Graft Chamber Mice Skin

" Continuous human cell line, contains Adl2-SV40. h Papilloma cell lines, contain activated c-rasHa. ' Phenotype dependent on high v-rasHa expression.

'' Cultured epithelial sheet implanted intact between dorsal musculature and skin.

Skin reconstitution or grafting is a more widely used method since it has the advantage of allowing the study of cells at intermediate stages of premalignant progression. In some studies the graft site on the host is prepared by inserting a ground glass disk (26 x 3 mm) between the skin and thoracic wall to induce a capsule of granulation tissue.84 The disk is first inserted at an incision at the base of the tail and moved up to the center of the back. After 3-4 weeks the disk is removed and a silicone grafting chamber is held in place using surgical clips. The grafting chamber (Renner GMBH, 6701 Dannstadt-Schauerheim 1, Riedstrasse 6 Germany) consists of an upper hat-shaped dome with a central 3-mm hole and a lower

Transplantation Chamber

Transplantation Chamber

Chamber Grafting Skin

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Fig. 3. Schematic cross section oi silicone grafting chamber held in place under dorsal skin with surgical clips.

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Fig. 3. Schematic cross section oi silicone grafting chamber held in place under dorsal skin with surgical clips.

chamber which fits into the dome that is an open cylinder with a broad rim95 (Fig. 3). More recent studies have shown that the granulation bed is not required for successful transplantation.95,96 In this method the assembled chamber is put in place immediately before adding the test cells. The dorsal epidermis of anesthetized mice is wiped with Betadine and 70% ethanol. An approximately 1-cm-diameter circle of dorsal epidermis is removed, using curved scissors, and the wound is treated with an antibiotic spray (Polysporin, Burroughs-Wellcome, Research Triangle Park, NC). The assembled grafting chamber is inserted directly, with the rim of the chamber underneath the host skin, and is held in place with surgical clips. In both methods the graft chambers are removed 1 week after the addition of the transplanted cells, and the grafts are allowed to heal.

Two types of cell preparation techniques have been employed: (1) transplantation of an organotypic culture directly to the graft site, or (2) transplantation of suspensions of keratinocytes and fibroblasts. In the first method, either mouse or human keratinocytes are plated in petri dishes on a thin collagen gel prepared within the lower part of the grafting chamber. Dermal fibroblasts can also be plated directly into the collagen gel prior to plating the keratinocytes. After 2 days in culture, the top of the grafting chamber is added and the culture is transplanted to the host.97 Alternatively,

95 J. E. Strickland, A. A. Dlugosz, H. Hennings, and S. H. Yuspa, Carcinogenesis (London) 14, 205 (1993).

96 W. C. Weinberg, L. V. Goodman, C. George, D. L. Morgan, S. Ledbetter, S. H. Yuspa, and U. Lichti, J. Invest. Dermatol. 100, 229 (1993).

97 A. Bohnert, J. Hornung, I. C. Mackenzie, and N. E. Fusenig, Cell Tissue Res. 244,413 (1986).

suspensions of epidermal cell lines or primary epidermal cultures and primary dermal fibroblasts96 can be combined, pelleted, and the thick slurry of cells added directly to the grafting chamber in place on the host, through the hole in the top dome.95 Routinely 5-6 x 106 epidermal cells are combined with 8 x 106 fibroblasts. It is important to use dermal fibroblasts that have been cultured for at least 1 week to avoid generation of hair in the graft96 and to remove any epithelial cells that could modulate the behavior of the test cells. Aseptic techniques must be used at all times in preparation of cells and during grafting since transplanted cells usually do not survive if the graft site becomes infected. Growth of transplanted tumor cells is usually apparent by 1 week after dome removal.

In addition to histological characterization, graft tumors can be analyzed using a series of markers that are associated with malignant progression of epidermal tumors. Tumors generated from oncogene-transduced grafted mouse keratinocytes have a similar pattern of marker expression as chemically induced mouse skin tumors.98 Specific polyclonal antibodies have been developed which allow in situ localization of protein markers.99 Keratins 1 and 10 are expressed in normal epidermis and well-differentiated papillomas, whereas keratin 13 is expressed during premalignant progression, often in cells that have lost expression of keratins 1 and 10.100 Keratin 8 is expressed only in highly dysplastic papillomas and in squamous carcinomas.100101 Additionally, the «6/34 integrin is a useful marker for the basal compartment which expands during the premalignant progression of mouse papillomas.100 TGF-/31 and TGF-/32 immunostaining is also lost during the premalignant progression of chemically induced and grafted mouse skin tumors.102 Human tumors also have a similar loss of keratins 1 and 10, and appearance of keratin 8.103 However, expression of markers in grafted oncogene-transduced keratinocytes has not been characterized. In general, sections from tissues frozen in OCT (Miles, Elkhart, IN) give the best results in immunohistochemistry, although Carnoy's fixative (60% ethanol:30%

9R T. Tennenbaum, S. H. Yuspa, A. Grover, V. Castronovo, M. E. Sobel, Y. Yamada, and

L. M. De Luca, Cancer Res. 52, 2966 (1992). "c) D. R. Roop, C. K. Cheng, L. Titterington, C. A. Meyers, J. R. Stanley. P. M. Steinert. and S. H. Yuspa, J. Biol. Chem. 259, 8037 (1984).

T. Tennenbaum, A. K. Weiner, A. J. Bélanger, A. B. Glick, H. Hennings, and S. H. Yuspa. Cancer Res. 53, 4803 (1993).

11.1 F. Larcher, C. Bauluz, M. Diaz-Guerra, M. Quintanilla. C. J. Conti, C. Ballestin, and J. L. Jorcano. Mol. Carcinog. 6, 112 (1992).

11.2 A. B. Glick, A. B. Kulkarni, T. Tennenbaum, H. Hennings, K. C. Flanders, M. O'Reilly, M. B. Sporn, S. Karlsson, and S. H. Yuspa, Proc. Natl. Acad. Sci. U.S.A. 90, 6076 (1993).

"" 1. M. Leigh, P. E. Purkis, A. Markey, P. Collins, S. Neill, C. Proby, M. Glover, and E. B. Lane, in "Skin Carcinogenesis in Man and in Experimental Models" (E. Hecker, E. G. Jung, F. Marks, and W. Tilgen, eds.), pp. 179-191. Springer-Verlag, New York, 1993.

chloroform: 10% v/v acetic acid) or ethanol fixation (70%) is also compatible with keratin immunostaining. Expression of the hair follicle-associated enzyme y-glutamyl transpeptidase also occurs in squamous carcinomas, and this can be detected with a histochemical stain.104 It is also useful to analyze cell proliferation in the tumors by immunohistochemical detection of bro-modeoxyuridine (BrdU) incorporated into DNA.105 Changes in number and the distribution of labeled nuclei are generally associated with premalignant progression and malignant conversion.100'102 For BrdU staining, tumor-bearing animals are injected 1 hr before sacrifice with 250 /xg/g of BrdU in 0.9% saline. Tissues fixed in either 70% ethanol or frozen in OCT can be used. Anti-BrdU antibodies are commercially available and suppliers provide the methods for use (Becton-Dickinson, San Jose, CA, Cat. No. 7580, and Zymed, San Francisco, CA, Cat. No. 93-3943).

Acknowledgment

The authors thank Margaret Taylor for secretarial assistance.

104 C. M. Aldaz, C. J. Conti, F. Larcher, D. Trono, D. R. Roop, J. Chesner, T. Whitehead, and T. J. Slaga, Cancer Res. 48, 3253 (1988).

105 H. S. Huitfeldt, A. Heyden, O. P. F. Clausen, E. V. Thrane. D. Roop, and S. H. Yuspa, Carcinogenesis (London) 12, 2063 (1991).

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