Cultures Of Oral Keratinocytes Research Articles

Genomic Engineering of Oral Keratinocytes to Establish In Vitro Oral Potentially Malignant Disease Models as a Platform for Treatment Investigation.

Precancerous cells in the oral cavity may appear as oral potentially malignant disorders, but they may also present as dysplasia without visual manifestation in tumor-adjacent tissue. As it is currently not possible to prevent the malignant transformation of these oral precancers, new treatments are urgently awaited. Here, we generated precancer culture models using a previously established method for the generation of oral keratinocyte cultures and incorporated CRISPR/Cas9 editing. The generated cell lines were used to investigate the efficacy of a set of small molecule inhibitors. Tumor-adjacent mucosa and oral leukoplakia biopsies were cultured and genetically characterized. Mutations were introduced in CDKN2A and TP53 using CRISPR/Cas9 and combined with the ectopic activation of telomerase to generate cell lines with prolonged proliferation. The method was tested in normal oral keratinocytes and tumor-adjacent biopsies and subsequently applied to a large set of oral leukoplakia biopsies. Finally, a subset of the immortalized cell lines was used to assess the efficacy of a set of small molecule inhibitors. Culturing and genomic engineering was highly efficient for normal and tumor-adjacent oral keratinocytes, but success rates in oral leukoplakia were remarkably low. Knock-out of CDKN2A in combination with either the activation of telomerase or knock-out of TP53 seemed a prerequisite for immortalization. Prolonged culturing was accompanied by additional genetic aberrations in these cultures. The generated cell lines were more sensitive than normal keratinocytes to small molecule inhibitors of previously identified targets. In conclusion, while very effective for normal keratinocytes and tumor-adjacent biopsies, the success rate of oral leukoplakia cell culturing methods was very low. Genomic engineering enabled the prolonged culturing of OL-derived keratinocytes but was associated with acquired genetic changes. Further studies are required to assess to what extent the immortalized cultures faithfully represent characteristics of the cells in vivo.

Calcitriol Treatment Decreases Cell Migration, Viability and β-Catenin Signaling in Oral Dysplasia.

Nearly 90% of oral cancers are characterized as oral squamous cell carcinoma (OSCC), representing the sixth most common type of cancer. OSCC usually evolves from oral potentially malignant disorders that, in some cases, are histologically consistent with a oral dysplasia. The levels of 1α,25 dihydroxyvitamin D3 (1,25-(OH)2D3; calcitriol), the active form of vitamin D3, have been shown to be decreased in patients with oral dysplasia and OSCC. Moreover, treatment with 1,25-(OH)2D3 has been proven beneficial in OSCC by inhibiting the Wnt/β-catenin pathway, a signaling route that promotes cell migration, proliferation, and viability. However, whether this inhibition mechanism occurs in oral dysplasia is unknown. To approach this question, we used dysplastic oral keratinocyte cultures and oral explants (ex vivo model of oral dysplasia) treated with 1,25-(OH)2D3 for 48 h. Following treatment with 1,25-(OH)2D3, both in vitro and ex vivo models of oral dysplasia showed decreased levels of nuclear β-catenin by immunofluorescence (IF) and immunohistochemistry (IHC). Consistently, reduced protein and mRNA levels of the Wnt/β-catenin target gene survivin were observed after treatment with 1,25-(OH)2D3. Moreover, 1,25-(OH)2D3 promoted membranous localization of E-cadherin and nuclear localization of vitamin D receptor (VDR). Functionally, DOK cells treated with 1,25-(OH)2D3 displayed diminished cell migration and viability in vitro.

Fibroblast encapsulation in gelatin methacryloyl (GelMA) versus collagen hydrogel as substrates for oral mucosa tissue engineering

PurposeOver the past decades, a variety of biomaterials have been investigated in terms of their suitability for oral mucosa tissue engineering. The aim of this study was to compare collagen and GelMA hydrogels as connective tissue scaffolds for fibroblasts and as substrates for seeding and culture of oral epithelial keratinocyte cells. MethodsHuman primary oral fibroblast and keratinocyte cells were isolated from gingival biopsies. The mixture of fibroblasts with GelMA or collagen gel were aliquoted within six-well tissue culture plate inserts and cross-linked using visible light or reconstitution buffer/heat, respectively. The viability of fibroblasts in the hydrogels was investigated after one and three days of cultivation using the PrestoBlue assay. Following the addition and culture of oral keratinocytes onto the connective tissue constructs, the tissue-engineered oral mucosa was assessed histologically. ResultsThe tissue viability assay shows that collagen hydrogels encapsulating fibroblasts displayed significantly higher cell viability than cell-laden GelMA constructs after 24 and 72 h (p < 0.05). A stratified and differentiated epithelium has formed on the surface of cell-laden collagen hydrogel but not on the surface of the GelMA-based substrate. ConclusionCollagen-based scaffold offers superior biological properties compared to GelMA hydrogel in terms of oral fibroblast growth, as well as epithelial cell adhesion and differentiation. Therefore, collagen-based hydrogels remain the preferred choice for oral mucosa tissue engineering.

Ex vivo culture of oral keratinocytes using direct explant cell culture technique.

Background:Culture of cells and tissues is a standard research method practiced in many laboratories. In most of the cases, these cultures are being used as substrates for cell products or as investigative tools for delving the mechanism of gene expression, cell proliferation and transformation. Primary monolayer cell culture has been beneficial to study the general biology of both oral and skin keratinocytes. There are two different techniques of primary cell cultures followed, which include direct explant and enzymatic techniques.Aims:The aim of the study was to optimize the culture of keratinocytes obtained from patients with normal oral mucosa by direct explant technique.Materials and Methods:Keratinocytes were isolated from 15 patients and were cultured in vitro and observed under an inverted microscope. The cultured cells were characterized by immunocytochemistry method using pan-cytokeratin.Results:The total success rate of primary culture of the oral epithelial cells by direct explant technique was 88.6%. No contamination of microorganisms in the primary cell cultures was obtained.Conclusion:Within the limited numbers of samples used in the current pilot study, we conclude that the direct explant technique appears to be a simple and successful technique for the isolation of oral mucosal keratinocytes if we follow the appropriate protocol.

Automated noninvasive epithelial cell counting in phase contrast microscopy images with automated parameter selection.

SummaryCell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a ‘destructive’ process requiring disruption of the cell monolayer resulting in the inability to follow cell growth longitudinally. This process is time consuming and utilises significant resource. In this study a relatively inexpensive, rapid and widely applicable phase contrast microscopy‐based technique has been developed that emulates the contrast changes taking place when bright field microscope images of epithelial cell cultures are defocused. Processing of the resulting images produces an image that can be segmented using a global threshold; the number of cells is then deduced from the number of segmented regions and these cell counts can be used to generate growth curves. The parameters of this method were tuned using the discrete mereotopological relations between ground truth and processed images. Cell count accuracy was improved using linear discriminant analysis to identify spurious noise regions for removal.The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum. The approach developed has broad applicability and utility for researchers with standard laboratory imaging equipment.

Potential role of periodontal pathogens in compromising epithelial barrier function by inducing epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire a mesenchymal-like phenotype and this may be induced by exposure to gram-negative bacteria. It has been proposed that EMT is responsible for compromising epithelial barrier function in the pathogenesis of several diseases. However, the possible role of EMT in the pathogenesis of periodontitis has not previously been investigated. The aim of this study therefore was to investigate whether gram-negative, anaerobic periodontal pathogens could trigger EMT in primary oral keratinocytes in vitro. Primary oral keratinocytes were harvested from labial mandibular mucosa of Wistar Han rats. Cells were exposed to heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis (100 bacteria/epithelial cell) and to 20μg/mL of Escherichia coli lipopolysaccharide over an 8-day period. Exposure to bacteria did not significantly change epithelial cell number or vitality in comparison with unstimulated controls at the majority of time-points examined. Expression of EMT marker genes was determined by semiquantitative RT-PCR at 1, 5, and 8days following stimulation. The expression of EMT markers was also assessed by immunofluorescence (E-cadherin and vimentin) and using immunocytochemistry to determine Snail activation. The loss of epithelial monolayer coherence, in response to bacterial challenge, was determined by measuring trans-epithelial electrical resistance. The induction of a migratory phenotype was investigated using scratch-wound and transwell migration assays. Exposure of primary epithelial cell cultures to periodontal pathogens was associated with a significant decrease in transcription (~3-fold) of E-cadherin and the upregulation of N-cadherin, vimentin, Snail, matrix metalloproteinase-2 (~3-5 fold) and toll-like receptor 4. Bacterial stimulation (for 8 days) also resulted in an increased percentage of vimentin-positive cells (an increase of 20% after stimulation with P.gingivalis and an increase of 30% after stimulation with F.nucleatum, compared with controls). Furthermore, periodontal pathogens significantly increased the activation of Snail (60%) and cultures exhibited a decrease in electrical impedance (P<.001) in comparison with unexposed controls. The migratory ability of the cells increased significantly in response to bacterial stimulation, as shown by both the number of migrated cells and scratch-wound closure rates. Prolonged exposure of primary rat oral keratinocyte cultures to periodontal pathogens generated EMT-like features, which introduces the possibility that this process may be involved in loss of epithelial integrity during periodontitis.

Metabolomic analysis identifies differentially produced oral metabolites, including the oncometabolite 2-hydroxyglutarate, in patients with head and neck squamous cell carcinoma

BackgroundMetabolomics represents a promising approach for discovering novel targets and biomarkers in head and neck squamous cell carcinoma (HNSCC). Here we used metabolomics to identify oral metabolites associated with HNSCC. MethodsTumor and adjacent normal tissue from surgical resections and presurgical oral washes as well as oral washes were collected from healthy participants. Metabolites extractions of these samples were analyzed by liquid chromatography-mass spectroscopy (LC/MS), LC/MS/MS and gas chromatography-MS (GC/MS). ResultsAmong 28 samples obtained from 7 HNSCC cases and 7 controls, 422 metabolites were detected (269 identified and 153 unidentified). Oral washes contained 12 and 23 metabolites in healthy controls and HNSCC patients, respectively, with phosphate and lactate being the most abundant. Small molecules related to energy metabolism were significantly elevated in HNSCC patients compared to controls. Levels of beta-alanine, alpha-hydroxyisovalerate, tryptophan, and hexanoylcarnitine were elevated in HNSCC oral washes compared to healthy controls (range 7.8-12.2-fold). Resection tissues contained 22 metabolites, of which eight were overproduced in tumor by 1.9- to 12-fold compared to controls. TCA cycle analogs 2-hydroxyglutarate (2-HG) and 3-GMP were detected exclusively in tumor tissues. Targeted quantification of 2-HG in a representative HNSCC patient showed increase in tumor tissue (14.7μg/mL), but undetectable in normal tissue. Moreover, high levels of 2-HG were detected in HNSCC cell lines but not in healthy primary oral keratinocyte cultures. ConclusionsOral metabolites related to energy metabolism were elevated in HNSCC, and acylcarnitine and 2HG may have potential as non-invasive biomarkers. Further validation in clinical studies is warranted.

Generation and Infection of Organotypic Cultures with Epstein-Barr Virus.

While numerous model systems are available to study EBV latency in B cells and have contributed greatly to our understanding of the role of these cells in the viral life cycle, models to study the EBV life cycle in epithelial cells in vitro are lacking. Epithelial cells are poorly infected in vitro, and EBV-infected cell lines have not been successfully obtained from epithelial tumors. Recently, we have demonstrated that organotypic cultures of oral keratinocytes can be used as a model to study EBV infection in the epithelial tissue. These "raft" cultures generate a stratified tissue resembling the epithelium seen in vivo with a proliferating basal layer and differentiating suprabasal layers. Here, we describe generation of EBV-infected raft cultures established from primary oral mucosal epithelial cells, which exhibit high levels of productive replication induced by differentiation, as well as methods to analyze EBV infection.

Evaluation of microRNA expression in head and neck squamous cell carcinoma cell lines and in primary culture of oral keratinocytes.

Functional in vitro studies are fundamental to understand the role of microRNAs, small non coding RNA molecules that function as post-transcriptional regulators, in cancer. The objective of this study was to determine the applicability of head and neck squamous cell carcinoma cell lines and human oral keratinocytes as models for functional studies on microRNAs previously identified as deregulated in head and neck squamous cell carcinomas. The expression level of four microRNAs was assessed in cell lines and in primary cultures of oral keratinocytes using specific real-time polymerase chain reactions. The identity of oral squamous cell carcinoma cell lines was confirmed by means of STR (short tandem repeats) profiling. The possible impact of feeder-layer gene expression in global microRNA expression results from keratinocyte primary culture was also evaluated. Significant differences in microRNA gene expression were observed among squamous cell carcinoma cell lines, particularly among cells lines from distinct subsites, as well as between primary culture of human keratinocytes and immortalized keratinocyte cell lines. Primary cultures of human keratinocytes and diverse tumor cell lines are relatively easy to obtain. However, each cell model possesses a characteristic phenotype; whereas one may be useful for a specific study, it may be inappropriate for another. Therefore, it is imperative that suitable cell lines are cautiously selected for functional studies in cancer.

Is it safe to utilize in vitro reconstituted human oral epithelium? An oncogenetic pathway study

Cell therapy is a therapeutic strategy used to replace or repair damaged tissue. The epithelium transplantation of cultivated keratinocytes has been applied to several modalities of reconstruction, like oral, urethra and ocular surface. Life and death signals work coordinately to ensure cellular quality control and the viability of an organism. The aim of this study is to verify that culture conditions did not induce genetic mutations through the analysis of the key genes: pAKT, Pten, p53 and MDM2 and investigate the presence of the related proteins in human oral keratinocytes obtained by primary culture and in vitro cultivated. Formalin fixed and paraffin embedded tissues from the oral cavity were utilized as control for normal expression of the related markers and two oral squamous cell carcinoma cell lines provided the expression pattern of the proposed markers in the event of cellular transformation. Akt, PTEN, p53 and MDM2 immunohistochemistry and Western-Blotting analyzes were performed. The results showed the expression levels and intracellular localizations of the four proteins evaluated. These analyzes confirmed that the produced in vitro epithelium is bio-compatible for its utilization as reconstruction and reparatory tissue, however further analyses and additional research on other biomarkers should be performed to analyse the long term engraftment of transplantable primary culture of oral keratinocytes and the long term resistance to cellular transformation.

Constitutive Release of Cytokines by Human Oral Keratinocytes in an Organotypic Culture

The Food and Drug Administration requires an accurate determination of the dose and potency of tissue-engineered or combination products as is required for drugs. This needs to be done as a rapid, quantitative, and noninvasive measurement of biologic function/activity in a way so as not to perturb the tissue-engineered product being developed. The aim of this study was to correlate constitutive release of cytokine(s) from unstimulated cells, at different stages of development, within a 3-dimensional (3D) organotypic ex vivo produced oral mucosa equivalent (EVPOME) to be used for intraoral grafting, with oral keratinocyte cell viability of the EVPOME. Tissue culture medium was assayed with an enzyme-linked immunosorbent assay from monolayer culture of oral keratinocytes and a 3D EVPOME to determine the constitutive release of interleukin (IL) 1alpha, IL-6, IL-8, and vascular endothelial growth factor (VEGF). VEGF messenger ribonucleic acid expression by oral keratinocytes within the 3D EVPOME was detected by in situ hybridization at days 4, 7, and 11. The number of viable oral keratinocytes within the EVPOME was extrapolated from VEGF release by use of a modified MTT assay. Both VEGF release level and the number of viable cells in the monolayer cultures and 3D EVPOME as measured by MTT assay significantly increased in a time-dependent manner (P < .001, r = 0.743). These results suggest that the increasing detectable levels of VEGF associated with the increasing number of viable cells in the EVPOME may provide a useful noninvasive/nondestructive means of assessing both cellular viability (dose) and biologic function/activity (potency) of a combination cell-based device such as the EVPOME.

Cytosolic serine proteases released from carcinoma induces nociception in mice

Nociceptive mediators involved in the generation of cancer pain remain poorly understood. Oral squamous cell carcinoma (SCC) produce high levels of proteases that are critical for carcinogenesis. The release of cytosol from damaged oral SCC cells, either during carcinogenesis or cancer treatment, may serve as a chemical trigger for nociception. To determine if cytosolic proteases released from cancer cells into the cancer microenvironment induce mechanical nociception, mechanically lysed cells derived from human oral SCC (HSC-3) and control human normal oral keratinocyte cultures or serum-free media (50 μl; n = 5/group) were injected into the hindpaw of female mice under light isoflurane anesthesia.

Metabolism and growth inhibition of four retinoids in head and neck squamous normal and malignant cells.

Isotretinoin (13- cis -retinoic acid, 13cRA) has proven to be active in chemoprevention of head and neck squamous cell carcinoma (HNSCC). Moreover, both all-trans-retinoic acid (ATRA) and 13cRA induce objective responses in oral premalignant lesions. After binding of retinoids to retinoic acid receptors (RARs and RXRs) dimers are formed that are able to regulate the expression of genes involved in growth and differentiation. We compared the metabolism and level of growth inhibition of 13cRA with that of ATRA, 9cRA and retinol in four HNSCC cell lines and normal oral keratinocyte cultures (OKC). These retinoid compounds are known to bind with different affinities to the retinoic acid receptors. We observed that all retinoids were similar with respect to their capacity to induce growth inhibition. One HNSCC line could be ranked as sensitive, one as moderately sensitive and the remaining two were totally insensitive; OKC were moderately sensitive. The rate at which the cells were able to catabolize the retinoid was similar for all compounds. Retinoid metabolism in HNSCC cells resulted in a profile of metabolites that was unique for each retinoid. These metabolic profiles were different in OKC. Our findings indicate that differences in retinoid receptor selectivity of these retinoids do not influence the level of growth inhibition and rate of metabolism. © 2001 Cancer Research Campaign http://www.bjcancer.com

Cloning, Mapping, Expression, Function, and Mutation Analyses of the Human Ortholog of the Hamster Putative Tumor Suppressor Gene doc-1

doc-1 is a putative tumor suppressor gene isolated and identified from the hamster oral cancer model. Here, we report the molecular cloning and the functional characterization of the human ortholog of the hamster doc-1 gene. Human doc-1 cDNA is 1.6 kilobase pairs in length and encodes for a 115-amino acid polypeptide (12.4 kDa, pI 9. 53). Sequence analysis showed 98% identity between human and hamster doc-1 protein sequences. DOC-1 is expressed in all normal human tissues examined. In oral keratinocytes, expression of DOC-1 is restricted to normal oral keratinocytes. By immunostaining of normal human mucosa, DOC-1 is detected in both the cytoplasm and nuclei of basal oral keratinocytes; while in suprabasilar cells, it is primarily found in the nuclei. Human oral cancers in vivo did not exhibit immunostaining for DOC-1. Like murine DOC-1, human DOC-1 associates with DNA polymerase alpha/primase and mediates the phosphorylation of the large p180 catalytic subunit, suggesting it may be a potential regulator of DNA replication in the S phase of the cell cycle. Using a human doc-1 cosmid as a probe, human doc-1 is mapped to chromosome 12q24. We identified four exons in the entire human doc-1 gene and determined the intron-exon boundaries. By polymerase chain reaction and direct sequencing, we examined premalignant oral lesion and oral cancer cell lines and found no intragenic mutations.

Prostaglandin E2 and interleukin-1 concentrations in nicotine-exposed oral keratinocyte cultures.

Oral keratinocytes are the first cells in contact with tobacco components and are capable of producing various inflammatory mediators, including PGE2 and IL-1. The purpose of this study was to examine PGE2 and IL-1 concentrations in nicotine-exposed oral keratinocyte cultures. Gingival keratinocyte cultures were established from healthy gingival tissues obtained from 7 subjects. Cultures were divided into 4 groups exposed to serum free medium (control), 0.1 microM, 10 microM or 1 mM nicotine for 4, 24 or 48 h. Using enzyme-linked immunosorbent assays, PGE2 and IL-1 alpha were quantified in culture supernatants; IL-1 alpha and beta were also measured in lysed cells. A repeated measures analysis of variance was used to identify significant differences over time and treatment. Nicotine exposure did not significantly alter PGE2 levels at any given time period; however, PGE2 quantities declined significantly (p = 0.0001) over time. At both 24 and 48 h, IL-1 alpha concentrations in lysates from 1 mM nicotine-exposed cells were significantly (p < 0.01) greater than those for all other treatments. Interleukin-1 alpha quantities also declined significantly (p = 0.037) over time in the cultures. Interleukin-1 beta concentrations were elevated, albeit not significantly, in the 1 mM treated cells at 24 and 48 h. Cell viability, mass and counts were not affected by nicotine treatment; these parameters increased significantly (p < 0.005) over time. In summary, nicotine treatment significantly increased IL-1 alpha concentrations in cultured keratinocytes; however, PGE2 synthesis was not altered. Elevated IL-1 production by keratinocytes may have implications in tobacco-induced lesions, given the central role IL-1 plays in tissue response to injury.

Three distinct keratinocyte subtypes identified in human oral epithelium by their patterns of keratin expression in culture and in xenografts

Abstract We have characterized the cells that form the human oral epithelia by analyzing their patterns of keratin expression in culture and in transplants. Keratinocytes of all oral regions synthesized high levels of keratins K5/K14 and K6/K16, K17, as expressed by cells of all stratified squamous epithelia in culture. However, cells from different regions varied in their expression in culture of retinoid-inducible (K19 and K13) and simple epithelial (K7, K8 and K18) keratins. By these criteria, all oral cells could be classified as belonging to one of three intrinsically distinct subtypes: “keratinizing” (gingiva, hard palate), “typical nonkeratinizing” (inner cheek, floor of mouth, ventral tongue) and “special nonkeratinizing” (soft palate), all of which differed from the epidermal kerdtinocyte subtype. Cells from fetal floor of mouth expressed a pattern of keratins in culture markedly different from that of adult floor of mouth cells but identical to that of the adult “special nonkeratinizing” subtype and similar to that of several oral squamous cell carcinoma lines. When cultures of oral keratinocytes were grafted to the dermis of nude mice, they formed stratified epithelial structures after 10 days. In some areas of the stratified structures, the basal layer recapitulated the K19 expression pattern of the oral region from which they had originated. Thus, regional differentiation of the oral epithelium is based on an intrinsic specialization of regional keratinocyte stem cells. Additionally, oral cell transformation either frequently involves reversion to the fetal keratin program or else oral cells that express this keratin program are especially susceptible to transformation.

Evidence for Autoregulation of Cell Division and Cell Transit in Keratinocytes Grown on Collagen at an Air-Liquid Interface

Oral and epidermal rat keratinocytes when cultured on a matrix of type I collagen fibrils at the interface between the gaseous and liquid phases of a culture form a highly ordered stratified squamous epithelium. Autoradiographic studies of cells labeled by tritiated thymidine indicate that the keratinocytes are capable of autoregulating cell division. Early confluent cultures exhibit 51% of basal cells labeled, a percentage that decreases to 18% when a full differentiated stratified squamous epithelium is formed. Such a decrease in labeling occurs in cultures where the mitotically active basal cells have unimpeded access to culture medium supplied from below and when no cell type other than the keratinocyte is present in the culture. Additionally, the transit of keratinocytes from the basal cell layer through other viable cell strata to the layer of terminally differentiated cells can be followed by tracking cells labeled with tritiated thymidine. In cultures of oral keratinocytes, cells move from the basal cell layer to the cornified layer at a maximum rate of 7 days, while virtually all labeled cells (91%) are localized in the terminally differentiated cell layer 14 days following labeling. Keratinocyte cultures grown in culture at an air-liquid interface exhibit tissue organization that closely resembles the native, parent tissue. Such cultures can be useful in studying the effects of pharmacologic mediators of cell division and cell transit.

The inter-relationship between anchorage independence and tumorigenicity in early cultures of oral keratinocytes

This study examines the expression of anchorage independence and tumorigenicity in early cultures of oral rat keratinocytes. The epithelial cell lines originated from the palatal and the lingual mucosa of rats that had been painted with the carcinogen 4-nitroquinoline N-oxide. The colony forming efficiency (CFE) in gel culture of the cell lines derived from five squamous cell carcinomas of the tongue and palate predominantly increased with passage in culture. Carcinoma-derived cell lines that had a relatively high CFE (greater than 2.5%) formed tumours when transplanted to athymic mice, but cells in which the CFE was less than 2.5% were non-tumorigenic. Keratinocytes from a dysplastic palatal lesion were immortal, anchorage dependent and non-tumorigenic. A lingual papilloma cell line consistently expressed a very low CFE but was tumorigenic at the higher culture passages. The results show that the routine passage of cells in culture leads to the emergence of the anchorage independent and tumorigenic phenotypes in keratinocytes of malignant origin and, further, suggest that anchorage independence and tumorigenicity may exist as distinct phenotypes, with anchorage independence preceding tumorigenicity.