ANTICANCER RESEARCH 35: 3253-3266 (2015) Differential Gene Regulation in Fibroblasts in Co-culture with Keratinocytes and Head and Neck SCC Cells MALIN HAKELIUS1, DANIEL SAIEPOUR1, HANNA GÖRANSSON2, KRISTOFER RUBIN3, BENGT GERDIN1 and DANIEL NOWINSKI1 Departments of 1Surgical Sciences, Plastic Surgery and 3Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden; 2Array Facility, Department of Medical Sciences, Uppsala University, Uppsala, Sweden Abstract. Background: While carcinoma-associated growth. In cancers, this microenvironment, or tumor stroma, fibroblasts (CAFs) support tumorigenesis, normal tissue constitutes the backbone of the tumor and is essential for the fibroblasts suppress tumor progression. Mechanisms behind cohesiveness of the tumor tissue the tumor’s ability to thrive conversion of fibroblasts into a CAF phenotype are largely (1). This stroma has considerable similarities with that of unrevealed. Materials and Methods: Transwell co-cultures non-malignant repair processes that are characterized by with fibroblasts in collagen gels and squamous-cell activation of fibroblasts and neoformation of stromal tissue, carcinoma (SCC) cells or normal oral keratinocytes (NOKs) which has led to the concept of a tumor as a "wound that in inserts. Differences in fibroblast global gene expression never heals" (2). were analyzed using Affymetrix arrays and subsequent A fibroblast phenotype characterized by expression of functional annotation and cluster analysis, as well as gene alpha-smooth muscle actin (SMA), platelet-derived growth set enrichment analysis were performed. Results: There were factor receptor-beta (PDGFR-β) and the pericyte marker 52 up-regulated and 30 down-regulated transcript IDs neuron glial antigen 2 (NG2) is regarded as a key cell in the (>2-fold, p<0.05) in fibroblasts co-cultured with SCC tumor stroma (3). Accordingly, this phenotype has been compared to NOKs. Functional analysis demonstrated an denoted “cancer-associated fibroblast”, CAF. It has been enrichment of collagen-related genes. There were similarities proposed that the CAF phenotype is the result of a reciprocal with gene sets reflecting a non-specific, innate–type response signaling between tumor cells and stromal fibroblasts (3). with activation of both interferon pathways and connective However, the nature of such signaling is only partially tissue turnover. Conclusion: There were distinct differences studied as there exist differences in signaling between benign in fibroblast gene expression between the co-culture types. proliferating keratinocytes and stromal cells in a healing Many were in genes related to an innate-type of response wound and the corresponding process in malignant tissue. and to connective tissue turnover. Two phenomena are worth mentioning. First, keratinocytes from the wound edge transiently exhibit many similarities to Tumors arise as consequences of genetic alterations in their transformed counterparts in squamous-cell carcinomas normal cells, which -in turn- result in a loss of cellular (SCCs) (4). Second, the stroma of a healing wound also growth control. In addition, tumor progression requires an contains cells with characteristics similar to those of the aberrant microenvironment that supports further tumor CAFs. Thus, similarities, as well as differences, in the interaction between the malignant keratinocyte phenotype and stromal cells, compared to the interaction between Abbreviations: CAF: Carcinoma associated fibroblasts; NOKs: benign keratinocytes and stromal cells, may be expected. normal oral keratinocytes; SCC: squamous cell carcinoma. The origin of the CAFs has not been established with certainty. Current information suggests that CAFs may be Correspondence to: Daniel Nowinski, MD, Ph.D., Department of derived from a variety of sources. Three main hypotheses Plastic and Maxillofacial Surgery, Uppsala University Hospital, 751 have been proposed. First, it has been suggested that CAFs 85 Uppsala, Sweden. Tel: +46 738664625, e-mail: daniel. [email protected] are recruited locally from resident cells in the vicinity of a growing and spreading tumor, including normal tissue Key Words: Head and neck cancer, keratinocytes, differential gene fibroblasts and tissue mesenchymal stem cells (MSCs). regulation, array. Second, bone marrow-derived cells with an ability to 0250-7005/2015 $2.00+.40 3253 ANTICANCER RESEARCH 35: 3253-3266 (2015) differentiate into fibroblast phenotypes have been shown to Co-culture. Fibroblasts were cultured in collagen gels. Briefly, a migrate to areas of proliferating tumor cells. Finally, other cold solution of 1.6 ml collagen type I (Vitrogen, Cohesion, Palo cell types, e.g. pericytes, myoepithelial cells and endothelial Alto, CA, USA), 0.15 ml (10×) Hank’s balanced salt solution (10xHBSS) and 0.15 ml fetal bovine serum (FBS) with or without cells, are thought to transdifferentiate towards a tumor fibroblasts (2×105/well) was pH-adjusted to pH 7.4 with 5 M NaOH fibroblast-like population (3). and added to 6-well plates. After polymerization at 37˚C, 2 ml Using co-culture systems we have previously shown that DMEM with 10% bovine calf serum (HyClone) was added to each normal keratinocytes from both skin and the oral cavity well. Normal oral keratinocytes or malignant keratinocytes (UT- decrease profibrotic characteristics of fibroblasts through SCC-87), in passage 16 or 17, were seeded in Falcon polyurethane humoral mechanisms, which -at least in part- involve release cell culture inserts (4.0 μm pore diameter). The inserts were pre- of interleukin-1α (IL-1α) from the epithelial cells (5-7). incubated with a mixture of 10 μg/ml bovine plasma fibronectin (Gibco BRL/Life Technology, Paisley, UK), 30 μg/ml bovine Furthermore, this down-regulation was more pronounced in collagen (Vitrogen) and 10 μg/ml bovine serum albumin (Sigma) co-cultures with normal oral keratinocytes (NOKs) than with for 2 h at 37˚C. NOKs and malignant oral keratinocytes were seeded oral SSC cells (5). This observation forms the basis for the in inserts, both in DMEM: HAM’s F12 (4:1) supplemented with present study, which investigates the differential effect of 5 μg/ml Zn-free insulin (Sigma Chemical Co.), 2 nM 3,3’,5-triido- NOKs and SCC on overall fibroblast gene expression D-thyronine (Sigma), 0.4 μg/ml hydrocortisone (Sigma), 0.1 nM through humoral communication, utilizing a conventional cholera toxin (Sigma), 10 ng/ml EGF (Austral Biologicals, San array approach. The aim of the study was to reveal to what Ramon, CA, USA), 24 μg/ml adenine, 10% fetal bovine serum (HyClone) and 50 μg/ml gentamicin. After 24 h, the medium was extent there exist differences in overall gene expression at a changed in wells and in inserts to 2 ml DMEM/Ham’s F12 (4:1) cluster level that reflects qualitative differences in fibroblast supplemented with 0.5% FCS in each. Inserts and wells with cell response after a humoral interaction with benign versus collagen gels were combined and propagated as co-cultures for an malignant epithelial cells. An additional aim is to determine additional 48 h. As controls, 0.15×106 fibroblasts were seeded in whether such differences exhibit similarities with publicly inserts instead of keratinocytes. Experiments were performed with presented gene expression profiles or molecular signatures. different seeding concentrations of NOKs and malignant keratinocytes and the number of cells was assessed with a cell- Materials and Methods counter (Z2 Cell and Particle Counter; Beckman Coulter AB, City, CA, USA) after 48 h. The reason for the different seeding-numbers for normal and malignant cells was to compensate for an observed Cells. A head and neck SCC (UT-SCC-87) cell line established at higher proliferation of malignant cells. After titration with different the University of Turku, Finland, was used in this study. The cell seeding densities, concentrations of 0.30×106 for NOKs and line was established from a previously untreated primary tumor of 0.16×106 for malignant cells were chosen for the experiments. In the mobile tongue in a 29-year-old female patient presenting with a this way, the average cell number at the termination of the co- T3N1M0 grade 1 SCC. The methods used in establishing and cultures was 0.45×106 for NOKs and 0.43×106 for malignant cells, characterizing the cell line have been described previously (8, 9). and a cell-density of about 80% was reached in all samples at the NOKs were obtained from the gingiva of a 28-year-old, previously end of the co-culture experiments. healthy, non-smoking male. Human primary dermal fibroblasts were obtained from patients undergoing reconstructive breast surgery at RNA extraction. Collagen gels were dissolved and RNA was the Department of Plastic Surgery, Uppsala University Hospital. extracted using a modification of the one-step-phenol-chloroform Fibroblasts from one healthy female were used for the experiments. method (11). Gels were dissolved in TRIzol reagent (Life Approval from the local ethics committee (Uppsala University) was Technology) under extensive vortex mixing. The amount and the obtained (Dnr 2005:332). This approval entails written informed quality of RNA were determined using the Agilent Bioanalyzer consent and written patient information. 2100 (Agilent Technology, Kista, Sweden). Cellular isolation and culture. Mucosal samples were treated with Microarray expression analysis. Three different experiments with dispase and the epithelium was mechanically separated from the duplicates were done with fibroblasts,