(CANCER RESEARCH 58. 2200-2208. May 15. 1998| Normal Keratinocytes Suppress Early Stages of Neoplastic Progression in Stratified Epithelium1 Ashkan Javaherian, Michael Vaccariello, Norbert E. Fusenig, and Jonathan A. Garlick2 Department of Oral Biology and Pathology. School of Dental Medicine. Westchester Hall, SUNY at Stany Brook. Stony Brook, New York 11794-8702 ¡A.J., M. V.. 3.A. G.¡.and German Cancer Research Center. Division of Differentiation and Carcinogenesis, D-69120 Heidelberg, Germany ¡N.E. F.] ABSTRACT The importance of interactions between potentially neoplastic cells and their normal neighbors on malignant progression of precancerous lesions is not well understood. In this study, we have established novel human tissue models that simulate intraepithelial neoplasia in stratified epithelia to investigate the fate and phenotype of neoplastic keratinocyte clones in normal cell context during clonal expansion and early malignant progres sion. This was accomplished by mixing genetically marked keratinocytes with malignant potential (II-4) with normal keratinocytes at ratios of 1:1, 4:1, 12:1, and 64:1 (normal:II-4) to visualize nests of marked, dysplastic cells in organotypic cultures and in cultures transplanted to nude mice. Four weeks after transplantation of 4:1 mixtures, grafts were normal and demonstrated no ß-galactosidase (ß-gal)-positive cells, suggesting that cells with malignant potential were eliminated from the tissue at this mixing ratio. However, grafted 1:1 mixtures demonstrated persistence of expanded foci of dysplastic cells (4 weeks) and invasion (8 weeks). This demonstrated that the capacity of a keratinocyte clone with neoplastic potential to persist and invade is directly related to the threshold number of such keratinocytes present in the tissue. To explain the failure of 11-4 to persist in vivo, the intraepithelial dynamics between the two populations were studied before grafting. Double-stain immunofluorescence for bro- modeoxyuridine/ß-gal and filaggrin/0-gal of mixtures grown in organo typic cultures for 7 days demonstrated that when increasing numbers of normal cells were added (12:1), II-4 ceased to proliferate and expressed filaggrin. This suggests a novel mechanism of tumor suppression wherein contact with normal cells induces cell cycle withdrawal and terminal differentiation of potentially malignant cells. These findings support the view that normal tissue architecture acts as a dominant suppressor of early neoplastic progression in stratified epithelium. INTRODUCTION Squamous cell cancer is initiated as a small nest of aberrant cells expands to dominate a tissue and form a macroscopic tumor. During early neoplastic progression, preinvasive lesions demonstrate dysplas tic foci that are initially surrounded by normal, undisturbed tissue (1, 2). There is in vivo (3-5) and in vitro (6-10) evidence that normal cells in the microenvironment can alter the neoplastic phenotype of carcinogen-initiated epithelium. These studies have proposed that normal cells limit the progression of initiated cells so that only a small fraction of potentially neoplastic cells express the neoplastic pheno type. However, mechanisms through which interactions with normal epithelial cells regulate the malignant potential, fate, and phenotype of transformed epithelial cells have not been elucidated. The role of normal cell context in controlling the growth of cells with malignant potential has been difficult to study in vivo. Most studies of in vivo carcinogenesis, including transgenic models, follow the progression of initiated cells that are surrounded by cells express- Received 11/21/97; accepted 3/18/98. The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the National Institutes of Dental Research (DE-11250-02). 2 To whom requests for reprints should be addressed, at Department of Oral Biology and Pathology. School of Dental Medicine, Westchester Hall, SUNY at Stony Brook. Stony Brook. NY 11794-8702. Fax: (516)632-9707; E-mail: jonathan.garlick@ sunysb.edu. ing the same phenotype (11). This does not accurately reflect the early progression of spontaneous tumors in stratified epithelial tissues be cause cells with neoplastic potential may not be contacting normal cellular neighbors. Investigation of the role of cell interactions in early neoplastic progression therefore requires the capacity to detect and characterize small numbers of cells with malignant potential in an environment of more normal cells. We have developed novel tissue models to study early neoplastic progression in stratified squamous epithelium in which normal cell context is respected and cells with malignant potential are genetically marked to study their fate and phenotype. We have used organotypic cultures in which normal human keratinocytes are grown on a colla gen matrix containing dermal fibroblasts at an air-liquid interface to generate fully stratified squamous epithelia with a basal rate of pro liferation ( 12). This culture system is optimal for studying cell inter actions and is advantageous over monolayer cultures, where kerati nocytes undergo limited differentiation and are hyperproliferative. The malignant cell line used in our studies (II-4) was derived by transfection of the spontaneously immortalized human keratinocyte line HaCaT (13) with an activated c-Harvey-ra.v oncogene (14). These cells have been shown previously to display severe dysplasia in organotypic culture and low-grade malignant behavior after in vivo transplantation (15). Upon grafting organotypic cultures to the dorsal surface of nude mice, we have generated normal and malignant tissues that manifest their characteristic behavior in vivo. By genetically marking these potentially malignant keratinocytes and mixing them with normal keratinocytes, we generated three dimensional tissues that simulate intrapeithelial neoplasia to study interactions between these cell types during early neoplastic progression. In this report, we study the role of epigenetic factors in the deter mination of the fate of cells with neoplastic potential and propose that normal tissue architecture acts as a dominant suppressor of early neoplastic progression in stratified epithelium. We demonstrate a mechanism wherein potentially invasive keratinocyte clones are nor malized by contact with surrounding normal keratinocytes and are eliminated from the tissue. The size of such clones is crucial in determining their ability to expand and invade, supporting a require ment for a high critical number of neoplastic cells to overcome growth suppression. This novel mechanism of tumor suppression was the induction of cell cycle withdrawal and terminal differentiation of potentially malignant cells by contact with normal cells and mainte nance of normal tissue organization. This suggests that the signaling network inherent in cell interactions in stratified epithelia may be effective in tumor control. Malignant behavior is, therefore, controlled by tissue architecture, and tissue phenotype can predominate over cellular genotype in early neoplastic progression. MATERIALS AND METHODS Cell Culture. Human epidermal keratinocytes were cultured from newborn foreskin by the method of Rheinwald and Green ( 16) in keratinocyte medium described by Wu et al. (17). Cultures were established through trypsinization of foreskin fragments and grown on irradiated 3T3 fibroblasts. 3T3 cells were maintained in DMEM containing 10% bovine calf serum. H-4 keratinocytes were grown in DMEM containing 5% FCS. Organotypic cultures were pre- 2200 Research. on February 7, 2016. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from