Morphological, Biological, and Biochemical Characteristics of a Benign Human Trichilemmoma Cell Line in Vivo and in Vitro'

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[CANCER RESEARCH 41, 2468-2475. June 1981] 0008-5472/81 /0041 -OOOOS02.00 Morphological, Biological, and Biochemical Characteristics of a Benign Human Trichilemmoma Cell Line in Vivo and in Vitro'

Tamotsu Kanzaki,2 Hikaru Eto, Akira Umezawa, Tohru Maeda, Hitoo Iwase, and Masatsugu Ito

Departments of Dermatology [T. K., H. E., A. U.Â¡,Obstetrics-Gynecology Â¡T.M.], Biochemistry [H. I.], and Plastic Surgery [M. I.], Kitasato University School of Medicine, Sagamihara 228. Japan

ABSTRACT but she had left it alone for over 40 years. The tumor did not change in size during this period. In June 1978, the tumor bled A cell line of a benign human tumor, trichilemmoma, was for the first time after a traumatic brushing with a comb and established in vitro and has been maintained in culture for 1.5 then started to grow aggressively. The tumor was elastic, soft, years with more than 30 passages. Plating efficiency was less and 7 x 7 x 3 cm in size (Fig. 1) in February 1979. The surface than 0.1%, and population doubling time was 10 days. Satu ration density was 106 cells/sq cm at the time of a monolayer of the tumor was eroded with telangiectasia. It appeared yel lowish and somewhat translucent. The eroded surface was with 98% cell viability. Ultrastructurally, tissue-cultured trichi coated with pus. The left cervical lymph nodes were softly lemmoma cells showed desmosome-tonofilament complexes at swollen and freely movable. cell-to-cell junctions. The tissue-cultured cells synthesized abundant glycogen (50 to 100 ^g/106 cells) as observed in Tissue Culture. The details of the tissue culture methods have been reported previously (14, 17, 18). Briefly, tumor vivo. Gas Chromatographie analysis revealed that extracted tissue obtained at surgery was minced into 1-cu mm pieces, glycogen was composed of glucose alone. Chromosome anal and primary culture was initiated in 35-mm plastic dishes yses with trypsin-Giemsa banding showed an abnormal kary- (Falcon Plastics, Los Angeles, Calif.) using Eagle's minimal otype with hypodiploid modal numbers of 44 and 45. There essential medium (Nissui Seiyaku Co., Ltd., Tokyo, Japan), were four marker chromosomes observed in 100% of cells in which contained glucose as a source of monosaccharide, 100 metaphase cells examined. Cells did not grow on fibroblast supplemented with 10% fetal bovine serum (Grand Island Bio monolayers or in soft agar in vitro but did induce tumors in logical Co., Grand Island, N. Y.), penicillin (100 units/ml), and athymic nude mice (12 of 15) after the s.c. injection of tissue- cultured cells (2.5 x 106 to 4.5 x 107 cells/mouse). The streptomycin (100 /Â¿g/ml) (Grand Island Biological Co.) in a humidified incubator (New Brunswick Scientific Co., Inc., Edi histological characteristics of the tumors in nude mice were son, N. J.) with 5% CO2 in air at 37Â°.Tumor cells migrated out similar to those of the original tumor. This is the first time, to from the primary explants on the third day of culture and then our knowledge, that a benign human tumor cell line has been propagated slowly but continuously in vitro. The culture be established in vitro which can induce tumors in nude mice. came free from fibroblasts after repeated treatments with 0.25% trypsin. Subculture was done with 0.25% trypsin in INTRODUCTION 0.5% EDTA solution (Toshiba Kagaku Kogyo Co., Tokyo, Ja pan) every 2 to 4 weeks. Growth factors, 3T3 fibroblast feeder Trichilemmoma (5, 6, 15, 20, 24) is a benign human tumor layers, or collagen gel substrates were not used in this culture of epidermal appendages, arising from or differentiating toward at any time except when special studies were performed (see the trichilemma (outer root sheath of the hair follicle). Although below). We did not have HeLa cells in our laboratory. This some tumors grow rapidly, it is well differentiated and well study was performed by using cells cultured in vitro for 7 to 10 organized histologically and is benign biologically (5, 6, 15, months (6 to 10 passages) after primary culture. Morphologi 20, 24). cal, biological, and biochemical characteristics have not been We had a case of trichilemmoma, misdiagnosed as basal cell changed since the study. epithelioma (carcinoma) because of its clinical appearance. Morphological Studies. The light-microscopic observations We initiated tissue culture of this tumor and have established of the original tumor were made after stains with HE,3 PAS with a benign human tumor cell line in vitro. This report describes and without amylase digestion, and Sudan III solutions. the morphological, biological, and biochemical characteristics For the electron-microscopic studies, the original tumor was of this trichilemmoma cell line in vivo and in vitro, including fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH tumorigenicity in nude mice. 7.4) and processed thereafter for electron microscopy as de scribed previously (14, 17). Thin sections were stained with MATERIALS AND METHODS uranyl acetate and with lead citrate (28) and observed in a Hitachi HU-12A electron microscope at 75 kV. Light-micro Clinical Summary. A patient, a 68-year-old Japanese fe scopic examination of tissue-cultured cells was made with a male, showed a large tumor on her scalp in February 1979. In Nikon phase-contrast microscope with an attached camera or her twenties, the patient had noted an asymptomatic, small (5 made after stains with HE or PAS solutions. For electron- x 5 mm in diameter), reddish tumor on the left temporal area, microscopic studies, cells were cultured on thin-sectionable plastic coverslips (Microbiological Associates, Inc., Bethesda, ' This work was supported by research grants from the Japanese Dermatolog ica! Association and the Lydia O'Leary Memorial Foundation. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: HE, hematoxylin and eosin; PAS. periodic acid- Received November 18. 1980; accepted February 5, 1981. Schiff.

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Md.), fixed in situ with 1% glutaraldehyde and processed However, cell propagation was very slow, and it took 10 weeks thereafter as described previously (14, 17). to make a 20-mm-diameter sheath. The first passage was Biological Studies. Population doubling time was estimated successfully done at the tenth week. Subculture was repeated by daily count of cells up to 14 days after seeding. Plating every 4 weeks until the sixth month in culture (fourth passage). efficiency was examined after seeding of 102 to 105 cells onto About this time, it was found that an extremely large number of 35-mm plastic dishes. Saturation density was obtained by cells (10s cells/sq cm) was required for subculture to make counting cells at the time of a monolayer. One hundred to 105 cells propagate well. By satisfying this requirement, cells prop cells/35-mm dish were seeded on fibroblast monolayers (1 ) or agated steadily, and subculture was done every 2 weeks. The plating efficiency was very poor, less than 0.1%. When 10" in soft agar (22) to observe cell growth in these conditions. Biochemical Studies. Since tissue-cultured cells synthe cells were seeded in 35-mm dishes, only a few (3 to 5) colonies sized abundant glycogen in vitro, glycogen content in cells was consisting of more than 50 cells were made in 4 weeks. Cell determined by the phenol-sulfic acid method (9). Extracted propagation greatly depended upon cell density. When 105 glycogen was analyzed by gas chromatography (34) to deter cells/sq cm were seeded, cell number became double on the mine the constituent carbohydrate. tenth day (the population doubling time was 10 days). The cell viability was 98%. When 5x10" cells/sq cm were seeded, it Chromosome Analyses. Chromosome analyses of 100 tis sue-cultured cells were done with conventional Giemsa staining took 4 to 8 weeks to reach a monolayer. The saturation density (12) and with trypsin-Giemsa banding by the method of Sea- at a monolayer was 10s cells/sq cm. When 10" cells/sq cm bright (30). were seeded, many cells died slowly with cytoplasmic vacuoli- Tumorigenicity. Fifteen athymic nude mice (NIH strain, Ni zation, and the propagation to death ratio of cells was one. non Clea, Tokyo, Japan) were inoculated s.c. with 2.5 x 106 Thus, these cells did not reach a monolayer in 4 months of to 4.5 X 107 tissue-cultured cells. Tumorigenicity was ob culture. When culture was continued after a monolayer, cells served for 6 to 9 months after inoculation. propagated continuously, making double to triple layers in 10 Preservation of Cells by Freezing. One to 2 million cells to 14 days from a monolayer. Cells started to pile up after were suspended in 1 ml of normal culture medium containing reaching triple layers. In the older culture, more than 4 weeks 5% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.) in culture, some cells of the uppermost layer became flat and and frozen at -80Â°. More than 90% of cells were viable at detached from the underlying cells. These cells looked like thaw, but all of them died with swelling in 24 hr when cultured stratum corneum cells in vivo. in normal culture medium. However, 30 to 50% of cells were Morphological Studies of Tissue-cultured Cells. Light mi viable and propagated when cells were cultured in Ca2+ (2.8 croscopically, cells were flat and epithelioid (Fig. 4, A and ÃŸ). mM)-added medium. Electron microscopically, tonofilaments and desmosome-ton- Mycop/asma Culture. Tissue-cultured medium containing 1 ofilament complexes were observed in cytoplasm and cell-to- x 106 cells was spread onto Mycop/asma agar and fluid broths cell junction sites, respectively (Fig. 4C). Half desmosomes (BRL Division of Becton, Dickinson, and Co., Cockeysville, were observed at the cell membrane where it was lying against Md.) and allowed to incubate at 37Â°for 4 weeks. plastic substrate. Glycogen granules of the dispersed type were observed in cytoplasm (Fig. 4C). Intranuclear inclusions RESULTS observed in the original tumor were not observed in tissue- Light- and Electron-microscopic Studies of the Original cultured cells. In older cultures, the cell membrane of flattened Tumor. The tumor was solid and lobulated (Fig. 2, A and ÃŸ). cells was highly invaginated. The inner cell membranes in these Tumor cells were well organized and well differentiated without cells were thickened; i.e., marginal bands (13) were formed, atypia (Fig. 2, ÃŸto E). Cells at the periphery of lobules were thus showing evidence of cellular keratinization in vitro. cylindrical and palisading on basal membrane (Fig. 2, ÃŸto E). Biochemical Studies (Glycogen Analysis). Virtually 100% The cell shape gradually became cuboidal toward the center of the cells synthesized abundant glycogen in vitro. Histochem- of lobules (Fig. 2, ÃŸto E) where microabscesses were present ically, cytoplasm stained strongly with PAS stain (Fig. 4/\) but (Fig. 2, ÃŸ,D, and E). There was little evidence of cellular did not stain after amylase digestion (Fig. 4ÃŸ).Electron micro keratinization or cyst formation. The cytoplasm of the tumor scopically, glycogen granules were observed in cytoplasm (Fig. cells was clear with HE stain (Fig. 2, B and C). The cytoplasm 4C). Qualitatively, the glycogen fraction extracted from these contained abundant PAS-positive granules which were di cells showed only one peak at the glucose position by gas gested by amylase (Fig. 2, D and E). No cytoplasmic lipid chromatography, meaning that the glycogen fraction was com droplets were detected with Sudan III stain. Thus, histological posed of glucose alone without other monosaccharides such appearances of the original tumor resembled those of trichilem- as fucose, mannose, or galactose. Quantitatively, 50 to 100 mal cells (outer root sheath of the hair follicle). Fibroblast jug of glycogen were present in 106 cells. proliferation, neutrophil infiltration, and hyalin substance were Chromosome Analyses. One hundred metaphase cells were observed in the stroma (Fig. 2, ÃŸto E). Electron microscopi examined with Giemsa and trypsin-Giemsa stains. Chromo cally, tonofilaments and glycogen granules of dispersed (ÃŸ) some number varied from 42 to more than 150 with hypodiploid type were observed in cytoplasm, and desmosome-tonofila- modal numbers of 44 and 45 (Table 1). Four marker chromo ment complexes were observed at cell-to-cell attachment sites somes were identified by trypsin-Giemsa banding (Fig. 5). All (Fig. 3). Particle-like inclusions (45 nm in diameter) were ob of these 4 marker chromosomes were present in 100% of served in nuclei (Fig. 3). 100 cells examined. A typical karyotype was 45,XX,1p + , Tissue Culture and Cell Kinetics. Many epithelial cells mi del(1 )(p13),del(3)(p13), 11 q + , -15,-18,-19,-21,-21, grated out from the primary explants on the third day of culture del(22Xq13), + mar1, + mar2, + mar3, + mar4 as shown in Fig. and made large sheaths (7 mm in diameter) on the fifth day. 5.

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Table 1 Distribution of chromosome numbers in trichilemmoma cells ChromosomeNo.

of metaphases42443124419452046147-59060-69770-9221>9216

Tumorigenicity. Tissue-cultured cells (2.5 x 106 to 4.5 x accelerates rather than declines after reaching a monolayer in 107) were injected s.c. into 15 nude mice. When less than 107 this cell line. cells (2.5, 5.0, and 7.5 x 106 cells each) were injected into 3 Very poor plating efficiency, slow propagation, and no mice, no mouse developed a tumor. However, when more than growth on fibroblast monolayers (1) or in soft agar (22) in vitro 1.5 x 107 cells (1.5, 3.0, and 4.5 x 107 cells, each in 4 mice) suggest that these cells are benign. A pile-up phenomenon were injected, tumors appeared in all mice (12 of 12). Small which was observed in this cell line does not necessarily tumors first appeared in 2 to 3 weeks, and these tumors then indicate cancer, because benign epidermal keratinocytes pile grew continuously. Tumors reached 1 to 2 cm in diameter in 2 up in vitro (8, 23, 29). It was therefore a surprise to observe a to 3 months (Fig. 6A). Tumor cells grew well when placed back successful growth of tumors in nude mice after the injection of in the culture. These tumors have been serially passed through these benign tumor cells. In the literature, however, benign 6 generations of nude mice and have maintained histological human tumors have been reported to grow or produce tumors patterns of growth which resembled those of the original tumor in athymic nude mice; a benign teratoma and 3 other benign (Fig. 68). tumors have been reported to have grown well in immunosup- No Mycoplasma was detected in repeated cultures. pressed mice (3). A fibrocystic disease with duct papillomatosis showed abundant growth in a nude mouse (26), and a papillary adenoma of the large bowel grew appreciably in a nude mouse DISCUSSION (36). Likewise, 3 cases of benign mixed tumors of the salivary gland grew in nude mice (32), and the RPMI 4098 cell line Trichilemmoma has been classified under benign pilar epi- derived from normal blood lymphocytes grew in nude mice theliomas of outer root sheath origin (5, 6, 15, 20, 24). Since (10). Therefore, our benign tumor is apparently not the first tumor growth is sometimes very rapid and since tumor cells one to show tumorigenicity in nude mice. However, all of the may be clear with HE stain, at one time, trichilemmoma was above-described benign solid tumors were made by the direct confused with clear cell carcinoma (21 ), sebaceous carcinoma transplantation of tumor tissues and not by the injection of (4, 31 ), or sweat gland carcinoma (33). A similar tumor, named tissue-cultured cells. In other words, none of these tumor cells proliferating trichilemmal cyst (35) or pilar tumor of the scalp was developed into a continuous line like ours. Taking advan (20), is also considered to originate from or differentiate toward tage of the tissue culture system, our original tumor and its cell the outer root sheath of the hair follicle, but this tumor shows line could be extensively investigated and thoroughly docu extensive keratinization and cyst formation (7, 20, 27, 35). mented clinically, morphologically, biologically, biochemically, Similar tumors and tumors seemingly identical to trichilem and karyologically. moma or proliferating trichilemmal cyst have been reported as Trichilemmoma cells in culture and in nude mice have syn trichochlamydocarcinoma (16). One of 7 cases was reported thesized abundant glycogen, as do normal hair follicle cells to have metastasized to a regional lymph node (16). Although and trichilemmoma cells in vivo. Glycogen synthesis in vitro our clinical impression was that the tumor was malignant, it appears to be unique to trichilemmal cells, because epidermal was benign histologically. No lymph node metastasis was pres keratinocytes and squamous cell carcinoma cells [COLO 16 (25)] do not synthesize glycogen appreciably in vitro." The ent despite such a long period of neglect. The patient was healthy and showed no local recurrence or distant metastasis glycogen metabolism in these cells in vitro has been reported 1.5 years after the surgical excision. elsewhere (19). Histogenesis of trichilemmoma is unknown, but it has been Chromosome analysis revealed the presence of 4 marker suggested that the cause is a papilloma virus infection (2, 11). chromosomes in 100% of the metaphases analyzed. One If so, the intranuclear inclusions observed in this tumor may be marker (M1) appears to be a metacentric. Banding pattern viral inclusions, although these particles (45 nm) were slightly indicated that the M1 marker was an 18p + . Other markers smaller than those of typical papilloma viruses (53 nm). Further (M2, M3, and M4) were too small to identify their origins. The studies are certainly needed to elucidate the oncogenesis of most interesting and significant finding is the presence of 4 pilar epithelioma. marker chromosomes in all cells examined. This result strongly The reason we have successfully established a cell line of a suggests that the large tumor observed clinically was mono benign human tumor without using epidermal growth factors, clonal in origin. collagen gel substrates, or 3T3 fibroblast feeder layers is the In summary, we have reported the establishment of a cell line finding that the cell propagation greatly depended upon the of a benign human tumor, trichilemmoma, in vitro. To the best cell density; more than 105 cells/sq cm (a monolayer of cells) of our knowledge, this is the first time a long-term cell line has were required for the subculture to make cells propagate well. been established from a benign tumor of the human skin. This phenomenon is extremely unusual in cell cultures, be cause the cell propagation is well known to cease or decelerate in the most benign or malignant cells after reaching a mono- 4T. Kanzaki, H. Eto, A. Umezawa,T. Maeda, H. Iwase, and M. Ito, unpublished layer. It is not known at present why the cell propagation observation.

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ACKNOWLEDGMENTS malignant melanoma cell lines in athymic nude mice. J. Nati. Cancer Inst., 62. 1151-1158, 1979. We wish to thank Haruo Takamiya for Mycoplasma cultures and Michiko 19. Kanzaki, T., Maki, I., Yonemoto, K., and Eto, H. Biochemical characteristics Ohno, Setsuko Sato, Shichiro Miyazawa, Tomoko Nakagawa, and Yasuo Moro- of trichilemmal cells in vitro. Clin. Res., 28: 570, 1980. 20. Lever. W. F., and Schaumburg-Lever. G. Tumors of the epidermal append hoshi for their excellent technical assistance. ages. In: W. F. Lever and G. Schaumburg-Lever (eds.), Histopathology of the Skin, Ed. 5. pp. 493-561. Philadelphia: J. B. Lippincott Co.. 1975. REFERENCES 21. Liu, Y. The histogenesis of clear cell papillary carcinoma of the skin. Am. J. Pathol., 25. 93-103, 1949. 1. Aaronson, S. A., Todaro, G. J., and Freeman, A. E. Human sarcoma cells in 22. Macpherson, I., and Montagnier, L. Agar suspension culture for the selective culture. Identification by colony-forming ability on monolayers of normal assay of cells transformed by polyoma virus. Virology, 23: 291 -294, 1964. cells. Exp. Cell Res., 61: 1-5, 1970. 23. Marcelo, C. L., Kim, Y. G., Kaine, J. L., and Voorhees, J. J. Stratification, 2. Ackerman, A. B. Trichilemmoma. Arch. Dermatol., 114: 286, 1978. specialization, and proliferation of primary keratinocyte cultures. Evidence 3. Bernbaum, M. C., Sheard, C. E., Reittie, J. R., and Bundick, R. V. The of a functioning in vitro epidermal cell system. J. Cell Biol., 79: 356-370, growth of human tumours in immunosuppressed mice and their responses 1978. to chemotherapy. Br. J. Cancer, 30. 13-32, 1974. 24. Mohlenbeck, F. W. Trichilemmoma. Eine Studie von 100 fallen. Z. Hautkr., 4. Bishop, E. L. Epidermoid carcinoma in sebaceous cysts. Ann. Surg., 93. 49:791-795, 1974. 109-112, 1931. 25. Moore, G. E., Merrick, S. B., Wood, L. E., and Arabasz, N. M. A human 5. Brownstein, M. H., and Shapiro, L. Trichilemmoma. Analysis of 40 new squamous cell carcinoma cell line. Cancer Res., 35: 2684-2688, 1975. cases. Arch. Dermatol., 107: 866-869, 1973. 26. Qutzen, H. C., and Custer, R. P. Growth of human normal and neoplastic 6. Burdick, C. O., Clearkin, K. P., Brown. R. K.. and Arakaki, S. Trichilemmoma mammary tissues in the cleared mammary fat pad of the nude mouse. J. of the scalp. Arch. Dermatol.. 95. 73-75, 1967. Nati. Cancer Inst., 55: 1461-1466, 1975. 7. Dabska, M. Giant hair matrix tumor. Cancer (Phila)., 28. 701-706. 1971. 27. Reed, R. J., and LÃ¡mar, L. M. Invasive hair matrix tumors of the scalp. 8. Delescluse. C., Fukuyama, K., and Epstein, W. L. Dibutyryl cyclic AMP- Invasive pilomatrixoma. Arch. Dermatol., 94: 310-316, 1966. induced differentiation of epidermal cells in tissue culture. J. Invest. Der 28. Reynolds, E. S. The use of lead citrate at high pH as an electron-opaque matol., 66. 8-13, 1976. stain in electron microscopy. J. Cell Biol., 17: 208-212, 1963. 9. Dubois, M., Cilles, K. A., Hamilton, J. K., Robert, P. A., and Smith, F. 29. Rheinwald, J. G., and Green, H. Serial cultivation of strains of human Colorimetrie method for determination of sugar and related substances. epidermal keratinocytes: the formation of keratinizing colonies from single Anal. Chem., 28: 350-356, 1956. cells. Cell, 6: 331-344, 1975. 10. Giovanella, B. C., and Fogh, J. Present and future trends in investigations 30. Seabright, M. The use of proteolytic enzymes for the mapping of structural with nude mouse as a recipient of human tumor transplants. In: J. Fogh and rearrangement in the chromosomes of man. Chromosoma (Beri.), 36: 204- B. C. Giovanella (eds.). The Nude Mouse in Experimental and Clinical 210. 1972. Research, pp. 291-312. New York: Academic Press, Inc., 1978. 31. Seff, J., and Berkowitz, S. Carcinomatous degeneration of sebaceous cysts. 11. Goldman, L., and Richfield, D. F. Trichilemmoma. Clinical lesions. Arch. Surg. Gynecol. Obstet., 23: 469-473, 1916. Dermatol., 113: 107-108, 1977. 32. Sharkey, F. E., Fogh, J. M., Hajdu, S. I., Fitzgerald, P. J.. and Fogh, J. 12. Harnden, D. G. A human skin culture technique used for cytological exami Experiences in surgical pathology with human tumor growth in the nude nations. Br. J. Exp. Pathol., 41: 31-37, 1960. mouse. In: J. Fogh and B. C. Giovanella (eds.), The Nude Mouse in Experi 13. Hashimoto, K. The marginal band. Arch. Dermatol., 703: 387-393, 1971. mental and Clinical Research, pp. 187-214. New York: Academic Press, 14. Hashimoto, K., and Kanzaki, T. Surface ultrastructure of tissue cultured Inc., 1978. keratinocytes. J. Ultrastruct. Res., 49: 259-269, 1974. 33. Stout, A. P., and Cooley, S. G. E. Carcinoma of sweat glands. Cancer 15. Headington, J. T., and French, A. J. Primary neoplasm of the hair follicle. (Phila.), 4:521-536, 1951. Histogenesis and classification. Arch. Dermatol., 86: 430-441, 1962. 34. Sweeley, C. C., Bentley, R., Makita, M., and Wells, W. W. Gas-liquid 16. Holmes, E. J. Tumors of lower hair sheath. Common histogenesis of certain chromatography of trimethylsilyl derivatives of sugars and related sub so-called "sebaceous cyst," acanthomas, and "sebaceous carcinomas." stances. J. Am. Chem. Soc., 85: 2497-2507, 1963. Cancer (Phila.), 21: 234-248, 1968. 35. Wilson Jones, E. Proliferating epidermoid cysts. Arch. Dermatol., 94: 11- 17. Kanzaki, T., Hashimoto, K., and Bath, D. W. Human malignant melanoma in 19, 1966. vivo and in vitro. J. Nati. Cancer Inst., 59: 775-785, 1977. 36. Wynn-Williams, A., and McCulloch, P. Human cancer and other transplants in the "nude" mouse. J. Pathol., Ã•22:225-228, 1977. 18. Kanzaki, T., Hashimoto, K., and Bath, D. W. Heterotransplantation of human

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'^. : â€¢rX^^-'fÃ•vi,,, â€¢-â€¢ ;?:^ :': .V V -^., / K â„¢ % "fÂ«k* Â»%â€¢ v 3& UÃ• Â»**."â€¢''.â€¢*i.."*^l-' -. "'-â€¢-_ 2E Fig. 1. The original tumor (trichilemmoma) of the head. Fig. 2. Histological sections of the original tumor. The tumor is solid and lobulated W and B). Tumor cells are well organized and well differentiated (B to Â£).Cells at the periphery of lobules are cylindrical and palisading on basal membrane (ÃŸtoE). Tumor cells are clear with HE stain (B and C). The cytoplasm stained with PAS (O) but did not stain after amylase digestion (E). Microabscess is present in the center of tumor lobules (ÃŸ,O,and E). Neutrophil infiltration, fibroblast proliferation, and hyalin substance are present in the stroma (B to E). A to C. HE stain, x. 3, x 160, and x 320, respectively 0 and E, PAS stain without (D) and after (E) amylase digestion, x 320 and x 320, respectively.

2472 CANCER RESEARCH VOL. 41

v-.^JrSF"- , *^* ^i *&mflifaitÃ e& ^ ,*. . ^ i ..

Fig. 3. An electron micrograph of the original tumor. Tonofilaments (7"), desmosome-tonofilament complexes (D-D, and glycogen granules (G) are seen. Intranuclear inclusions (/c) which are magnified in the inset are observed, x 12,600. Inset, x 67,000.

JUNE 1981 2473

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Fig. 4. Tissue-cultured trichilemmoma cells. A and B, PAS stainin without W)(A) and after amylase digestion (B).(B), x 320 andanc x 320, respectively. C, an electron- icroscopic photograph showing tonofilaments (T), desmosome-tonofilament complexes (D-7),- and glycogen (G), x 40.000

2474 CANCER RESEARCH VOL. 41

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fcÂ» ft 11 13 14 15 16

* â€¢*

19 20 21 22 - ,<â€¢â€¢â€¢â€¢â€¢;'-â€¢-â€¢ Ml M2 M3 M4

Fig. 5. Metaphase of a tissue-cultured trichilemmoma cell with 45 chromosomes, XX,1p+,del(1Xp13),del(3Xp13),11q+, â€”15,â€”18,â€”19,â€”21,â€”21, del(22Xq13), + mar1(M1),+rnar2(M2), + mar3(M3) and +mar4(M4). M), M2, M3, and M4 are marker chromosomes. Trypsin-Giemsa stain, x 8,000. Fig. 6. In A. tissue-cultured trichilemmoma cells (3 x 107) were inoculated into a nude mouse. Ten weeks after the injection, a large tumor developed at the site of inoculation. B, the histological appearance of the tumor in a nude mouse. Note the remarkable similarity of the tumor histology in the nude mouse to that of the original tumor in Fig. 2. HE stain, x 320.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1981 American Association for Cancer Research. Morphological, Biological, and Biochemical Characteristics of a Benign Human Trichilemmoma Cell Line in Vivo and in Vitro

Tamotsu Kanzaki, Hikaru Eto, Akira Umezawa, et al.

Cancer Res 1981;41:2468-2475.

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