An Electron Microscope Study of Squamous Cell - Cancer Research

(CANCER RESEARCH 26, 172-182, January 1966]

An Electron Microscope Study of Squamous Cell Carcinoma in MerinoSheep Associated with Keratin-filled Cysts of the Skin

R. BORLAND AND A. J. WEBBER

Department of Veterinary Pathology and Bacteriology and the Electron Microscope Unit, University of Sydney, Sydney, Australia

Summary

The fine structure of a naturally occurring cystic condition ofthe skin in Merino sheep and associated squamous cell carcinomawas studied and compared with the fine structure of the normalinterfollicular epidermis of the sheep. A number of obvious differences were observedâ€”namely, large intercellular spaces, mi-crovillus formation, gaps in the basement membrane, fewerdesmosomes, and in the tumor cells only, 2 large nucleoli with aprominent nucleolonema.

Introduction

Recently Carne et al. (8) have shown that squamous cell carcinoma of the wool-bearing skin in a certain strain of Merinosheep in Australia was closely associated with a characteristicand highly inherited cystic condition of the skin. Most of thetumors were observed to arise from the walls of the cysts, although only a relatively small proportion of cysts gave rise totumors. Lloyd (18) demonstrated that the formation of keratin-filled cysts in susceptible animals was due to grass seeds penetrating the skin, dislodging epidermal cells, and carrying theminto the dermis, where they multiplied to form cysts.

This paper records a study of the epidermal cell in its transition from normality to malignancy. Although differences canbe recognized between the histologie structure of normal epidermis, the walls of cysts, and tumor tissue, the light microscopedoes not reveal obvious differences between individual cells fromthese 3 sources. The electron microscope has been used in anattempt to discern any changes in ultrastructure that may occurin this stepwise transition from normality to malignancy. Forthis purpose an examination was made of normal interfollicularepidermis, cyst lining, and the squamous carcinoma arising fromthe cyst wall.

Materials and Methods

Specimens of skin, cyst lining, and tumor were excised from 3normal sheep and 8 cyst-bearing sheep, 6 of which had spontaneous squamous cell carcinomata on the wool-bearing areasof the body.

The tissue was fixed in either chilled 1'/Ã³osmium tetroxidesolution buffered to pH 7.2 (24) for 3-4 hr or ice-cold 5% glu-taraldehyde in cacodylate buffer at pH 7.2 (25) for 1-2 hr andthen in 1% osmium tetroxide for 2 hr. After they were rinsed

Received for publication April 2(1,1905.

and dehydrated through a graded series of alcohols, the specimenswere embedded in Araldite (14). In some cases the tissue wasstained with a saturated solution of phosphotungstic acid (PTA)in absolute alcohol prior to infiltration.

The skin and cyst lining were cut as nearly perpendicular totheir surfaces as possible. Sections were prepared on an L.K.B.microtome with glass knives (17) and collected on nitrocellulose-coated copper grids. Sections from material not previouslystained with PTA were stained with Karnovsky's lead stain,

Method A (16).Examination of the sections was carried out in either a Siemens

Elmiskop I or a Philips KM200 electron microscope.

Results

Light Microscopy

THE INTERFOLLICULAREPIDERMIS. On wool-bearing areas ofthe body this was often only 3-4 cells thick and consisted of awell-defined stratum germinativum, an ill-defined stratumspinosum, an ill-defined stratum granulosum, and a stratumcorneum (Fig. 1).

THECYSTWALL. Cysts varied from 2 mm to 3 cm in diameter,and their walls were composed of stratified squamous epithelium,usually 3-4 cells thick. However, the thickness of the wall wasoften made quite variable by long, finger-like processes of cellsderived from the cyst lining projecting into the dermis. Thecells of the cyst wall were flattened tangentially, and there was avery distinct stratum granulosum, the cells of which had largerkeratohyaline granules in their cytoplasm. Concentric lamellaeof keratin formed the center of the cyst (Fig. 2).

TUMORS. These took the form of mushroom-shaped masses,2-12 cm in diameter, raised above the surface of the skin (Fig.3). In the depths of the tumor there were numerous smallislands of epithelial cells surrounded by dermal connective tissue, and nearer the surface the groups of squamous epithelialcells were larger and had keratin pearls at the center of the cellmasses (Fig. 4).

Electron Microscopy

EPIDERMIS. The interfollicular epidermis was selected forstudy as it was that portion of the integument that most closelyresembled the cyst lining in gross structure. Ultrastructuralstudies of human skin by Selby (26, 27), Brody (6, 7), Ã–dland(23), and Zelickson (32) have shown the epidermis to be relatively thick and to have a more obvious stratification than was

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FIG. 1. Normal interfollicular epidermis of the sheep showing the stratum germinativum (G), the stratum corneum (C), and thedermis (D). II & E, X 2000.

FIG. 2. Cyst wall showing layers of keratin (K), a prominent stratum granulosum (Gr), and tangentially elongate nuclei (Ar). H

& E, X 2000.FIG. 3. A squamous cell carcinoma showing the mushroom-like growth, the exposed surface (Su) of the tumor, and the adjacent

epidermis (E) and dermis (D). H & E, X 4.FIG. 4. Groups of squamous carcinoma cells with keratin "pearls" (Kp) showing the relatively large tumor cell nuclei (N) with their

prominent nucleoli (No). H & E, X 2000.

JANUARY I960 173



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Squamous Cell Carcinoma with Keratin-filled Skin Cysts

found in sheep. The intracellular structures were essentiallythe same as those described in human epidermis. In the basalcells the nuclei tended to be round or slightly oval and theirlong axes were usually perpendicular to the skin surface, but inthe region of the stratum corneum the nuclei were more elongateand their long axes were parallel to the skin surface. Numerousnucleoli were seen in many of the epidermal cell nuclei examined.Relatively few mitochondria were present, and the cytoplasmcontained a number of free ribosomes and little obvious endo-

plasmic rcticulum.The plasma membranes of adjacent cells were closely applied

to one another; they formed 3 electron-dense layers separatedby 2 clear zones, the center dense line being the "intercellularcontact layer" described by Ã–dland (23). Many desmosomes

were present on the plasma membranes of adjacent cells (Fig.7), and they were particularly numerous on the external andbasal surfaces, as compared with the lateral borders, of the cells.Groups of tonofilaments, approximately 40-60 A in diameter,were seen to be inserted into the attachment plates of the desmosomes. The basement membrane formed a regular smooth barrier between the dermis and the epidermis (Figs. 1,7), and itselectron translucent area was firmly attached to the basal cellsby numerous desmosomes. Within the basal cells the tonofilaments were not as electron dense as they were in the cellsnear the keratinized layer of the epidermis (Fig. 7).

CYSTLINING. The cells of the cyst lining and their nuclei wereflattened tangentially, particularly near the keratin center ofthe cyst.

In contrast with the epidermis, there was a very loose contactbetween cells, the plasma membranes of adjacent cells beingseparated by obvious intercellular spaces and the "intercellularcontact layer" being absent (Fig. 8). This effect was most

pronounced between the basal cells of the cyst and became lessobvious nearer the center of the cyst (Fig. 6). Associated withthis loose contact the cell surfaces showed numerous villus-likeprojections that extended into the intercellular spaces (Figs.8, 10) (cf. human stratum spinosum and embryonic skin).

The basement membrane surrounding the cysts was throwninto numerous folds by the irregular borders of the basal cells(Figs. 6, 10), and although the contact between cyst cells andbasement membrane was normally very close, as in the epidermis,a number of apparent breaks were found. At these breaks, thecytoplasm of the cyst cells protruded through into the dermis(Figs. 16, 17). Tonofilaments were numerous and highly keratinized in all the cyst cells including the basal cells (cf. epidermalbasal cells) (Figs. 7, 8), but they seemed to be distributed in arather haphazard fashion. A very obvious stratum granulosumwas present in the cyst lining, where the cells contained numerous large, electron-dense, keratohyaline granules 0.3-0.5 p indiameter (Fig. 6). Also present in these cells were a number ofprominent mitochondria and ribonucleic protein granules (Fig.10).

TUMOR. The surface of the tumor cells was very irregular andformed numerous microvilli (Figs. 11, 13, 14). There were largespaces between adjacent tumor cells, complete absence of intercellular contact, and no formation of intercellular contact layerexcept at the desmosomes, which appeared to be reduced innumber compared with the epidermis and cyst lining.

All the clumps of tumor cells were surrounded by a basementmembrane separating them from the dermis (Fig. 12). As inthe cyst wall, apparent breaks were found in the basement membrane in 1 tumor, and cell cytoplasm flowed through into thesurrounding dermal tissue (Fig. 15).

In cells selected from the surface areas of the tumors therewere numerous heavily keratinized tonofibrils that seemed tohave a perinuclear arrangement (Figs. 11, 13). The bundlesof tonofilaments were less distinct and not so heavily keratinizedin cells taken from the deeper portions of the tumor. Mitochondria were relatively numerous, and there was some roughendoplasmic reticulum present in the tumor cell cytoplasm (Fig.11). A few free ribosomes were seen, but they tended to bearranged in rows unassociated with any obvious membranousstructure rather than to be scattered throughout the cytoplasm(Fig. 14). The large, round tumor cell nuclei invariably contained 2 very large and obvious nucleoli, each with a distinctnucleolonema (Fig. 11).

Discussion

In this study of a naturally occurring inherited cystic conditionof the sheep skin associated with spontaneous tumor formation,a number of changes were observed in the fine structure of thecyst lining and the squamous cell carcinoma when compared withthe normal interfollicular epidermis of the sheep.

Large intercellular spaces were a feature of both the cyst liningand the squamous carcinoma; since the only close contact wasat the desmosomes, there seemed to be a lack of adhesion betweenadjacent cells. Mercer (21), in a review of the cancer cell, suggested that in the case of skin tumors this separation could bedue to failure to synthesize adequate amounts of surface adhesivelayers. SetÃ¤lÃ¤et al. (28), in an electron microscopic examinationof the effect of locally applied carcinogen on the interfollicularepidermis of the mouse skin, demonstrated abnormal cell-to-cellsurface contact in the treated epidermis. It has long been considered that neoplastic cells are less adhesive and have differentsurface properties, such as lack of "contact inhibition," whencompared with normal cells (1-4, 10-12). An ultrastructuralexamination of the normal epidermis of the sheep failed to demonstrate any such intercellular spaces (Fig. 5). According to somestudies involving the normal epidermis of humans, rats, and mice(7, 23, 27), intercellular spaces do exist in the stratum spinosum,although Hibbs and Clark (15) found only a small true intercellular space in an ultrastructural examination of the humanepidermis.

FIG. 5. Interfollicular epidermis showing the keratin (K), the round to oval cell nuclei (A"),the basement membrane (BM), and the

collagen fibers (C). X 7500.FIG. 6. Cyst-wall epithelium with keratin (K), the flattened cells and nuclei (N), and the prominent keratohyalin granules (Kh).

Note the obvious intercellular spaces (S) and the finger-like projections of the basal cells with associated basement membrane (BM).X 8500.

JANUARY 1900 175



R. Borland and A. J. Webber

The plasma membranes of cells of the cyst lining and the squa-mous carcinoma formed numerous microvillous projections thatextended into the intercellular spaces and were most numerousand pronounced in the tumor tissue (Figs. 8,14). Other workers,in electron microscope studies that have included the stratumspinosum of normal human epidermis (7, 23, 31), have describedthe cell surfaces as being markedly plicated and having numerousmicrovilli projecting into the intercellular spaces, whereas inthe sheep the main feature of this region of the epidermis is theconvoluted nature of the cell surfaces and the close apposition ofadjacent surfaces (Fig. 7).

In addition to the above surface alterations, there appeared tobe fewer desmosomes between the tumor cells than in the epidermis or the cyst wall; although this finding was not confirmedquantitatively by serial sectioning, it was common to a largenumber of sections examined. A similar apparent decrease inthe number of desmosomes in human epidermis was described byCaulfield and Wilgram (9) in an ultrastructural examination ofDarier's disease; it is interesting to note that this disease is also

an inherited condition.Two large nucleoli with filamentous nucleolonemata were an

almost constant feature of the tumor cell nuclei. Zbarsky et al.(30) and Bernhard and Granboulan (5), in studies of tumor cellnuclei, described similar structures that varied even within agiven neoplasm but did not find that they were constant featuresof the tumor cells. Care would have to be taken in interpretingthe variety of these structures owing to the normal pleomorphismof the nucleolus (22).

Breaks in the basement membrane were found only in thecysts and tumor (Figs. 15-17) and were not seen in the normalepidermis. It is thought that these gaps could be related tomalignant transformation and to a possible mechanism for tissueinfiltration in the tumor. Luibel et al. (19), in a study of invasive carcinoma of the cervix in humans, and Frei (13), in anexamination of carcinogen-induced epidermal tumors in mice,described similar defects of the basement membranes; in both

cases the defects were associated with local tissue invasion by thetumors.

In this study the main distinguishing features of the pre-malignant and malignant tissues when compared with the normalepidermis were changes in the cell surfaceâ€”namely, microvillusformation, large intercellular spaces, poorer cell-to-cell contacts,and basement membrane defects.

The main distinguishing features of the ultrastructure of themalignant tissue as outlined above were suggestive of the characteristics of malignant cells described by Abercrombie andAmbrose (1) and othersâ€”namely, alteration in surface propertiesand lack of "contact inhibition." It is important to note, how

ever, that each one of the fine structural changes observed incyst lining and tumor cells has been described for other animalsas occurring separately in normal skin and embryonic skin (20)and in other pathologic conditions of the skin (9, 29). However,it would appear to be significant that all occur together only inthe cyst wall and in tumor cells.

The finding that cyst-lining cells have many of the fine structural features of the carcinoma cells implies that morphologicallythere is a very narrow gap between the premalignant and themalignant tissue. On the other hand, a number of features discernible by the electron microscope indicate that both the cystwall and the squamous carcinoma differ markedly from the normal epidermal cell.

Acknowledgments

We wish to thank Professor H. R. Carne for his help and interestin this work, Dr. E. H. Mercer for helpful suggestions and discussion, and Dr. D. G. Drummond for affording us the facilities of theElectron Microscope Unit. We should also like to thank Mrs. S.MacLeman for cutting the sections and Mr. L. Whitlock and staffand Mr. R. F. Jones for the preparation of the photography of thehistopathologic specimens. One of us (R. B.) carried out thiswork while holding an N. S. W. State Cancer Council ResearchFellowship.

FIG. 7. Basal cell of the epidermis showing the oval nucleus (-V),the relatively regular basement membrane (BM) with the associatedhalf-desmosome (d), and a few tonoh'laments (T), some attached to an intercellular desmosome (D). X 15,000.

FIG. 8. Basal cells of the cyst wall showing the irregular shaped nuclei (N), the prominent intercellular spaces (S) with cellularprojections (P), electron-dense bundles of tonofilaments (T), and the irregular projections of the basal border of the cells with theassociated basement membrane (BM). X 15,000.

FIG. 9. Adjacent epidermal cells at higher magnification showing the involuted cell borders and closely applied plasma membranes(Pm) with an obvious intercellular contact layer (Cl). Desmosomes (D) with attached bundles of tonofilaments (T) can be seen.X 60,000.

FIG. 10. Adjacent cyst-wall cells at higher magnification showing the prominent intercellular spaces (S) with villous like cell projections (P) extending into the spaces. Other cytoplasrnic constituents present include mitochondria (A/), ribonucleoprotein granules(R), and tonofilaments (T). X 60,000.

FIG. 11. Tumor cells showing the large nuclei (N) with prominent nucleoli (Aro), intercellular spaces (S), and microvillus projections (Mv). Mitochondria (A/), eridoplasmic reticulum (ER), and tonofilaments (T) are also present. X 12,500.

FIG. 12. A cell from the periphery of a tumor cell group showing the relatively large nucleus (N), basement membrane (BM), andcollagen fibers (C) in the dermis. X 15,000.

FIG. 13. Tumor cell showing a perinuclear arrangement of tonofilaments (T). X 28,500.FIG. 14. Adjacent tumor cells at a higher magnification showing the very prominent microvilli (Mv), the intercellular space (S), and

the numerous free ribonucleoprotein granules (R), some of which are lined up unassociated with a cytomembrane (ÃŸi). X 46,500.FIG. 15. Part of the basement membrane (BM) surrounding a group of tumor cells showing three apparent breaks (Br) where tumor

cell cytoplasm (Cm) flows through into the dermis (D). X 28,500.FIGS. 16, 17. Portions of basal cells from 2 cysts each showing an apparent break (Br) in the basement membrane (BM). Fig. 16,

X 30,000; Fig. 17, X 38,500.





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References

1. Abercrombie, M., and Ambrose, E. J. The Surface of CancerCells: A Review. Cancer Res., Â£Â£.-525-48,1962.

2. Abercrombie, M., and Heaysman, J. E. Observations on theSocinl Behaviour of Cells in Tissue Culture. 1. Speed of Movement of Chick Heart Fibrophiats in Relation to their MutualContacts. Exptl. Cell Res., 5:111-31, 1953.

3. â€” â€”.Observations on the Social Behaviour of Cells in TissueCulture. 11. "Monolayering" of Fibroplasts. Ibid., 6:293-306,

1954.4. Ambrose, E. J., and Easty, G. C. Differences between the Sur

face Properties of Normal and Tumour Cells. Acta UniÃ³.Intern, contra Cancrum, Ã•6.-36-40,1960.

5. Bernhard, W. and Granboulan, N. The Fine Structure of theCancer Cell Nucleus. Exptl. Cell Res., Suppl. 9, pp. 19-53,1963.

6. Brody, I. An Ultrastructure Study on the Role of the Kerato-hyalin Granules in the Keratinization Process. J. Ultrastruct.Res., 3:84-104, 1959.

7. â€” â€”.The Ultrastructure of the Tonofibrils in the Keratinization Process of Normal Human Epidermis. Ibid. 4:267-97,I960.

8. Carne, H. R., Lloyd, L. C., and Carter, H. B. Squamous Carcinoma Associated with Cysts of the Skin in Merino Sheep.J. Pathol. Bacterio!., Â«6:305-15,1963.

9. Caulfield, J. B., and Wilgram, G. F. An Electron MicroscopeStudy of Dyskeratosis and Acantholysis in Darier's Disease.J. Invest. Dermatol. 41.-57-65, 1963.

10. Coman, D. R. Decreased Mutual Adhesiveness, a Property ofCells from Squamous-cell Carcinomas. Cancer Res., 4:625-29,

1944.11. â€” â€”. Mechanisms of the Invasiveness of Cancer. Science,

^05:347-48, 1947.12. . Cellular Adhesiveness in Relation to the Invasiveness

of Cancer: Electron Microscopy of Liver Perfused with aChelating Agent. Cancer Res., 14:519-21, 1954.

13. Frei, J. V. The Fine Structure of the Basement Membrane inEpidermal Tumours. J. Cell Biol., Ã•5.-335-42,1962.

14. Glauert, A. M., Rogers, G. E., and Glauert, R. H. A NewEmbedding Medium for Electron Microscopy. Nature, /78.-803,

1956.15. Hibbs, R. G., and Clark, W. H. Electron Microscope Studies of

the Human Epidermis. The Cell Boundaries and Topographyof the Stratum Malpighii. J. Biophys. Biochem. Cytol., 6:71-76, 1959.

16. Karnovsky, M. J. Simple Methods for Staining with Lead atHigh pH in Electron Microscopy. Ibid., 11:729-32, 1961.

17. Latta, H., and Hartmann, J. F. Use of a Glass Edge in ThimSectioning for Electron Microscopy. Proc. Soc. Exptl. Biol.Med., 74:436-39, 1950.

18. Lloyd, L. C. The Aetiology of Cysts in the Skin of Some Families of Merino Sheep in Australia. J. Pathol. and Bacterio!.,.88:219-27, 1964.

19. Luibel, F. J., Sanders, E., and Ashworth, C. T. An ElectronMicroscopic Study of Carcinoma in Situ and Invasive Carcinoma of the Cervix Uteri. Cancer Res., Â£0:357-61,1960.

20. Menefee, M. G. Some Fine Structure Changes Occurring in theEpidermis of Embryo Mice during Differentiation. J. Ultrastruct. Res., 1: 49-61, 1957.

21. Mercer, E. H. The Cancer Cell. Brit. Med. Bull., Ã•8.-187-92,

1962.22. Montgomery, P. O., Jr. Experimental Approaches to Nucleolar

function., Exptl. Cell Res., Suppl. 9, pp. 170-75, 1963.23. Ã–dland, G. F. The Fine Structure of the Inter-relationships of

Cells in the Human Epidermis. J. Biophys. Biochem. Cytol.,.4:529-38, 1958.

24. Palade, G. E. A Study of Fixation for Electron Microscopy.J. Exptl. Med., 95:285-98, 1952.

25. Sabatini, D. D., Beliseli, K., and Barrnett, R. J. Cyto-chemis-try and Electron Microscopy. The Preservation of CellularUltrastructure and Enzymatic Activity by Aldehyde Fixation.J. Cell Biol., Ã•7.-19-58,1963.

26. Selby, C. C. An Electron Microscope Study of the Epidermis-ot Mammalian Skin in Thin Sections. Dermo-EpidermalJunction and Basal Cell Layer. J. Biophys. Biochem. Cytol.,/.-429-43, 1955.

27. â€” â€”. The Fine Structure of Human Epidermis as Revealed

by the Electron Microscope. J. Soc. Cosmetics Chemists,7:584-99, 1956.

28. SetÃ¤lÃ¤,K., Merenmies, L., Niskanen, E. E., Nyholm, M., andStjernvall, L. Mechanism of Experimental Tumorigenesisâ€”VI. Ultrastructural Alterations in Mouse Epidermis Causedby Locally Applied Carcinogen and Dipole Type TumourPromoter. J. Nati. Cancer Inst., Â£5:1155-90,1960.

29. Wyllie, J. C., More, R. H., and Harsst, M. D. Electron Microscopy of Epidermal Lesions Elicited during Delayed Hyper-sensitivity. Lab. Invest., Ã•3.-137-51,1964.

30. Zbarsky, I. B., Dmitrieva, N. P., and Yermolayeva, L. P.Structure of Tumour Cell Nuclei. Exptl. Cell. Res., Â£7:573-76,1962.

31. Zelickson, A. S. Normal Human Keratinization Processes asDemonstrated by Electron Microscopy. J. Invest. Dermatol.,37:369-79,1961.

32. â€” â€”. Electron Microscopy of Skin and Mucous Membrane.Springfield, 111.,Charles C Thomas, 1963.




1966;26:172-182. Cancer Res R. Borland and A. J. Webber Merino Sheep Associated with Keratin-filled Cysts of the SkinAn Electron Microscope Study of Squamous Cell Carcinoma in

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