmiumtetroxide in 0.1 M phosphate-saccharose. After dehydration in graded ethanol series (35–100%), specimens were dried using a critical point dryer. Spheroids were then coated with carbon, sputtered with gold palladium particles and viewed with an SEM (DSM 962; Zeiss, Oberkochen, Germany).

 Statistical analysis

All statistical analyses and graphs were performed with GraphPad Prism 4 (GraphPad Software, Inc., La Jolla, CA, USA).

 Results Morphology of spheroids and expression of crucial genes

Cells (5,000) of each cell line (Table I) were seeded into 96 ULA U-bottom well plates. Fig. 1 shows 72 h old spheroids originating from 5,000 cells of each cell line.

Fig. 1 demonstrates the morphological differences between spheroids of different monolayer origin. While HLaC78 and HSmC78 form small and very tight, nearly indestructible spheroids (even by harsh pipetting upon trypsin incubation), FADU, HNO210 and SCC-4 spheroids are larger in diameter, but also round shaped. Cal27 and PE/CA-PJ41 are typically surrounded by a halo of dead/nonaggregated cells. HPaC79, Hep2/Hep2-Tax and HLaC79/HLaC79-Tax cell lines rather aggregate into loose clusters. Spheroid size ranges from ~250 μm for tight, compact spheroids (HLaC78 and HsmC78) till diameters >1 mm (HPaC79).

In general spheroid sizes are both determined by tightness of cell-cell contacts and proliferation rates of the cell lines within the aggregates. Tightness of the spheroids and the proliferation rates within were measured by qRT-PCR expression analysis of E-cadherin (as a marker for adhesion) and Ki67 (as a proliferation marker; Fig. 2) and opposed to expression of these markers in monolayers.

It is noticeable, that tight spheroids show either strong upregulation of E-cadherin upon aggregation (as HLaC78, zHSmC78 Cal27 or Pe/CA-PJ41). or a basic high E-cadherin expression in monolayer cultures with moderate increase/decrease (HNO210 and SSC4) following spheroid formation. Cell lines, agglomerating into intermediate or loose spheroids featured a basically low E-cadherin expression, increasing only weakly or decreasing upon loss of attachment to 2D surfaces (Fig. 2). Proliferation decreased in all cell lines except FADU, most markedly in cell lines forming tight spheroids. Since usually E-cadherin expression causes a decrease in proliferation (13) it is noticeable that the cell lines HPaC79, Hep2 and Hep2-Tax on the one hand decreased E-cadherin expression upon spheroid formation (log fold-changes HPaC79 −1.06, Hep2 −1.62, Hep2-Tax −2.20 and on the other hand also proliferation diminished (log fold-changes Ki67: HPaC79 −4.02, Hep2 −0.48 and Hep2-Tax −1.44; see Table IV). Wang et al (14) described an enhancement of proliferation by downregulation of E-cadherin in HNSCC monolayer cell lines. The enhanced proliferation was mediated by upregulation of EGFR. To check this in the cell lines HPaC79, Hep2 and Hep2-Tax we performed qRT-PCR for EGFR. In these cell lines E-cadherin was downregulated along with EGFR in Hep2 (EGFR log fold-change −0.90) and Hep2-Tax (EGFR log fold-change 1.35) following aggregation. HPaC79 did not express EGFR in monolayer or in the spheroid culture.

 Gene expression

Microarray analysis was used to create a gene expression array profile of head and neck cancer cell line growing as monolayer and in a 3D context. We selected the HLaC78 cell line due to its spontaneous aggregation into tight, well-shaped spheroids with a smooth, tissue-like surface with apical microvilli protrusions typical for epithelia (Fig. 3). A global upregulation of gene expression in HLaC78 larynx carcinoma cells line growing in a tissue-like 3D structure was primarily related to cell adhesion, cell junctions and cytochrome P450-mediated metabolism of xenobiotics. Expression downregulation could be predominantly associated with cell cycle, DNA replication and DNA mismatch repair.

A summary of selected differentially expressed probe sets is shown in Table II, subdivided into functional groups.

To transfer data obtained from HLaC78 expression analysis and to determine key changes in switching between 2D and 3D conditions, the expression of selected genes of each functional group were analyzed by quantitative RT-PCR in monolayer and spheroid cultures of 12 different HNSCC cell lines.

Genes selected for TaqMan® qRT-PCR expression quantification in monolayer and spheroid cultures of HNSCC cell lines are summarized in Table III, log fold-changes for spheroids of each cell line in comparison with monolayer culture are shown in Table IV.

Common expression shifts occurred in cell lines forming tight spheroids under 3D conditions. Thus, expression of cell junction and cell adhesion genes, as well as cytochrome P450 detoxification genes increased (GJB6, E-cadherin, AKR1C1, Cyp1a1 and 1b1), while expression of genes important for proliferation and DNA-replication (MKI and RCF3) decreased. In the DNA mismatch repair group no common tendency of expression shift was visibleX (at least for the tested FEN1/MLH1 genes). In cell lines, forming only loose aggregates, no upregulation of GJB6 or E-cadherin occurred under 3-dimensional conditions. Cell proliferation and DNA replication genes, however, were downregulated independent of spheroid compaction in the 3D context. Notably, the tested DNA mismatch repair genes were uniformly downregulated in cell lines aggregating loosely.

 Discussion

The present study provides an overview of spheroid formation properties of 12 HNSCC cell lines. Spheroids differed enormously in size and tightness. The cell lines were divided in those forming tight spheroids or intermediate/loose aggregates. Since the size of spheroids is in general dictated by cell adhesion and proliferation rates within spheroids, expression profiles of Ki67 (proliferation) and E-cadherin (adhesion) were generated. For generation of tight, regular spheroids a distinct level of E-cadherin expression in monolayers was necessary and underwent a further significant upregulation upon aggregation. Alternatively, this further significant upregulation seemed to be expendable, when E-cadherin expression in monolayer cell lines was exceptionally high as in HNO210 or SCC4 cell lines, respectively. The cell lines expressing only low levels of E-cadherin under two-dimensional (2D) conditions were not able to gain a sufficient increase in E-cadherin expression to generate compact spheroidal tissue-like aggregates. Most of these cell lines even decreased E-cadherin expression under non-adherent conditions. Proliferation, however, decreased in all cell lines (except FADU) when the attachment to cell culture surfaces was suspended and 3-dimensional growth was initiated. The observation that spheroid tightness in HNSCC is partially determined by the expression of E-cadherin in monolayer culture is in agreement with the observation in other systems (15). In general upregulation of E-cadherin causes a β-catenin-ediated contact inhibition and subsequent decrease in proliferation (13). Furthermore, it has been shown that knock-down of E-cadherin enhances the proliferation of HNSCC cell lines by upregulating epidermal growth factor (EGFR) receptor. Despite the usual pathway of E-cadherinmediated contact inhibition, in the cell lines HPaC78, Hep2 and Hep2-Tax, all forming only loose aggregates, E-cadherin was downregulated, but yet proliferation decreased. Obviously pathways initiated in monolayer cell cultures are not uniformly conferrable to the 3D context.

A comparison of overall gene expression between the monolayer culture of the human larynx carcinoma cell line HLaC78 and the corresponding 72 h old spheroids was performed. HLaC78 cells form tight spheroids spontaneously with a smooth, ciliated surface. Gene expression changed towards more tissue-like features. Changes in overall gene expression was organized in main functional groups, concerning cell junctions, cell adhesion, cytochrome P450 metabolism, cell cycle, DNA mismatch repair and DNA replication. While cell adhesion and cell junction gene expression increased, DNA replication/cell cycle genes as well as base excision DNA repair genes expression decreased in HLaC78 cell line. These principal observations roughly conform with a study on prostate cancer in 2007 (16), with respect to cell cycling, cell adhesion and DNA repair genes. In contrast, no angiogenesis genes were upregulated in HLaC78 upon spheroid aggregation, and vascular endothelial growth factor was slightly downregulated. It has to be considered, however, that in older spheroids an upregulation of angiogenesis genes may also occur, when central hypoxia increases (in the present study HIF-1 was upregulated only non-significantly after 72 h). To transfer data obtained from HLaC78 expression analysis line and to determine key changes in switching between 2D and 3D conditions, the expression of selected genes of each functional group were analyzed by quantitative RT-PCR in monolayer and spheroid cultures of different HNSCC cell lines.

The decrease in cell cycling/DNA synthesis seemed to be a common mechanism also in HNSCC cell lines, as shown by the expression analysis of Ki67 and RCF3. Cell junction and adhesion gene upregulation, however, was restricted to those cell lines with distinct E-cadherin expression already under two-dimensional conditions. These cell lines usually experienced a further upregulation of E-cadherin upon spheroid aggregation. Cell lines with low E-cadherin levels in monolayer cultures, however, further decreased expression under 3D conditions. This is somewhat divergent from the findings of Kim et al (17), who unveiled E-cadherin directly regulating the hippo signaling pathway controlling cell proliferation. E-cadherin mediates contact inhibition, which is essential for tissue maintenance. In the cell lines producing only loose aggregates, proliferation decreased despite downregulation of E-cadherin, indicating further pathways, regulating proliferation under tissue-like conditions.

Upregulation of cytochrome P450 metabolism genes has been solely reported several times for hepatocellular carcinoma cell lines. Thus, overall gene expression was analysed in HepG2 liver carcinoma monolayer cells and corresponding spheroids (18). The authors reported upregulation of cytochrome p450 1A1 (CYP1A1) as well as aldo-keto reductase C1 (AKR1C1), both factors involved in cytochrome P450-mediated metabolism of xenobiotics in spheroids. Hsiao et al (19) showed that CYP1A1 and CYP1B1 activity increased in hepatocytes with assembly of spheroids and decreased with disassembly of spheroids on collagen coated surfaces.

Notably, Masood et al (20) reported downregulation of CYP1A1 mRNA in head and neck cancer specimens compared to normal mucosal tissue. Upregulation of the cytochrome P450 enzymes or AKR1A1 in spheroids may therefore reflect a regression to a more regulated cell type in a tissue-like context.

Francia et al (21) performed a differential display analysis of monolayers and spheroids of EMT-6 mammary carcinoma cells and compared differentially expressed genes with EMT-6 tumours selected in vivo to be resistant to alkylating agents. They identified the DNA mismatch repair-associated mismatch repair endonuclease 2 (PMS2) gene to be associated with resistance. Downregulation of PMS2 caused a multicellular resistance of tumour cell spheroids against alkylating agents. They hypothesized, that a deficiency in DNA mismatch repair may explain some forms of multicellular resistance, particularly to alkylating agents.

In HLaC78 genes of the functional group, DNA mismatch repair was significantly downregulated. We have not tested resistance of monolayers and spheroids of our cell lines against alkylating agents. Furthermore, it seems, that downregulation of DNA mismatch-repair genes is not a general mechanism of HNSCC cancer cell lines upon aggregation into spheroids, but it is notable, that the tested genes where more commonly downregulated in those cell lines forming loose clusters instead of tight spheroids.

Summarizing our results, it seems that in head and neck cancer cell lines (HNSCC) the 3D context reverses monolayer cells to growth and behavior of cells in a more tissue-like controlled condition with enhanced cell-cell adhesion and decreased proliferation. The formation of tight regular spheroids is dependent on distinct E-cadherin expression levels in monolayer cultures, usually resulting in further upregulation upon aggregation into 3D structures. Cell lines expressing only low levels of E-cadherin produce only loose cell clusters, frequently decreasing E-cadherin expression further. These cell lines diminish proliferation despite decreased E-cadherin. Common shifts in gene expression between 2D and 3D growth of HNSCC cell lines include the functional groups cell junctions, cell adhesion, cell cycle, DNA replication as well as genes concerning the metabolism of xenobiotics.

 Acknowledgments

We would like to thank Dr Piet Tas (PhD) for critical reading of the manuscript.

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Spheroids of 10 different HNSCC cell lines established from 5,000 cells in ULA 96-well plates with conical bottom 72 h after seeding. Spheroids were grouped according to tightness.

Expression of E-cadherin and Ki67 in monolayer cultures compared to spheroids of 12 different HNSCC cell line. For adequate presentation of E-cadherin cell lines were divided into groups expressing high or low levels of E-cadherin in monolayer cultures.

Scanning electron microscopy (SEM) display of a typical HLaC78 spheroid surface. Bar, 1 μm.

HNSCC cell lines used in the present study.

Cell line Origin
FADU Hypopharynx carcinoma, LGC standards (Wesel, Germany)
HLaC78 Larynx carcinoma (5)
Hep-2 Larynx carcinomaa, LGC standards (Wesel, Germany)
Hep-2-Tax Larynx carcinoma, paclitaxel-resistant clone
HLaC79 Larynx carcinoma (5)
HLaC79-Tax Larynx carcinoma, paclitaxel-resistant clone
HPaC79 Parotis carcinoma (6)
HSmC78 Submandibular gland (5)
Cal27 Tongue, LGC standards (Wesel, Germany)
PE/CA-PJ41 Tongue, Sigma-Aldrich (Taufkirchen, Germany) (7)
SCC4 Tongue, LGC standards (Wesel, Germany)
HNO210 Larynx carcinoma, CLS (Eppelheim, Germany)

Supposed to be HeLa contaminated, http://www.lgcstandards-atcc.org/products/all/CCL-23. HNSCC, head and neck cancer.

Log fold-changes in gene expression in spheroids vs. monolayer cultures of HLaC78 cell line.

Gene symbol Gene logFC
Cell junctions
GJB6 Gap junction protein, β6, 30 kDa 4,588
CDH2 Cadherin 2, type 1, N-cadherin (neuronal) −3,963
GJB2 Gap junction protein, β2, 26 kDa 3,547
CLDN10 Claudin 10 3,364
DSC2 Desmocollin 2 3,254
DSG3 Desmoglein 3 2,727
NOTCH3 Notch 3 2,723
GJA1 Gap junction protein, α1, 43 kDa 2,488
DSC3 Desmocollin 3 2,238
CLDN7 Claudin 7 2,222
NOTCH1 Notch 1 1,991
F11R F11 receptor 1,887
CLDN8 Claudin 8 1,878
CAV1 Caveolin 1, caveolae protein, 22 kDa −1,730
JUP Junction plakoglobin 1,657
CDH1 Cadherin 1, type 1, E-cadherin (epithelial) 1,589
ITGB1 Integrin, β1 (fibronectin receptor) −1,195
DSP Desmoplakin 1,181
ITGB6 Integrin, β6 1,099
GJB5 Gap junction protein, β5, 31.1 kDa 1,041
Cell adhesion
HLA-DRA Major histocompatibility complex, class II, DRα 7,514
HLA-DRB1 Major histocompatibility complex, class II, DRβ1 4,842
HLA-DMA Major histocompatibility complex, class II, DMα 4,692
HLA-DPA1 Major histocompatibility complex, class II, DPα1 4,391
HLA-F Major histocompatibility complex, class I, F 4,050
HLA-DMB Major histocompatibility complex, class II, Dβ 3,924
HLA-DRB5 Major histocompatibility complex, class II, DRβ5 3,864
HLA-DPB1 Major histocompatibility complex, class II, DPβ1 3,464
CLDN10 Claudin 10 3,364
HLA-B Major histocompatibility complex, class I, B 2,965
HLA-C Major histocompatibility complex, class I, C 2,618
HLA-DQB1 Major histocompatibility complex, class II, DQβ1 2,477
HLA-E Major histocompatibility complex, class I, E 2,468
CLDN7 Claudin 7 2,222
PTPRM Protein tyrosine phosphatase, receptor type, M 2,192
CD58 CD58 molecule 2,076
HLA-A Major histocompatibility complex, class I, A 2,058
PVRL1 Poliovirus receptor-related 1 (herpesvirus entry mediator C) 2,016
F11R F11 receptor 1,887
CLDN8 Claudin 8 1,878
CDH1 Cadherin 1, type 1, E-cadherin (epithelial) 1,589
CDH3 Cadherin 3, type 1, P-cadherin (placental) 1,566
NEO1 Neogenin 1 1,518
CD86 CD86 molecule 1,424
ITGB8 Integrin, β8 1,370
GLG1 Golgi glycoprotein 1 1,368
CNTN1 Contactin 1 1,366
CD40 CD40 molecule, TNF receptor superfamily member 5 1,359
SDC1 Syndecan 1 1,340
SDC2 Syndecan 2 1,265
PTPRF Protein tyrosine phosphatase, receptor type, F 1,159
CD276 CD276 molecule 1,100
Cytochrome P450 metabolism
AKR1C1 Aldo-keto reductase family 1, member C1 3,798
AKR1C3 Aldo-keto reductase family 1, member C3 3,364
CYP1B1 Cytochrome P450, family 1, subfamily B, polypeptide 1 2,837
CYP1A1 Cytochrome P450, family 1, subfamily A, polypeptide 1 2,327
GSTA4 Glutathione S-transferase α4 2,299
ALDH3B2 Aldehyde dehydrogenase 3 family, member B2 2,039
MGST2 Microsomal glutathione S-transferase 2 1,196
GSTK1 Glutathione S-transferase κ1 1,131
GSTZ1 Glutathione S-transferase ζ1 1,112
Cell cycle
CDKN3 Cyclin-dependent kinase inhibitor 3 −5,795
CDK1 Cyclin-dependent kinase 1 −5,556
BIRC5 Baculoviral IAP repeat containing 5 −5,257
CCNA2 Cyclin A2 −5,205
CDC20 Cell division cycle 20 homolog (S. cerevisiae) −5,19
CDC6 Cell division cycle 6 homolog (S. cerevisiae) −5,104
MAD2L1 MAD2 mitotic arrest deficient-like 1 (yeast) −4,665
CCNB1 Cyclin B1 −4,573
CDC25A Cell division cycle 25 homolog A (S. pombe) −4,500
AURKB Aurora kinase B −4,439
CCNB2 Cyclin B2 −4,387
MKI67 Antigen identified by monoclonal antibody Ki67 −4,028
BRCA1 Breast cancer 1, early onset −3,798
RAD51 RAD51 homolog (S. cerevisiae) −3,543
KNTC1 Kinetochore associated 1 −3,023
MCM3 Minichromosome maintenance complex component 3 −2,973
CKS2 CDC28 protein kinase regulatory subunit 2 −2,919
STMN1 Stathmin 1 −2,783
AURKA Aurora kinase A −2,778
GTSE1 G-2 and S-phase expressed 1 −2,719
CHEK1 Checkpoint kinase 1 −2,687
CKS1B CDC28 protein kinase regulatory subunit 1B −2,636
CCND2 Cyclin D2 −2,488
TFDP1 Transcription factor Dp-1 −2,242
BCCIP BRCA2 and CDKN1A interacting protein −2,239
TFDP1 Transcription factor Dp-1 −2,219
BRCA2 Breast cancer 2, early onset −2,147
CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) 2,116
CCNF Cyclin F −2,103
CDK2 Cyclin-dependent kinase 2 −1,949
RBL1 Retinoblastoma-like 1 (p107) −1,905
SKP2 S-phase kinase-associated protein 2, E3 ubiquitin protein ligase −1,580
MDM2 Mdm2, p53 E3 ubiquitin protein ligase homolog (mouse) 1,454
RAD1 RAD1 homolog (S. pombe) −1,416
CCNE1 Cyclin E1 −1,379
CCNC Cyclin C −1,336
MAD2L2 MAD2 mitotic arrest deficient-like 2 (yeast) −1,263
NBN Nibrin −1,249
CDK4 Cyclin-dependent kinase 4 −1,245
RB1 Retinoblastoma 1 −1,219
MCM4 Minichromosome maintenance complex component 4 −1,203
STMN1 Stathmin 1 −1,193
CDK5R1 Cyclin-dependent kinase 5, regulatory subunit 1 (p35) −1,177
CCNG2 Cyclin G2 1,102
CDK6 Cyclin-dependent kinase 6 −1,094
BCCIP BRCA2 and CDKN1A interacting protein −1,085
CCND1 Cyclin D1 −1,064
DNA mismatch repair
FEN1 Flap structure-specific endonuclease 1 −2,869
NEIL3 Nei endonuclease VIII-like 3 (E. coli) −2,787
POLE2 Polymerase (DNA directed), ε2, accessory subunit −2,760
PCNA Proliferating cell nuclear antigen −2,279
HMGB1 High mobility group box 1 −2,076
PARP1 Poly(ADP-ribose) polymerase 1 −1,931
LIG1 Ligase I, DNA, ATP-dependent −1,724
POLE Polymerase (DNA directed), ε, catalytic subunit −1,621
POLD3 Polymerase (DNA-directed), δ3, accessory subunit −1,387
MBD4 Methyl-CpG binding domain protein 4 −1,378
MLH1 mutL homolog 1, colon cancer, non-polyposis type 2 (E. coli) −1,369
POLD2 Polymerase (DNA directed), δ2, accessory subunit −1,248
POLD1 Polymerase (DNA directed), δ1, catalytic subunit −1,182
NEIL2 Nei endonuclease VIII-like 2 (E. coli) −1,143
UNG Uracil-DNA glycosylase −1,132
DNA replication
RFC3 Replication factor C (activator 1) 3, 38 kDa −3,710
POLA2 Polymerase (DNA directed), α2, accessory subunit −3,076
MCM3 Minichromosome maintenance complex component 3 −2,973
PRIM1 Primase, DNA, polypeptide 1 (49 kDa) −2,948
FEN1 Flap structure-specific endonuclease 1 −2,869
MCM6 Minichromosome maintenance complex component 6 −2,859
MCM7 Minichromosome maintenance complex component 7 −2,853
POLE2 Polymerase (DNA directed), ε2, accessory subunit −2,760
MCM4 Minichromosome maintenance complex component 4 −2,616
DNA2 DNA replication helicase/nuclease 2 −2,429
RNASEH2A Ribonuclease H2, subunit A −2,381
MCM2 Minichromosome maintenance complex component 2 −2,344
PCNA Proliferating cell nuclear antigen −2,279
RFC5 Replication factor C (activator 1) 5, 36.5 kDa −2,227
RFC4 Replication factor C (activator 1) 4, 37 kDa −2,086
RFC2 Replication factor C (activator 1) 2, 40 kDa −1,798
LIG1 Ligase I, DNA, ATP-dependent −1,724
POLA1 Polymerase (DNA directed), α1, catalytic subunit −1,671
MCM5 Minichromosome maintenance complex component 5 −1,622
POLE Polymerase (DNA directed), ε, catalytic subunit −1,621
POLD3 Polymerase (DNA-directed), δ3, accessory subunit −1,387
RPA3 Replication protein A3, 14 kDa −1,368
RNASEH2B Ribonuclease H2, subunit B −1,260
POLD2 Polymerase (DNA directed), δ2, accessory subunit −1,248
RFC1 Replication factor C (activator 1) 1, 145 kDa −1,235
PRIM2 Primase, DNA, polypeptide 2 (58 kDa) −1,185
POLD1 Polymerase (DNA directed), δ1, catalytic subunit −1,182
POLE3 Polymerase (DNA directed), ε3, accessory subunit −1,123

Genes tested for expression in monolayer and spheroid cultures of different HNSCC cell lines.

Functional group Gene
Cell junctions GJB6, gap junction protein β6
Cell adhesion CDH1, E-cadherin
Cytochrome P450 metabolism AKR1C1, aldo-keto family 1 reductase member
CYP1B1, cytochrome P450 1B1
CYP1A1, cytochrome P450 1A1
Cell cycle MKI, Ki67 proliferative antigen
DNA mismatch repair FEN1, flap endonuclease 1
DNA replication RFC3, replication factor C3

HNSCC, head and neck cancer.

Log fold-changes in gene expression of selected genes in spheroids vs. monolayer cultures of HNSCC cell lines.

GJB6 CDH1 AKR1C1 CYP1A1 CYP1B1 MKI FEN1 MLH1 RCF3
Tight spheroids
HLaC78 3.06 2.17 4.64 2.83 1.86 −5.55 −1.42 −0.37 −3.30
HSmC78 5.93 3.04 6.58 5.02 5.23 −3.71 0.73 0.81 −0.95
Cal27 5.05 1.81 4.04 4.69 2.55 −1.17 −0.22 1.34 −0.61
PE/CA-PJ41 0.49 1.35 2.76 6.78 4.88 −4.35 1.51 −0.04 −0.65
HNO210 5.75 −0.44 5.99 1.49 0.12 −6.48 3.31 0.40 −2.65
SCC4 0.59 0.79 0.15 5.71 0.75 −2.41 −0.92 0.91 −7.64
Loose/intermediate aggregates
FADU −0.68 0.06 0.04 0.45 0.79 0.43 −0.38 −0.52 −0.22
Hep2 −2.52 −1.62 −0.77 1.21 −2.21 −0.48 −1.85 −0.68 −1.57
Hep2-Tax −2.23 −2.20 2.36 1.26 0.07 −1.44 −1.71 −3.17 −0.65
HPaC79 −1.16 −1.06 −1.27 −1.47 −3.89 −4.02 −1.56 −1.40 −5.58
HLaC79 −0.29 1.04 0.90 0.11 0.15 −0.13 −0.34 −1.09 −0.55
HLaC79-Tax −5.22 −3.24 −7.64 −2.55 −0.99 −0.01 −5.95 −0.28 −0.68

HNSCC, head and neck cancer.