Engineering skin to study human disease

Adv Biochem Engin/Biotechnol (2006) 103: 207–239 DOI 10.1007/b137206 © Springer-Verlag Berlin Heidelberg 2006 Published online: 5 January 2006 Engineering Skin to Study Human Disease – Tissue Models for Cancer Biology and Wound Repair Jonathan A. Garlick Division of Cancer Biology and Tissue Engineering Department of Oral and Maxillo- facial Pathology, Tufts University, 55 Kneeland Street, Room 116, Boston, Massachusetts 02111, USA Jonathan.Garlick@Tufts.edu 1Introduction 208 2 Engineered human tissue models used to study early cancer progression in stratified squamous epithelium 211 2.1 Cell–cell interactions inherent in 3-D tissue architecture suppress early cancer progression by inducing astateofintraepithelialdormancy 213 2.2 Factors altering cell–cell and cell–matrix interactions abrogate the microenvironmental control onintraepithelialtumorcellsandpromotecancerprogression 217 2.2.1 The tumor promoter TPA enables expansion ofintraepithelialtumorcells 217 2.2.2 Immortalization of adjacent epithelial cells cannotinduceintraepithelialdormancyoftumorcells 218 2.2.3 UV-B Irradiation is permissive for tumor cell expansion by inducing a differential apoptotic and proliferative response betweentumorcellsandadjacentnormalcells 221 2.2.4 Basement membrane proteins promote progression of early cancer by rescuing tumor cells from intraepithelial dormancy through their selective adhesion to laminin 1 and Type IV collagen andsubsequentexpansion 223 3 Three-dimensional skin-equivalent tissue models to study wound reepithelialization of human stratified epithelium 227 3.1 Morphologyofwoundedskinequivalents 227 3.2 Proliferationinskinequivalentsinresponsetowounding 231 3.3 Migrationinskinequivalentsinresponsetowounding 232 3.4 Growth factor responsiveness and synthesis inwoundedskinequivalents 233 3.5 Matrixmetalloproteinaseactivityinwoundedskinequivalents 235 3.6 Keratinocytedifferentiationinwoundedskinequivalents 236 References 237 Abstract Recent advances in the engineering of three-dimensional tissues known as skin equivalents, that have morphologic and phenotypic properties of human skin, have provided new ways to study human disease processes. This chapter will supply an overview of two such applications – investigations of the incipient development of squamous 208 J.A. Garlick cell cancer, and studies that have characterized the response of human epithelium during wound repair. Using these novel tools to study cancer biology, it has been shown that cell-cell interactions inherent in three-dimensional tissue architecture can suppress early cancer progression by inducing a state of intraepithelial dormancy. This dormant state can be overcome and cancer progression enabled by altering tissue organization in response to tumor promoters or UV irradiation or by modifying the interaction of tumor cells with extracellular matrix proteins or their adjacent epithelia. By adapting skin equivalent models of human skin to study wound reepithelialization, it has been shown that several key responses, including cell proliferation, migration, differentiation, growth- factor responsiveness and protease expression, will mimic the response seen in human skin. In this light, these engineered models of human skin provide powerful new tools for studying disease processes in these tissues as they occur in humans. Keywords Tissue engineering · Human skin equivalents · Intraepithelial neoplasia · Wou nd re pa ir · Squamous cell carcinoma Abbreviations IE Intraepithelial ECM Extracellular matrix β-gal β-Galactosidase BM Basement membrane 1 Introduction Engineered human tissue models designed to advance our understanding of human disease processes require the ability to fabricate tissues that faithfully mimic their in vivo counterparts. In vitro studies using human cells have of- ten been limited by their inability to simulate the characteristic morphology, phenotype and behavior found in vivo. For example, monolayer cultures of human skin keratinocytes demonstrate limited stratification, partial differentiation and hyperproliferative growth [1], and have been of limited use when studying the complex cellular responses seen in intact tissues due to their lack of three-dimensional (3-D) tissue architecture. Biologically-meaningful signaling pathways, mediated by the linkage of adhesion and growth, function optimally when cells are spatially organized in 3-D tissues, but are uncoupled and lost in two-dimensional culture systems [2–4]. It is therefore essential to generate 3-D cultures that display the architectural features seen in vivo in order to engineer tissue models that will allow the study of human diseases in their appropriate tissue context. During the last decade, the development of tissue-engineered models that mimic human skin, known as skin equivalents (SE), have provided novel experimental systems to study the behavior of normal and altered human stratified squamous epithelium. The SE is cultured at an air-liquid inter- Engineering Skin to Study Human Disease 209 face on a collagen matrix populated with dermal fibroblasts to generate 3-D, organotypic tissues that demonstrate in vivo-like epithelial differentiation and morphology, as well as rates of cell division similar to those found in human skin [5, 6]. Organotypic, SE tissue models have previously been adapted to study epithelial and skin biology on a variety of connective tissue substrates that have served as dermal equivalents [7, 8]. A well-stratified epithelium was seen when cultures were grown on dermal equivalents fabricated as Type I collagen gels which were populated with fibroblasts [5, 9, 10]. Porous membranes seeded with fibroblasts or coated with extracellular matrix proteins have been used to generate skin-like organotypic cultures [11]. Alternatively, fibroblasts have been incorporated into a three-dimensional scaffold, where these cells could secrete and organize an extracellular matrix [12]. While organotypic cultures of stratified epithelium have been shown to express basement membrane components in organotypic culture [13–17], limited success has been achieved in attaining structured basement membrane [8, 18, 19]. Since it is known that basement membrane components play a functional role in the regulation of epidermal growth and differentiation [20], it is important to generate cultures that have a well-structured basement membrane. Furthermore, it has previously been shown that the correct spatial organization and polarity of basal cells was associated with functional hemidesmosomes and basement membrane integrity [21]. Since the goal in the fabrication of SEs of human skin has been to fabricate and maintain a stratified epithelium that demonstrates in vivo-like features of epidermal morphology, growth and differentiation, it is critical to optimize these features in 3-D cultures. This has been accomplished by combining the three components thought to be critical in epidermal normalization – keratinocyte stem cells with high proliferative potential, viable dermal fibroblasts and structured basement membrane. Epidermal stem cells have been shown to be present in SE cultures as transplants of these cultures have shown the persistence of genetically-marked progenitor cells in these tissues up to one year after grafting. Dermal fibroblasts are required to stimulate epithelial growth and to promote its stratification while the presence of pre-existing basement membrane components were required to initiate and promote the rapid assembly of structured assembly BM in SE cultures [6], which is needed to sustain keratinocyte growth and to optimize epithelial architecture. Our laboratory has optimized the growth and differentiation of skin-like, organotypic cultures by growing keratinocytes on an acellular, human dermal substrate (AlloDerm) that was repopulated with human fibroblasts to generate SEs with a high degree of tissue normalization forming a structured, mature basement membrane (Fig. 1). This human tissue model recapitulates the morphology of skin to a large degree and has facilitated further clarification of the contributions made by basement membrane components and dermal fibroblasts to normal epidermal morphogenesis. 210 J.A. Garlick Fig. 1 Appearance of skin equivalents grown in organotypic culture at an air–liquid interface. Skin equivalent cultures of normal human keratinocytes were grown at the air– liquid interface for ten days on a deepidermalized human dermis (AlloDerm) containing basement membrane components (layer B). The epithelium generated (layer A) demonstrated in vivo-like tissue architecture, characterized by the presence of all morphologic strata and epithelial rete pegs at the interface with the connective tissue. The contracted collagen gel (layer C) containing dermal fibroblasts on which the AlloDerm was layered is seen at the bottom of the tissue, where fibroblasts have migrated into the lower part of the AlloDerm Such optimally-engineered human tissues can be adapted to study a variety of human disease processes that simulate events that occur in human skinandotherstratifiedsquamousepithelia.AsexamplesofhowSEscan be adapted to study human disease, this chapter will describe how SE cultures have been used to characterize the response of human skin-like tissues following wounding and during early cancer development in a premalignant tissue. First, studies that have utilized these 3-D tissues to demonstrate the critical role of tissue architecture and cell–cell interactions during the earliest stages in the development of cancer in stratified squamous epithelium will be reviewed. Secondly, the response of human tissue models designed to mimic the in vivo reepithelialization of wounded human skin, from the initiation of keratinoctye activation until restoration of epithelial integrity. This will be accomplished by reviewing previous studies that have defined key response parameters, such as growth, migration, differentiation, growth-factor responsiveness and protease expression after wounding SEs. These applications demonstrate the utility of these engineered, human epithelial tissues in the study of responses that characterize the switch from a normal to a regen- erative epithelium during wound reepithelialization and during the earliest, intraepithelial stage of carcinogenesis. It is hoped that by describing human Engineering Skin to Study Human Disease 211 tissue models that recapitulate tissue regeneration and carcinogenesis in vivo, furtherstudyofthenatureoftheseprocesseswillbefacilitated. 2 Engineered human tissue models used to study early cancer progression in stratified squamous epithelium Squamous cell cancer is initiated as a small nest of aberrant, dysplastic cells that expand to dominate a tissue and form a macroscopic tumor. During the earliest, intraepithelial (IE), premalignant stages of cancer progression, before the onset of cancer cell invasion, premalignant lesions demonstrate dysplastic cell foci, with abnormal nuclear and cytoplasmic morphologic features, that are initially surrounded by normal, undisturbed tissue [22, 23]. However, the role of 3-D tissue architecture, as characterized by interactions between potentially neoplastic cells and their normal neighbors, in the progression of human cancer during these early IE stages is not known. While it has been shown that normal cells can alter the phenotype of transformed cells in vitro [24–28], these studies were performed in conventional 2-D cul- turesthatdonotaccountfortherolethattissuearchitectureplaysincancer development [29, 30]. Furthermore, the role of normal cell context in control- ling 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 cells with malignant potential that are surrounded by cells manifesting similar properties of transformation [31]. This does not accurately reflect the early progression of spontaneous tumors in stratified epithelial tissues, since cells with neoplastic potential are usually in contact with normal cellular neighbors during the incipient stages of tumor development. In recent years, evidence has been accumulating that cancer is a disease of altered tissue organization. The view that cancer development and progression is a consequence of altered interactions between tumor cells and their immediate tissue microenvironment has recently been named the “tissue organization field theory of carcinogenesis” [32]. This theory proposes that cancer development disrupts normal interactions between adjacent cells or stroma, which dramatically modifies the ability of cells to sense normal regulatory signals that are inherent in tissue architecture. A corollary of this theory is that cells with neoplastic potential can be reprogrammed to behave like normal cells if found in normal tissue context. In this light, investigation of the role of cell–cell interactions in early neoplastic progression requires the capacity to detect and characterize small numbers of cells with malignant potential in the context of a 3-D network of more normal cells. Engineered human tissues that display 3-D human tissue architecture are therefore essential tools for accomplishing this goal. 212 J.A. Garlick Over the last decade, novel tissue models have been engineered to study early neoplastic progression in stratified squamous epithelium in which normal cell context is respected and cells with malignant potential are marked to study their fate and phenotype. To adapt these 3-D cultures to simulate premalignant disease as it occurs in human tissues, SEs with varying degrees of dysplasia were fabricated by mixing normal keratinocytes with tumor cells. The tumor cells used were a cell line (HaCaT-II-4) that was de- Fig. 2 A human tissue model for premalignant disease of stratified squamous epithelium. Normal keratinocytes (NHK) form a well-stratified epithelium with normalized tissue architecture when grown in skin-equivalent culture (A) , while II-4 cells that were labeled with the gene for β-galactosidase generate a disorganized and dysplastic tissue (B). When these two cell types are mixed in a 1 : 1 ratio, expanded clusters of β-gal-positive cells are randomly distributed among normal cells (C). The presence of such large numbers of II-4-gal cells has disrupted normal tissue architecture. When II-4 cells are mixed in a 12 : 1 ratio (NHK : II-4 cells), β-gal-positive intraepithelial tumor cells do not expand and remain as individual cells in the context of the well-preserved tissue architecture of normal cells (D) Engineering Skin to Study Human Disease 213 rived by transfection of the spontaneously-immortalized human keratinocyte line (HaCaT) [33] with an activated c-Harvey-ras oncogene [34]. These cells have previously been shown to display severe dysplasia in organotypic culture and low-grade malignant behavior after in vivo transplantation [35]. By genetically-marking these potentially-malignant keratinocytes with a retro- viral vector encoding β-glactosidase (β-gal) and mixing them at varying ratios with normal keratinocytes, epithelial tissues with varying degrees of dysplasia were generated to directly study the intraepithelial dynamics between NHK and adjacent, intraepithelial tumor cells (Fig. 2). These SEs were then transplanted into the dorsa of nude mice as surface transplants to allow the earliest events in neoplastic progression to be studied in vivo, by generating a stratified epithelium which evolved from a preinvasive, focally dysplastic tissue to one demonstrating tumor cell invasion into the connective tissue. 2.1 Cell–cell interactions inherent in 3-D tissue architecture suppress early cancer progression by inducing a state of intraepithelial dormancy Using the approach described above to engineer precancerous lesions in humantissues,itwasfoundthatnormaltissuearchitectureactedasadom- inant suppressor of early cancer progression in stratified epithelium [36]. This occurred as interactions with adjacent normal keratinocytes induced intraepithelial tumor cells to withdraw from cell cycle and undergo terminal differentiation. These findings showed that the signaling network inherent in cell interactions in stratified epithelia was effective for tumor control and that a higher level of tissue organization, such as that seen in intact 3-D tissues, could predominate over cellular genotype in early cancer progression. This was first shown in premalignant tissues that were generated as mixtures of NHK and II-4 cells, in which normal cells were the predominant cell type (12 : 1, NHK : II-4), that were clinically and morphologically normal four weeks after grafting to nude mice and showed no β-gal positive. This suggested that cells with malignant potential were eliminated from the tissue at this 12 : 1 mixing ratio. II-4 cells were only detected in 1 : 1 mixtures after grafting and they demonstrated larger foci of dysplastic cells that in- vaded into the underlying connective tissue. These findings suggested that a relatively high, critical number of cells with malignant potential needed to be present for these cells to persist, undergo clonal expansion and form a focally dysplastic and early invasive tumor in vivo. To explain the obser- vation that II-4 cells failed to persist in the tissue when a greater number of normal cells were present in the mixture, the fate and distribution of cells with malignant potential was studied after in vitro growth in organotypic 214 J.A. Garlick cultures that were generated before transplantation. Tissues constructed by mixing cells at 1 : 1 and 12 : 1 ratios were analyzed by immunohistochemi- cal staining to determine if cells with malignant potential were undergoing changes in their biologic behavior due to interactions with adjacent, normal cells that could explain their subsequent loss from the tissue after grafting. Tissue dynamics were studied after growth for one week in organotypic culture by assessing the proliferation and differentiation of II-4 cells in these mixtures. Double immunofluorescent staining of 12 : 1 mixed cultures for β- gal and BrdU demonstrated that no proliferation was seen in the individual β-gal positive cells, which were only present above the basal cell layer, sug- gesting that II-4 cells were growth-suppressed when surrounded by normal cells. In contrast, 1 : 1 mixtures demonstrated numerous, suprabasal β-gal positive clusters which were BrdU-positive, showing that these cells were able to continue to proliferate and expand. This demonstrated that clusters containing larger numbers of II-4 cells continued to proliferate and that cell growth was not affected when the number of contiguous II-4 cells was sufficiently high. In addition, when mixed with normal keratinocytes in a ratio of 12 : 1, II-4-gal cells underwent terminal differentiation as evidenced by the colocalization of β-gal and filaggrin, a marker of keratinocyte terminal differentiation. This showed that II-4 cells were being normalized by adjacent normal keratinocytes which were undergoing terminal differentiation. In contrast, II-4 cells grown in cultures at a ratio of 1 : 1 were not induced to express filaggrin by neighboring cells. Furthermore, since most II-4 cells mixed with NHK at growth-supressive ratios were detected as individual cells in the suprabasal layers, it was important to determine how this sorting occurred. When the distribution of II-4 cells was examined in 12 : 1 mixtures (NHK : II-4) shortly (16 hours) after seeding, a monolayer of keratinocytes was seen on collagen gels that contained a small number of basal β-gal-positive cells amidst a large number of NHK. Within two days, all β-gal-positive, II-4 cells had been displaced to a position above the basal cell layer. It appeared that NHK could actively compete with II-4 cells for basal position and displace them as they preferentially attached to this Type I collagen matrix. This supports the view that the suprabasal distribution of II-4 cells was due to an active sorting process through which these cells were displaced from their initial basal position, leading to their ultimate loss from the tissue. These findings are shown schematically in Fig. 3. When tumor cells were mixed in a 1 : 1 ratio with normal cells, IE clusters were able to form in vitro (Fig. 3A) and tumor cells were able to invade into the connective tissue after in vivo transplantation (Fig. 3C). In contrast, tumor cells grown in the context of a majority of normal cells demonstrated individual, differentiated β-gal-positive cells that were growth-suppressed in vitro (Fig. 3B) and desquamated from the tissue after grafting in vivo (Fig. 3D). By using 3-D tissues that mimic the early stages of epithelial cancer in humans, these Engineering Skin to Study Human Disease 215 Fig. 3 Cancer progression can only occur when intraepithelial dormancy is overcome through the presence of elevated numbers of tumor cells in the tissue. Skin equivalent cultures were grown as mixtures of II-4 and normal keratinocytes at ratios of either 1 : 1 (A)or12:1(B) and grafted to nude mice. 1 : 1 mixtures demonstrated clusters of II-4 cells that that underwent intraepithelial expansion in vitro (A)andinvadedintotheun- derlying connective tissue after transplantation to nude mice (C). However, when 12 : 1 mixtures were grown (B), tissues demonstrated individual β-gal-positive cells that did not expand while grown in vitro and underwent desquamation of II-4 cells from the tissue after grafting (D). This demonstrated that a state of intraepithelial dormancy was induced by surrounding normal cells that suppressed the growth of II-4 cells and prevented their persistence within the tissue at this suppressive ratio. Only when tumor cell clusters of sufficiently large size were present, as in the 1 : 1 mixtures, were II-4 cells able to evade this local growth-suppression and invade into the connective tissue findings demonstrated a novel mechanism for elimination of cells with malignant potential that could suppress early neoplastic progression, namely a tissue-based growth control induced by interactions between adjacent cells that leads to normalization and elimination of sufficiently small numbers of potentially malignant cells. The size of an initiated clone is therefore crucial in determining its survival potential during development of early neoplasia, and progression to malignancy from a premalignant state requires the presence of a number of potentially malignant cells above a critical threshold. In larger tumor cell clusters, greater numbers of cells can interact and can escape this environmental growth suppression. When individual tumor cells were present, they entered a quiescent state known as “intraepithelial dormancy”, in which the full, neoplastic potential of the tissue was not realized. These cells are therefore held in a conditionally-suppressed state and can undergo one of two ultimate fates – either being eliminated from the tissue together 216 J.A. Garlick with adjacent normal cells, or overcoming this dormant state when tumor cells interact with each other in sufficiently large clusters. Further proof of this “conditional” state of growth suppression was shown by determining whether II-4 cells could resume growth when this tissue was disaggregated and single cells regrown in submerged monolayer culture. To isolate only suprabasal II-4 cells from mixtures, SEs were grown in low-calcium media in order to strip all suprabasal cell layers while leaving basal cells attached to the collagen matrix. Suprabasal cells detached as a sheet, which was then trypsinized and single cells grown at clonal density in submerged culture on a 3T3 feeder layer. Expanded colonies of II-4 cells were seen throughout these cultures, proving that growth-inhibited II-4 cells had only transiently withdrawn from the cell cycle when contacting NHK in 12 : 1 mixtures in organotypic culture. These findings supported earlier observations made in the “classical”, two-stage theory of skin carcinogenesis in experimental animals, which had shown that application of a tumor promoter to previously initiated skin pro- duces tumors, while “subcarcinogenic” initiation alone results in no tumors. Initiated skin must therefore contain altered cells that cannot be identified microscopically since they do not form discrete foci and are “operationally normal” in the absence of promotion and in the appropriate cell microenvironment [36]. It has been theorized that these “repressed single mutant cells” are held in a nonproliferating state by feedback from normal, differentiated cells [37]. An epithelium exposed to subcarcinogenic levels of initiating effects may therefore contain large numbers of repressed, initiated cells that may never progress to neoplasia. This may help explain why premalignant lesions such as actinic keratosis of skin, cervical dysplasia, oral leukoplakia and lobular carcinoma of the breast can contain initiated or dysplastic cells that do not always advance to invasive cancer. These studies on the role of 3-D tissue architecture in cancer development directly implicate tissue architecture and the cellular milieu as dominant reg- ulators of the neoplastic phenotype, and support the “tissue organization field theory of carcinogenesis” [32]. In this light, maintenance of normal tissue architecture can constrain potentially malignant tumor cells in a conditionally- suppressed state and this is sufficient to abrogate cancer progression through an intrinsic, tissue-based elimination of tumor cells by normal cell neighbors. Using a 3-D culture system, it has been shown that this phenomenon also occurs in human breast tumorigenesis, where interactions between extracellular matrix (ECM) proteins and their receptors could normalize tissue architecture and revert malignant cells to a normal phenotype [38]. Neoplas- tic progression could occur only if the microenvironment was changed to allow growth of initiated cells so that this suppressive tissue-barrier could be overcome. Clearly, studies on the role of 3-D tissue architecture in tumor biology would not have been feasible without the capacity to adapt engineered human SEs to mimic early cancer progression. [...]... modifications to its microenvironment is central to the induction of clonal expansion and early neoplastic change By facilitating clonal expansion, cells are now more likely to acquire additional genetic changes, thereby leading to future malignant progression Engineering Skin to Study Human Disease 227 3 Three-dimensional skin- equivalent tissue models to study wound reepithelialization of human stratified... normal cells The use of these 3-D tissue models to model the effects of UV-B-induced sun-damage in skin thus supports current theories for malignant progression in UV-damaged skin that propose that UV-B exposure can preferentially induce apoptotic cell death in cells that are not resistant to apoptosis, while adjacent tumor Engineering Skin to Study Human Disease 223 cells that harbor p53 mutations can... levels of these soluble factors as determined by ELISA analysis of supernatants from wounded SEs Taken together, these studies demonstrated the utility of SEs in determining the presence of and response Engineering Skin to Study Human Disease 235 to soluble factors in patterns that simulate wound repair events known to occur in vivo 3.5 Matrix metalloproteinase activity in wounded skin equivalents The matrix... wound repair, as they do not provide the proper tissue architecture to study wound response as it occurs in vivo SEs adapted to study wound repair in human epithelium have been found to simulate the chronology of events that occur during reepithelialization in human skin and have advanced our understand of the healing of wounds in human skin and other stratified epithelia [60] This tissue model has allowed... thus provides an opportunity to study the appearance of wound Engineering Skin to Study Human Disease 229 keratinocytes during the various stages of reepithelialization and epithelial reconstitution (Fig 10) At 8 h, a wedge-shaped epithelial tongue 2–3 cells in thickness was seen at the edge of the wound margin (Fig 10) in a tissue in 230 J.A Garlick Fig 10 Morphology of skin equivalents at various... Since ethical reasons have limited the ability to directly study the effects of UVB irradiation in the skin of human volunteers, the use of skin- like, 3-D SE tissue models containing human keratinocytes offers an attractive alternative for 222 J.A Garlick studying the effects of UV-B irradiation on human skin Using the 3-D tissue models for premalignant disease described above, it has recently been... centrally into the elongating epithelial tongue that partially covers Engineering Skin to Study Human Disease 237 the wound floor At the same time, nondividing cells continually migrated and together with the progeny of proliferating cells, advanced to completely cover the wound floor These early changes mark a phenotypic switch from a proliferating, unwounded epithelium to one in which cell migration is predominant... wounded SEs compared to in vivo wound repair Interestingly, the proliferation of wound epithelium is subject to environmental regulation as the growth response was shown to be sensitive to growth factor regulation, as described below 3.3 Migration in skin equivalents in response to wounding In vivo, wound response is known to alter the temporal and spatial patterns of integrin receptor expression and... thrombin [61] The pres- Engineering Skin to Study Human Disease 233 ence of fibrin was found to accelerate keratinocyte activation and it reduced the time of wound closure when compared to controls that did not contain fibrin This promotion of reepithelialization was associated with the de novo synthesis of α5-integrin, which is not expressed in mature epithelium and is known to be upregulated during... resistant to apoptosis and that the observed expansion of II-4 cells when the two cell populations were mixed and irradiated with UV-B was apparently due to the differential induction of apoptosis of NHK relative to II-4 cells Thus, the differential sensitivity of normal keratinocytes and the early-stage II-4 tumor cells to induction of growth arrest and apoptosis was associated with the expansion of apoptosis-resistant . have been feasible without the capacity to adapt engineered human SEs to mimic early cancer progression. Engineering Skin to Study Human Disease 217 2.2 Factors altering cell–cell and cell–matrix. seen in human skin. In this light, these engineered models of human skin provide powerful new tools for studying disease processes in these tissues as they occur in humans. Keywords Tissue engineering. resistant to apoptosis, while adjacent tumor Engineering Skin to Study Human Disease 223 cells that harbor p53 mutations can undergo clonal expansion. Since it is known that sun-exposed skin contains