Histopathogenesis of Malignant Skin Melanoma Induced in Genetically
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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8822-8826, October 1993 Medical Sciences Histopathogenesis of malignant skin melanoma induced in genetically susceptible transgenic mice (melanocytdc hyperplasa/nevi/ulceratdon/invaslon/metastss) BEATRICE MINTZ*, WILLYS K. SILVERS, AND ANDRES J. P. KLEIN-SZANTO Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111 Contributed by Beatrice Mintz, June 28, 1993 ABSTRACT Animal models ofhuman malignant skin mel- Unexpectedly, melanocytes in the grafted skin proliferated anoma were created in melanoma-susceptible Inbred-strain excessively and selectively, close to areas of greatest wound transgenic mice by grafting skin from donors of high- healing, especially near the graft margins (3), thereby strongly susceptibiliblines to hosts of a low-susceptibility line, thereby suggesting a causal role of growth factors and cytokines overcoming the problem ofearly death ofthe more susceptible associated with wound repair (4). The melanocytic lesions animals from eye melanomas. As already descbed [Mintz, B. progressed to malignant melanoma in all skin grafts from the & Silvers, W. K. (1993) Proc. Nadl. Acad. Sci. USA 90, more susceptible of two transgenic donor lines tested, and in 8817-8821], melanocs within the grad selectively prolifer- one-fourth of the grafts from another line; no melanomas of ated in close proimity to areas of greatest wound healing, host onrgin developed. We have proposed that the induction of presumably in reponse to mitogenic factors fom ceils con- this maliancy in susceptible skinby wound healing is merely tributing to wound repair. An orderly sequence of externally a caricature or exaggeration of the same basic biological and visible events ed in malinant melanoma. We emne molecular events as ordinarily underlie the genesis of mela- here the hisogenetic concomitants of these changes and find noma, without skin grafting, and that the exaggeration makes that they deflne a sepwie sequence striknly comparable to those events more accessible to analysis (3). that leding to human cutaneous melanoma. Moreover, the From the externally visible evidence ofprogressive changes histological detais suggest some ofthe underlying mechanisms. in the lesions, we concluded that the development of the While the early lesions are first seen in the superficial dermis transgenic melanomas bears a stiking resemblance to that of in the mouse, and in the basal layer of the epidermis in the human skin melanomas and is an excellent in vivo model ofthe human, both progress by radial growth followed by vertical human malignancy (3). This model has the further advantages growth. Melanocytic hyperplasa resulted in nests of densely ofgenetic uniformity and ofthe absence ofany other kinds of melanied fisform cells which were lodng their dendrites. skin tumors in the animals. In the present report, we describe Some discrete lesons in the deep dermis appered as blue nevi. the histogenetic evidence in support of a multistep tumor As radial proferation advanced, ceilular atypia insed and progression paralleling the sequence in human cutaneous the preiouly independent melanocytes cohered dosely and melanomas. In addition, details visible only at the microscopic formed a smail solid tumor; the ceils were usua_ly then hy- level suggest mechanisms on which some of the changes pomelanotic or amelanotic. Ulceration of tumor through the leading to malignancy may depend. epidermis ourrd early. The tumor mass grew rapidly in the deep dermis and invaded and destroyed subcutaneous tissue MATERIALS AND METHODS and muscle. Primary tumors in the skin were often heteroge- neous, with lobules or regions differing in pigmentation or The same transgenic mouse lesions as in the companion atypia. However, the cells in crulating emboli, or in metas- report (3) were examined histologically here. These orgi- tases in lymph nodes and lungs, appeared relatively homoge- nated in donor body-skin grafts from line 8 [hemizygous for neous. These genetically uniform transgenic mouse models the transgene (Tag!-), and line 9 (Tag/Tag homozygotes), provide experimental access to the multistage genesis of mel- each developing in a line 12 (Tag!-) host. Wild-type body- anoma. skin grafts, experimentally wounded but ungrafted transgenic skin, and unmanipulated wild-type and transgenic skin were The Tyr-SV40E transgene targets expression of the simian also included. Tissues were fixed in neutral formalin, em- virus 40 oncogenic sequences to pigment cells in transgenic bedded in paraffin, and stained with hematoxylin/eosin. mice and results in spontaneous malignant melanomas of the Some sections were immunostained as described (1) to vi- eyes and various internal organs; skin melanomas tend to sualize the S-100 or HMB-45 proteins associated with ma- arise later (1, 2) and have only rarely attained malignancy lignant melanoma. Staining for 3,4-dihydroxyphenylalanine before the mice die-usually of invasive and metastatic eye (dopa) was used in some cases to detect nonmelanized melanomas. This problem was recently overcome, and ma- pigment cells (along with melanized ones). lignant skin melanomas were obtained, by taking advantage experimentally of the fact that different inbred lines of the RESULTS AND DISCUSSION mice differ in level of expression of the transgene and hence in degree of susceptibility to melanoma; yet all are members Melanocytes in C57BL/6 Wild-Type and Transgenic Body ofthe same standard strain, C57BL/6. Thus, by grafting skin Skin. Pigmented melanocytes in the fur-bearing body skin of from high-susceptibility transgenic donors to low-suscepti- the wild-type C57BL/6 mouse occur chiefly within the hair bility transgenic hosts, the skin outlived the donors in im- follicles. They are virtually absent in the body skin outside munocompetent hosts tolerant of the transgene product (3). the follicles, although they are present in the skin of other regions such as the ears and tail (5). Some dopa-positive The publication costs ofthis article were defrayed in part by page charge unpigmented melanocytes (or melanoblasts) persist in the payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 8822 Downloaded by guest on September 27, 2021 Medical Sciences: Mintz et al. Proc. Natl. Acad. Sci. USA 90 (1993) 8823 epidermis and superficial dermis ofbody skin and synthesize most densely pigmented areas ofthe grafts, discrete blue nevi melanin when the mice are exposed to ultraviolet light (6). In were not apparent externally; however, similar lesions were contrast, pigmented melanocytes occur in the epidermis and found histologically. Among the several dozen melanocytes dermis ofhuman body skin, especially in the basal layer ofthe in such nevi, some were spindle shaped and others were epidermis (7). The epidermis itself is much thinner in the epithelioid (Fig. 1 c and d). The admixture with dense dermal haired areas of mouse skin than in human skin and has collagen fibers, other connective tissue elements, and mela- less-defined strata. nin-containing macrophages (or melanophages) was similar The body skin of Tyr-SV40E transgenic control mice was to the description of human blue nevi (13). found to differ from wild-type controls of the same inbred Melanomas. In no case did a discrete and isolated blue strain in having occasional pigmented melanocytes in the nevus, situated outside the regions of wound-associated superficial dermis and mid-dermis. In older transgenics, the dense melanocytic hyperplasia, become a melanoma in the cells were seen in the mid-dermis and deep dermis. Small flat course of the graft experiment, spanning over 1 year. It is black spots or macules also appeared spontaneously in the therefore likely that many of these are indolent lesions older transgenic mice. The macules had regular borders and generally destined to remain benign, as is often the case in the consisted of groups of loosely associated pigment cells in the human cellular blue nevus (13). Nevertheless, the very grad- dermis. The accumulation of pigmented cells in unusual num- ual nature ofthe series ofchanges seen in our skin grafts, and bers or sites has also been described in various internal organs the physical contiguity of nevi, blue nevi, atypical nevi (14), of the transgenic mice and was referred to as melanosis (2). and melanoma, support the view that certain nevi are in fact Melacytic Hyperp_sa In Grafted Transgec Skin. After the precursors ofthe mouse melanomas, as is likely in human transplantation, the number of isolated melanocytes in the melanomas (15). In Fig. ld, this contiguity is apparent in the dermis increased, in comparison with ungrafted transgenic deep dermal blue nevus (right arrowhead), the transitional skin of similar age, so that scattered dendritic pigment cells area (immediately above) with decreasing cellular melaniza- were not difficult to find (Fig. la). A much more striking tion, and the early melanoma (left arrowhead). It seems change appeared within a few weeks after grfting and was possible that paracrine influences are needed for nevi to make readily apparent externally as a blackened localized ringjust this transition; such factors may be more readily available inside the margins of the graft and occasionally as a more where injury and wound repair are in progress. irregular black tracery in the interior of the graft (3). These As documented in Fig. 1 c, d, and g, atypical