Melanocytes: from Morphology to Application

Skin Pharmacol Physiol 2009;22:114–121 Published online: February 4, 2009 DOI: 10.1159/000178870

Melanocytes: From Morphology to Application

a a a, b a A. Santiago-Walker L. Li N.K. Haass M. Herlyn a b Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, Pa. , USA; Immune Imaging Program, Centenary Institute of Cancer Medicine and Cell Biology, University of Sydney, Sydney, N.S.W. , Australia

Key Words skin is organized into two layers: the dermis, comprised M e l a n o c y t e ؒ K e r a t i n o c y t e ؒ M e l a n o m a ؒ Model ؒ S k i n ؒ of fibroblasts and endothelial cells, provides structure Three-dimensional reconstruct ؒ Drug and strength; and the epidermis, containing basal layer melanocytes, Langerhans cells and stratified layers of keratinocytes which form the protective barrier between Abstract skin and the environment. The epidermis and dermis are Melanocytes in human skin are intricately regulated by kera- separated by the basement membrane which is formed by tinocytes and the surrounding stroma. The development of protein secretions of fibroblasts and keratinocytes. Each melanoma is thought to arise from disrupted melanocyte ho- melanocyte, through dendritic projections, interacts meostasis. It is now known that microenvironment plays a with approximately 36 keratinocytes in what is termed major role in maintenance of cellular homeostasis and can the epidermal melanin unit [1]. The control of melano- contribute to tumor initiation and tumor progression. Histor- cyte homeostasis and the process of melanoma develop- ically, melanocyte studies have been performed in two-di- ment are areas of active research. Here we highlight the mensional culture systems, and often with melanocytes cul- importance of using three-dimensional/organotypic cultured in the absence of keratinocytes. Here we present the tures in these studies. biological basis for the use of organotypic, three-dimensional model systems in the study of melanoma, and highlight the features of the most utilized organotypic model sytems. Melanocyte Homeostasis and Regulation by Copyright © 2009 S. Karger AG, Basel Keratinocytes

The proliferating cells of the skin are found primarily Introduction at the basal layer of the epidermis, where the melanocyte

to keratinocyte ratio is approximately 1: 5. During child- Melanocytes are specialized, pigment-producing cells hood, melanocytes proliferate as needed during growth derived from the neural crest. Melanocytes respond to to maintain proper skin architecture. In adults, however, ultraviolet (UV) radiation by synthesizing melanin which melanocytes are for the most part quiescent and rarely forms a photoprotective cap over the nucleus of keratino- proliferate, though they maintain the capacity to do so. cytes, protecting skin cells from DNA damage. Human Melanocyte proliferation is primarily under the control

© 2009 S. Karger AG, Basel Meenhard Herlyn, MD 1660–5527/09/0222–0114$26.00/0 The Wistar Institute Fax +41 61 306 12 34 3601 Spruce Street, Room 489 E-Mail [email protected] Accessible online at: Philadelphia, PA 19104 (USA) www.karger.com www.karger.com/spp Tel. +1 215 898 3950, Fax +1 215 898 0980, E-Mail [email protected] Keratinocyte-derived DecouplingDivision Migration Recoupling factors

SCF, ET-1, GM-CSF E-cadherin

Proliferation ACTH, ␣-MSH Melanogenesis

Melanogenesis Color version available online E-cadherin SCF CCN3 E-cadherin Desmoglein 1 bFGF DDR1 ET-1 ET-3 HGF CCN3

Fig. 1. Dynamics of melanocyte proliferation in human skin. De- Melanocyte coupling: secretion of endothelin-1 (ET-1) by keratinocytes and DDR1 hepatocyte growth factor/scatter factor (HGF) by fibroblasts re- Integrin sults in the downregulation of E-cadherin expression. Division: bFGF, SCF, HGF Basement Collagen proliferation is induced by secreted factors including stem cell Laminin factor (SCF) secreted by keratinocytes and fibroblasts, basic fibro- membrane blast growth factor (bFGF) by keratinocytes and ET-3 by fibroblasts. Migration: expression of the matricellular protein CCN3 Fibroblast-derived and its receptor discoidin domain receptor 1 (DDR1) is required factors for proper melanocyte localization on the basement membrane. Recoupling: expression of E-cadherin is increased, recoupling to keratinocytes and dendrite extension occurs. Fig. 2. Melanocyte microenvironment. Keratinocytes secrete factors that promote melanocyte proliferation and melanogenesis including stem cell factor (SCF), endothelin-1 (ET-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), adreno- corticotrophic hormone (ACTH) and ␣ -melanocyte stimulating ␣ of keratinocytes. Cell-cell contacts with keratinocytes are hormone ( -MSH). Fibroblasts also contribute to melanocyte regulation by secretion of basic fibroblast growth factor (bFGF), known to be important for the maintenance of melano- SCF and hepatocyte growth factor (HGF). Melanocytes maintain cyte homeostasis. In order for a melanocyte to proliferate, proper localization by binding to collagen and laminin on the the following steps are proposed (fig. 1): (1) retraction of basement membrane. Fukunaga-Kalabis et al. [9, 10] recently re- dendrites and decoupling of melanocytes from surround- ported a role for CCN3 and discoidin domain receptor 1 (DDR1) ing keratinocytes and the basement membrane; (2) pro- in melanocyte basement membrane localization. liferation; (3) repositioning via migration along the basement membrane; (4) dendrite extension and recoupling to keratinocytes [2] . Keratinocytes signal to melanocytes to control proliferation via cell-cell contacts mediated by undergo an E- to N-cadherin class switch, allowing me- cadherins as well as by the secretion of signaling mole- lanocytes to escape keratinocyte control as well as pro- cules and growth factors ( fig. 2). moting binding to dermal fibroblasts and endothelial The cadherins are a family of cell surface glycopro- cells. Not only are melanoma cells free of keratinocyte teins which mediate calcium-dependent cell adhesion, control over proliferation under these conditions, but in- and represent the transmembrane components of cell- teractions with fibroblasts and endothelial cells allow cell adhesion junctions. They are homophilic binding melanoma cells to invade and survive in the dermis [5] . proteins allowing cells of the same type, or same cad- N-cadherin induces melanoma cell migration, and pro- herin expression pattern, to bind and organize into tis- motes survival via activation of Akt, increased ␤ -catenin sues [3] . In skin, cadherin expression is cell type specific expression and inactivation of the proapoptotic protein and critical for maintenance of skin architecture and reg- Bad [6] . The importance of this cadherin class switch is ulation of homeostasis. E-cadherin is expressed on mela- supported by experiments showing that re-expression of nocytes, keratinocytes and Langerhans cells of the epi- E-cadherin in human melanoma cells restored keratino- dermis, while N-cadherin is expressed on dermal fibro- cyte coupling and inhibited invasive potential [7] . blasts and endothelial cells [4] . This differential cadherin Integrin-mediated attachment also plays a role in me- expression is exploited in melanoma, where melanocytes lanocyte maintenance. Integrins are cell surface recep-

Melanocytes: From Morphology to Skin Pharmacol Physiol 2009;22:114–121 115 Application tors for extracellular matrix (ECM) proteins such as lam- hesion molecule (Mel-CAM/MUC18) and ␤ 3 integrin inin and collagen. Integrins, which are comprised of het- [13] . Melanocyte phenotype can be rescued by the addi- erodimers of ␣ - and ␤ -subunits, connect the ECM to the tion of keratinocytes to melanocyte cultures. Keratino- actin cytoskeleton and are required for melanocyte at- cytes regain control over melanocyte proliferation such tachment to and release from the basement membrane that when keratinocytes and melanocytes are seeded at during migration. In addition to their role in attachment, fixed ratios, these ratios are maintained as the cells pro- integrins impact multiple aspects of cell physiology in- liferate. The multidendritic morphology of melanocytes cluding proliferation, differentiation and survival. Al- is restored upon coculture. Most interestingly, expression tered integrin expression is thought to help melanocytes of melanoma-associated antigens is lost within 3–4 days survive in the dermis, specifically integrins ␣v ␤3 and of coculture [14] . In support of this, in vivo nevocytes ␣5 ␤1 are indicative of melanoma progression [8] . (nests or groupings of melanocytes) residing in the der- Recently, work from our laboratory has revealed a role mis, separated from keratinocytes, express melanoma- for the matricellular protein CCN3 in melanocyte ho- associated antigens while nevocytes localized in the epi- meostasis. CCN3 (nephroblastoma overexpressed) was dermis do not. Coculture of dermal nevocytes with kera- found to be upregulated in melanocytes and secreted in tinocytes can restore normal melanocytic phenotype response to coculture with keratinocytes. CCN3 expres- [13] . Further evidence for the role of keratinocytes on me- sion inhibited melanocyte proliferation and was required lanocyte homeostatis comes from a recently developed for proper melanocyte localization on the basement mouse model in which the retinoid X receptor ␣ (RXR ␣ ) membrane. CCN3, via the receptor tyrosine kinase dis- was knocked out selectively in keratinocytes. Keratino- coidin domain receptor 1 (DDR1), promotes adhesion to cyte-selective RXR␣ -null mice developed an increased collagen IV on the basement membrane [9] (fig. 2). Inter- number of melanocytic nevi, of which some progressed estingly, CCN3 expression was found to be downregu- to human melanoma-like tumors [15] . lated in advanced melanoma. Overexpression of CCN3 in 1205Lu, a mestastatic melanoma cell line, resulted in decreased expression and activation of matrix metallopro- Organotypic Models in Melanoma teinases and inhibited invasion [10] . Keratinocytes secrete multiple factors that impact me- Disruption of melanocyte homeostasis is considered lanocyte proliferation and melanogenesis. Keratinocyte- to be the basis for melanoma development and progres- derived endothelin-1 (ET-1), granulocyte-macrophage sion. It is now understood that the cellular environment colony-stimulating factor (GM-CSF) and stem cell factor significantly contributes to both normal cellular func- (SCF) regulate both proliferation and melanogenesis. Ba- tion and tumor growth. In order to truly elucidate the sic fibroblast growth factor (bFGF/FGF2) expression can function and regulation of a particular cell type, the con- regulate melanocyte proliferation, while secretion of tributions of the surrounding stroma and nearby cell ␣-melanocyte stimulating hormone and adrenocortico- types should be considered. This is particularly impor- trophic hormone impact melanogenesis and dendrito- tant for the study of tumorigenesis, where the tumor mi- genesis [11, 12] . Fibroblasts can also participate in mela- croenvironment plays a major regulatory role [16] . For nocyte regulation by the secretion of bFGF, SCF and he- the study of melanocytes, and in particular melanoma, patocyte growth factor/scatter factor which stimulates several model systems have been utilized. proliferation and migration via the hepatocyte growth The development of melanoma occurs in a stage-wise factor receptor c-Met. progression. The first stage includes the development of The intricate regulation of melanocytes by keratino- common acquired and congenital nevi with structurally cytes begs the use of organotypic cultures in studying normal melanocytes. The second stage is defined by dys- melanocyte function. Indeed, melanocytes cultured in plastic nevi exhibiting abnormal melanocytic structure the absence of keratinocytes exhibit a melanoma-like and architecture. The third-stage, radial growth phase phenotype. Unlike what is observed in vivo, cultured me- (RGP) melanomas are nontumorigenic and nonmetastat- lanocytes proliferate steadily with a doubling time of 48– ic. Stage 4 consists of vertical growth phase (VGP) mela- 96 h [13] . The dendritic phenotype is lost in monolayer, nomas which are tumorigenic, invasive and have the ca- with most cells adopting a bi- or tripolar morphology. pacity to metastasize. The fifth and final stage is meta- Importantly, cultured melanocytes express multiple mel- static melanoma [17] . An important feature of a mela- anoma-associated antigens including melanoma cell ad- noma model system is that melanoma cells recapitulate

116 Skin Pharmacol Physiol 2009;22:114–121 Santiago-Walker /Li /Haass /Herlyn

their proliferative, migratory and invasive properties in harvested and mixed with a suspension of collagen type I. vitro. The ability to maintain features of the primary tu- These cells are then plated in culture dishes containing a mor can aid in the selection of a model system in which cell-free collagen layer. Once the cellular collagen layer has to study explanted melanoma cells. The goal is to recre- polymerized, culture medium is finally added [19] . ate, as closely as possible, the features of the tumor in the Using the collagen-implanted spheroid model, we have proper microenvironmental context. However, it is also observed differences in cell phenotype between two- and important to consider factors such as ease of use, data in- three-dimensional culture. Cells representing the 3 ma- terpretation, cost and applicability issues such as genetic jor stages of melanoma, RGP, VGP and metastatic, exhib- manipulation and drug delivery when selecting a model ited similar phenotype and motility in two-dimensional system. The following will highlight the pros and cons of culture. However, when the same lines were grown as col- the major model systems and their application in the lagen-implanted spheroids, they reproduced the poten- study of melanoma. tial for motility and invasion characteristic of the original tumor type such that RGP-derived cells were poorly in- Two-Dimensional Culture vasive, VGP cells moderately invasive and metastatic In the preceding paragraphs, the limits of culturing melanoma-derived cells were aggressively invasive [19, melanocytes alone in monolayer have been described. 20] . Differential response to MEK and PI3K inhibition These can be circumvented by coculture with keratino- was also observed. Under two-dimensional conditions, cytes which are seeded at a melanocyte to keratinocyte inhibition of either the MAPK or PI3K pathways inhib- ratio of approximately 1: 5 to 1: 10. Studies using two-di- ited proliferation of cells from all three melanoma stages. mensional melanocyte cultures, with or without kerati- When the same cells were grown as spheroids, only the nocytes, have greatly advanced our knowledge of mela- early melanoma lesions were responsive to single inhibi- noma and will continue to be used extensively. However, tion of these pathways exhibiting decreased growth and results generated in two-dimensional culture can be fur- survival, while the metastatic lines were insensitive. In the thered by the use of model systems that mimic the natural same study, the significance of ECM interaction for tumor tumor environment and may result in the development survival was also reinforced. Collagen had a protective ef- of treatments with better therapeutic outcomes. fect on spheroid survival, as spheroids plated on agar were more susceptible to apoptosis after inhibitor treatment Collagen-Implanted Melanoma Spheroid than spheroids grown on a collagen matrix [19] . This A model which is particularly amenable to drug-screen- model can be adapted and modified as needed. For ex- ing studies is the collagen-implanted spheroid. These con- ample, in order to more accurately represent the tumor sist of cell aggregates grown in a collagen matrix, as col- stroma, fibroblasts can be added to the collagen layer. lagen is the most abundant ECM protein in human skin. The collagen-implanted spheroid can be utilized to Melanoma spheres mimic an in vivo tumor with cells hav- study invasion and cell-cell adhesion, and is readily ap- ing differing access to oxygen and nutrients depending on plied to inhibitor studies. It has been proposed to use position in the spheroid. Similar to a tumor in vivo, the three-dimensional models to develop individualized proliferating cells are found on the outside of the spheroid, therapies. Patient samples cultured three-dimensionally while the innermost cells have downregulated extracellu- and treated with therapeutic drugs can potentially pre- lar signal-regulated kinase and cyclin-dependent kinase dict patient response [21] . activity [18] . Cells grown three-dimensionally exhibit increased expression of drug transporters and are less acces- Human Skin Xenografts on Mice sible via diffusion than two-dimensional cultures, making Melanocytes are differentially localized in human and them suitable for the study of drug resistance. mouse skin. Human melanocytes are found primarily at While there are multiple methods for generating spher- the basal layer of the epidermis and in the hair follicle. In oids, our laboratory has primarily used the liquid overlay mice, however, melanocytes reside deep in the hair folli- method. A cell suspension is plated on top of solidified cle and are not typically present in the dermis. Mice do (1.5%) agar which prevents adhesion to the tissue culture not develop spontaneous melanoma. Developing a mouse plastic. After 48–72 h, the cells aggregate to form spher- model relevant to human disease is therefore a challenge. oids. These spheroids can be used directly in drug toxicity Several mouse models of melanoma have been developed studies. Alternatively, an additional step of collagen im- and are reviewed elsewhere [22] . Undoubtedly, studies plantation can be performed, in which the spheroids are using these models have significantly contributed to the

Melanocytes: From Morphology to Skin Pharmacol Physiol 2009;22:114–121 117 Application Color version available online

a b

Fig. 3. Reconstructed skin. Foreskin keratinocytes and melanocytes plated on a dermal matrix of foreskin fibroblasts and collagen I. a HNE staining of reconstructed skin. The stratified, differentiated keratinocytes are visible as layered epithelial sheets. b Staining of the basement membrane, separating the epidermal and dermal compartments, with antibody to collagen IV. c Mela- nocytes in reconstructed skin stained for the melanocytic marker c S100. Scale bar = 100 ␮ m .

field. Primarily, these models rely on genetic modifica- is infrequent compared with the frequency of squamous tion, with or without chemical or UV stimuli, to induce cell carcinomas in normal skin treated with the carcino- melanoma. Human melanomas are diverse and a partic- gen DMBA and exposed to UV radiation [23]. Alterna- ular mouse model, namely a ras point mutant, may rep- tively, normal skin from melanoma patients or those ge- resent only 1 type of melanoma. Alternative strategies are netically predisposed to melanoma can be grafted, and considered due to the differences in skin architecture be- may result in an increased incidence of melanoma le- tween the mouse and human, low incidence of melanoma sions. The xenograft model can also be used to study mel- development, specific genetic modifications required anoma metastasis in vivo. Melanoma cells injected into and the time-consuming nature of these models. grafted skin proliferate and recapitulate the phenotype of The human skin xenograft model addresses the differ- the original tumor, and metastasize to the lymph nodes ences in architecture between mouse and human skin. In and lungs [24] . Thus, the effect of various treatments and this model, human skin is grafted onto the back of im- genetic manipulations on melanoma progression, inva- munodeficient mice. Skin architecture is preserved using sion and metastasis can be observed. this method. This model can be utilized to determine the effect of environmental factors such as UV irradiation Three-Dimensional Skin Reconstructs and carcinogens, on tumor initiation and development. Of the previously described in vitro models, the or- The development of melanoma or melanoma-like lesions ganotypic skin reconstruct is the most advanced. Recon-

118 Skin Pharmacol Physiol 2009;22:114–121 Santiago-Walker /Li /Haass /Herlyn

Color version available online

Fig. 4. Melanoma cells recapitulate original tumor phenotype in dermis. Vertical growth phase (VGP) cells cross the basement reconstructed skin. Melanocytes and melanoma cells were stained membrane, invade and proliferate in the dermis. Cells from met- for S100. Foreskin melanocytes (FOM) reside at the dermal-epi- astatic (MET) melanoma rapidly invade deep into the dermal dermal junction. Radial growth phase (RGP) melanoma cells pro- compartment. Scale bar = 100 ␮ m . liferate and form nests in the epidermis, but do not invade the

structed skin closely resembles human skin in architec- formed in reconstructed skin, as melanocytes extend ture and composition, with all major cell types represent- dendrites into the upper layers of epidermis and are able ed in physiologically relevant ratios. The reconstructs to transfer pigment to keratinocytes. feature a stratified, differentiated epidermal layer com- There are several options for generation of the der- prised of keratinocytes and melanocytes, a dermal com- mal compartment. Intact, de-epidermized human der- partment containing fibroblasts in a collagen matrix as mis can be used as the basis for the reconstruct. Skin well as a functional basement membrane (fig. 3). As in obtained from surgery or post-mortem is sterilized, and vivo, melanocytes in reconstructed skin are localized at the epidermis removed. The subcutaneous side is seeded the basement membrane interspersed with basal layer with fibroblasts which integrate into the dermis, while keratinocytes. A functional epidermal melanin unit is the epidermal side is seeded with keratinocytes [25] .

Melanocytes: From Morphology to Skin Pharmacol Physiol 2009;22:114–121 119 Application Color version available online

Fig. 5. Two-photon microscopy of reconstructed skin. Melano- ages from 3 angles are shown: (1) top of reconstruct; (2) diagonal cytes labeled with green fluorescent protein were incorporated view; (3) side view showing melanocytes at the dermal-epidermal into reconstructs and imaged using two-photon microscopy; sec- j u n c t i o n . ond harmonic generation signals highlight collagen I in blue. Im-

Synthetic polymers such as polylactic acid, polyglycolic ments with transfected/infected cells and therefore are a acid and polyglactin have been used in dermal recon- powerful tool in which to study protein function. Fig- struction [25] . Alternatively, as done in our laboratory, ure 5 depicts melanocytes labeled with green flurores- dermal equivalents are constructed from a mixture of cent protein incorporated into a skin reconstruct. Recon- fibroblasts and collagen type I plated on a layer of acel- structs were used to assess the role of melanoma-derived lular collagen. After approximately 5 days, the fibro- bFGF in melanoma progression. Unlike normal melano- blasts contract the collagen gel forming a concave area cytes and early RGP melanomas, VGP melanoma cells on which keratinocytes and melanocytes can be seeded. produce bFGF. RGP melanoma cells transduced to ex- The use of de-epidermized dermis allows for the conser- press bFGF took on the characteristics of more advanced vation of the original basement membrane and papil- VGP melanoma acquiring the ability to invade and sur- lary morphology of the skin. In organotypic cultures vive in the dermis and exhibiting tumorigenicity in vivo made from reconstructed dermis, the basement mem- [26]. Genetically modified cells can also be used to deter- brane is formed by keratinocytes and fibroblasts during mine how changes in tumor microenvironment impact the culturing process, and epidermal-dermal junction is melanoma progression, invasion and metastasis. The ef- straight. fect of ␤ 3 integrin on melanoma invasion and survival After formation of the dermal compartment, the der- has been assessed in this way. RGP melanoma cells trans- mis is seeded with melanocytes and undifferentiated ke- duced to overexpress ␤ 3 integrin took on a VGP-like phe- ratinocytes at a ratio of 1: 3 to 1: 20. The cells are incubat- notype and invaded the dermal compartment of skin re- ed for 3–6 days, submerged in medium under low calci- constructs, while control RGP cells only spread horizon- um and low serum conditions. Keratinocyte differentia- tally in the epidermis. ␤ 3 integrin-overexpressing RGP tion and stratification are induced upon exposure to air, melanoma cells were able to survive in the dermis, while when the inserts upon which the reconstructs are plated control cells at the epidermal-dermal border showed are lifted. At some point during the culturing process, signs of apoptosis [27] . melanocytes home to the basement membrane and dis- Skin reconstructs have a life span of approximately 1 tribute amongst basal layer keratinocytes [25] . month, which can be extended to months or years if re- Melanoma cells cultured in this model system exhibit constructs are grafted onto immunodeficient mice, al- the same characteristics as the original tumor ( fig. 4 ). lowing long-term studies to be performed. In this way, RGP cells grow as single cells or clusters in the epidermis, reconstructs containing genetically modified cells can be while VGP or metastatic melanoma-derived cells cross monitored for effects in vivo after grafting. the basement membrane and invade the dermal compartment [26] . Skin reconstructs are suitable for experi-

120 Skin Pharmacol Physiol 2009;22:114–121 Santiago-Walker /Li /Haass /Herlyn

S u m m a r y may not adequately reproduce human tumors or drug re- sponses [21]. This is the case in melanoma where a de- Melanoma development does not occur in isolation, finitive model of human melanoma has yet to be devel- but rather is the result of deregulated signaling between oped. Considering the differences in melanocyte archi- melanocytes, keratinocytes and the surrounding stroma. tecture between mice and humans, the expense and time With this in mind, methods such as three-dimensional constraints associated with the use of mouse models, spheroid cultures, human skin mouse xenografts and re- and the inability of two-dimensional cultures to mimic constructed skin have been applied to the study of mela- tumor microenvironment, three-dimensional spheroid noma. Two recent reviews recognize three-dimensional cultures and organotypic skin reconstructs may, in some in vitro models as the link between two-dimensional cul- cases, represent more easily applied and physiologically tures and whole animal models [21, 28] . Animal models relevant models of melanoma.

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