The ultrastructure and virus-like particles in feline mammary carcinomas

J. COMP.

THE

PATH.

1974. Vol.

84.

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ULTRASTRUCTURE AND VIRUS-LIKE PARTICLES IN FELINE MAMMARY CARCINOMAS BY D. VON BOMHARD and S. G. DE S. WETTIMUNY* Faculty

Institute for General Pathology and Pathological Anatoq, of Veterinary Medicine, lJnirers+v of Munich, IVest Germany

INTRODUCTION

Recent work in our Institute on the ultrastructure of mammary tumours of bitches (von Bomhard and vcn Sandersleben, 1973, 1974) suggested a study of the ultrastructural morphology of mammary tumours of cats. No attention seems to have been paid hitherto to the ultrastructure of mammary carcinoma in cats. In view of the reported occurrence of virus-like particles in feline mammary carcinomas by Feldman and Gross (1971), their existence is of special interest in view of the occurrence of viruses and virus-like particles in spontaneous and experimentally induced tumours in many species. Mammary tumours of mice are caused by viruses (Bernhard, Bauer. GuCrin and Oberling, 1955). Virus-like particles have also been observed in mammary tumours of women (Dmochowski, Seman and Gallager, 1969; Thomssen, Bandlow and Stankovid, 1972), in a Rhesus monkey (Chopra and Mason, 1970) and 1970), but their significance in the aetiology of the in rats (Chopra and Taylor, tumours has not been conclusively established. Seman, Guillon, Proenca and M&y (1968) saw enterovirus-like particles in 2 of 8 canine mammary tumours, while Feldman and Gross (1971), in an electron microscopic study of 11 feline and 11 canine mammary tumours, reported the presence of virus-like particles in 5 of the tumours from cats, but none from dogs. This paper describes the ultrastructure of 4 mammary carcinomas in the car and records the presence of virus-like particles in them. These particles are compared with those seen in spontaneous mammary tumours of mice. MATERIALS

AND

METHODS

Four mammary carcinomas (Nos 66, 117, 160,204) from 3 cats whose ages ranged from 8 to 13 years were studied by light and electron microscopy. All tumours occurred in one of the right posterior mammary glands and were obtained for examination by surgery. The right superficial inguinal lymph node of the first cat, bearing tumour 66, was also removed, but was found to be free of tumour deposits histologically. Tumour 160 was removed from the second cat which had developed a recurrent tumour (204) at the same site, 1 month after surgery. Tumour metastases were detected in the right inguinal and right axillary lymph nodes in this cat, 4 months after removal of the recurrent tumour. Autopsy revealed, in addition, secondary tumours in the lung and kidney. The third cat, bearing tumour 117, had also shown tumour recurrence at the same site and was destroyed, but was not available for autopsy. For comparison of the morphology of the virus-like particles, 5 spontaneous mammary carcinomas of CBA and C3H mice were examined. Routine histological preparations were made after fixation in 7 per cent, neutral formaldehyde and paraffin sections were stained with haematoxylin and eosin. For * Stipendiate

of the Alexander

van

Humboldt

Foundation.

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electron microscopy, pieces of tumour tissue of about lmm3, taken immediately after surgery and pre-fixed for 2 h. in 6.25 per cent. glutaraldehyde at 4 “C., were then washed for at least 2 h. in O-1 M phosphate buffer containing sucrose, post-fixed in 1 per cent. osmium tetroxide and stained with O-25 per cent. aqueous uranyl acetate. They were then dehydrated in alcohols and embedded in Durcupan ACM. Semithick and ultra-thin sections were cut on a Reichert Ultramicrotome OMU 2. The semi-thick sections were stained with Toluidine Blue and Safranin and the ultra-thin sections contrasted with uranyl acetate and lead citrate. Stained sections were examined with a Siemens Electron Microscope IA. RESULTS Histology

The 4 tumours have a uniform histological structure and all are adenocarcinomas with papillary cribriform and solid areas. The acini-like structures formed by tumour cells contain an eosinophilic homogeneous secretory material. Marked necrosis of tumour tissue is seen in all cases. There is very little connective tissue stroma. Invasion of vessels by tumour cells is evident in 3 of the tumours. Electronmicroscopy The electron microscopical investigation shows that the carcinoma is, in general, made up of groups of epithelial cells arranged around central lumina.

Fig.

1. Feline mammary carcinoma; cylindrical and polyhedral villi (Mv) ; hardly distinguishable virus-like particles centrioles (C) . X 3800.

dark cells lining a lumen (L) ; micro(arrowed) ; basal membrane (Bm) ;

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These epithelial formations are enclosed by an intact basal lamina (lamina densa) which separates them from the surrounding stroma. Their luminai surface is frequently lined by microvilli. Two cell types can be distinguished, a dark or electron-dense cell, cylindrical or polyhedral in shape (Fig. 1) and a light or electron-lucent cell of variable shape and size (Fig. 2). Both types of cell

Fig.

2. Feiine

mammary

carcinoma;

light

cells

(LC)

and

portions

ofdark

cells

(DC).

60UO.

may occur in close connection to one another. The dark type has a relatively large nucleus, often irregular and sometimes indented, showing a clumpy distribution of chromatin inside the nucleoplasm and around the nuclear membrane. Numerous mitochondria of varying sizes and shapes are present in the cytoplasm. The Golgi apparatus located near the nucleus is inconspicuous. The endoplasmic reticulum is formed by slightly dilated cisterns bearing ribosomes on their outer surface. The cytoplasm is rich in ribosomes and polysomes, and sometimes contains centrosomes and tonofibrils. The adjacent cell membranes are joined together by desmosomes, and sometimes by terminal bars and iuterdigitating microvilli. The light type is less common and is mostly seen at t he luminal surface. It has an electron-lucent cytoplasm and a relatively small round or oval nucleus with diffusely distributed chromatin. These cells contain fewer rihosomes and other cell organelles than the dark cells. Intermediate cells between these 2 types can also be recognized in the carcinoma. Numerous cells in mitosis are seen among carcinoma cells (Fig. 3). Secretory granules are absent, but the lumina contain a fine granulated material. All turnouts revealed the presence of doughnut-shaped virus-like particles composed of two shells with an outer diameter of 93*0&8*2 nm and an electronlucent core 38-O&6.45 nm. The inner membrane is always more distinct and denser than the outer. The particles are seen most often in the cisternae of the endoplasmic reticulum (Figs 3 and 4) and occasionally in the perinuclear space

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(Fig. 5). At times it is not always possible to say with certainty whether the particles are intracisternal or intracytoplasmic (Fig. 6) since the membranes of the endoplasmic reticulum cannot always be identified. The particles occur

Fig. 3. Feline mammary plasmic reticulum

carcinoma; (arrowed)

Fig. 4. Feline

carcinoma:

mammary

mitosis (M) of tumour ; small lumen (L) with virus-like

Fig. 5. Feline mammary carcinoma; virus-like reticulum; nucleus (N). x 39 000. Fig. 6. Feline mammary (T). x 57 000.

carcinoma;

virus-like

particles particles particles

cell with microvilli.

virus-like particles x 12 000.

in the endoplasmic in the perinuclear probably

lying

reticulum.

in the endox

40 000.

space and in the endoplasmic in the cytoplasm;

tonofibrils

singly or in clusters of up to 40. In tumour 117, morphologically similar particles are also present in the intercellular space [Fig. 7(a)]. Budding into the cisternae of the endoplasmic reticulum is observed occasionally [Fig. 7(b)], but not into the extracytoplasmic space. The number of particles seen in any single tumour is variable. In tumours 66 and 117 there are only a few isolated particles. In the

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recurrent tumour 204, dense clusters of particles are seen in close connection with the endoplasmic reticulum and perinuclear space. In the 5 mammary carcinomas of mice examined, numerous intracytoplasmic and intravacuolar A type doughnut-shaped particles with 2 concentric shells, and also both mature and immature B particles are seen. In one tumour there is a large accumulation of intracytoplasmic A particles (Fig. 8). The outer diameter of the intracytoplasmic particles in our study is 86.5h3.9 nm, the electron-lucent inner core being 55.7kl3.3 nm. The corresponding measurements for the intravacuolar particles is 115*0&9*2 nm, with the electron-lucent core 63*7+7-l nm, with the electron-lucent cvre 63.7 67-l nm. Budding is seen to occur frequently.

Fig.

7a. Feline

mammary

carcinoma;

extracellular

Fig. 7b. Feline

mammary

carcinoma;

budding

Fig. 8. Mammary cisternal

virus-like into a cistern

particles.

x 35 000.

of the endoplasmic

reticulum.

x 39 000.

carcinoma of the mouse. Accumulation of intracytoplasmic particles (CP) ; intraparticles (IP) ; budding (arrowed) ; B-particles (t) ; Desmosome (D). x 12 000. DISCUSSION

The object of the investigation was to study the fine structure of the commonly occurring adenocarcinoma in the cat (Cotchin, 1957; Misdorp, 1964). A careful search for virus-like entities was also made in view of their reported occurrence in feline mammary carcinomas (Feldman and Gross, 1971). The light microscopical appearance in all 4 tumours was that of adenocarcinoma with papillary, cribriform and solid components. This appearance is also not uncommon in this type of tumour in women and bitches. The dark cells seen in feline mammary carcinoma have many ultrastructural features in common with A-cells described in human mammary carcinomas by Haguenau and Arnoult (1959). These features are an electron-dense and ribosome-rich cytoplasm with well developed moderately dilated endvplasmic reticulum, numerous mitochondria, an inconspicuous Golgi apparatus and a nucleus that is often irregular and sometimes indented. The high ribosome

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content of these cells may indicate a high protein synthesis for replication of cells and viruses. Secretory granules similar to those described by Archer and Omar (1969) and Wellings and Roberts (1963) in mammary tumours of women were not seen. The carcinoma cells of the cat do not possess intracytoplasmic lumina lined by microvilli as are seen in human mammary carcinoma (Schafer, 1967; Murad, 1971). The light cells in the cat mammary tumour, fewer in number, have certain characteristics similar to the B-cells of Haguenau and Arnoult (1959). They have an electron-lucent cytoplasm, reduced nuclear-cytoplasmic ratio, a distinctly reduced number of organelles and a diffused distribution of chromatin in the nucleus. In the cat carcinomas, neither “normal” nor neoplastic myoepithelial cells were seen. These cells have been described in breast tumours in man (Hamperl, 1939; Murad, 1971) and in the so-called mixed mammary tumours of bitches (Pulley, 1973; von Bomhard and von Sandersleben, 1973, 1974). As seen by light and electron microscopy the adenocarcinoma of the cat is poor in connective tissue stroma. The epithelial formations are well demarcated from the surrounding connective tissue stroma by an intact limiting basement membrane, whereas in the medullary (Murad and Scarpelli, 1967) and non-infiltrating duct carcinoma (Schafer, 1967) of the human mammary gland this membrane is not intact. The cat tumours always show a basement membrane in spite of their malignant characteristics and their tendency to infiltrate into the blood and lymph vessels. The necrosis which is common in these tumours is often severe and is doubtless due to the scanty stroma and the rapid growth of the carcinomas as suggested by the clinical history and the high mitotic rate. Weijer, Head, Misdorp and Hampe (1972) suggested that the feline mammary carcinoma might serve as a suitable model for the study of human mammary carcinoma because of their similar clinical behaviour and light microscopic structure. Our electronmicroscopic study confirms that there are many morphological similarities between feline and human carcinoma. Structures resembling virus particles were demonstrated in all 4 feline tumours examined. Their electron-lucent centre and outer shell are not characteristics of other normal or carcinoma cell constituents, but are features of some known tumour viruses. These particles, present in the cisternae of the endoplasmic reticulum and perinuclear space, we have designated “intracisternal A-like”, according to the classification proposed for oncogenic viruses by Dalton, de Harven, Dmochowski, Feldman, Haguenau, Harris, Howatson, Moore, Pitelka, Smith, Uzman and Zeigel (1966). Type A particles are the most common in tumour tissue and are known to occur in mammary carcinomas and many other tumours (for references see Bernhard, 1960). Feldman and Gross (197 1) found virus-like particles in 5 of 11 mammary carcinomas in cats examined. Three of the tumours revealed particles with 2 (seldom 3) concentric shells (intracisternal A) and one with 4 shells (immature B) : in the fifth tumour they found all three types of particles. The intracisternal and probably also the intracytoplasmic particles seen in our study resemble the intracisternal particles with two shells described by Feldman and Gross. In tumour 117 we observed a few particles morphologically similar to type A

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particles, but located in the extracellular space. No particles with 3 or more shells were seen in our material. Feldman and Gross observed occasional budding into the intracisternal and extracellular space in feline mammary carcinoma and we also observed occasional budding into the intracisternal, but not into the extracellular spaces. In the mouse mammary tumours that we have examined budding was a common occurrence into both intracisternal and extracellular spaces. The aetiological importance of the particles in mammary tumours of cats is not known, but their distinct morphological resemblance to RNA oncogenic type viruses may be significant. The particles are similar in structure to those in the mouse, but their outer and inner diameters differ significantly in size (P
Four mammary adenocarcinomas of cats which were examined by electron microscopy, reveal that the tumours are formed of two types of epithelial cell, often arranged to form central lumina. The electron-dense cell is morphologically similar to the A cell of Haguenau and Arnoult and the electron-lucent cell to the B cells. All the tumours showed doughnut-shaped particles with two shells in the cisternae of the endoplasmic reticulum and occasionally in the perinuclear space. In one tumour particles were also seen in the intercellular space. Occasional budding occurred within cisternae of the endoplasmic reticulum. The particles resemble the type A intracisternal ones seen in the 5 mouse mammary tumours investigated for comparison. ACKNOWLEDGMENT

We are grateful to Mrs H. Schulze and Miss A. Eble for technical

assistance.

REFERENCES

Archer, F., and Omar, M. (1969). The fine structure of fibroadenoma of human breast. Journal of Pathology and Bacteriology, 99, 113-l 17. Bernhard, W. (1960). The detection and study of tumor viruses with the electron microscope. Cancer Research, 20, 7 12-727. Bernhard, W., Bauer, A., G&in, M., and Oberling, Ch. (1955). Etude au microscope Clectronique de corpuscules d’aspect virusal dans des Cpitheliomas mammaires de la souris. Bulletin de l’dssociation Franfaise pour l’ktude du Cancer, 42, 163-178. Bomhard, D. v., and Sandersleben, J. v. (1973). cber die Feinstruktur von Mammamischtumoren der Hiindin. I. Das Vorkommen von Myoepithelzellen in myxoiden Arealen. Virchows Archiv A, Pathologische Anatomie und Histologie, 359, 87-96.

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Bomhard, D. v., and Sandersleben, J. v. (1974). Uber die Feinstruktur von Mammamischtumoren der Htindin. II. Das Vorkommen von Myoepithelzellen in chondroiden Arealen. Virchows Archiv A, Pathologische Anatomie und Histologie, 362,157-167. Chopra, H. C., and Mason, M. M. (1970). A new virus in a spontaneous mammary tumor of a rhesus monkey. Cancer Research, 30, 2081-2086. Chopra, H., and Taylor, D. J. (1970). V irus particles in rat mammary tumors of varying origin. Journal of the national Cancer Institute, 44, 1141-1147. Cothin, E. (1957). Neoplasia in the cat. Veterinary Record, 69, 425-434. Dalton, A. J., de Harven, E., Dmochowski, L., Feldman, D., Haguenau, F., Harris, W., Howatson, A. F., Moore, D. H., Pitelka, D., Smith, K., Uzman, B., and Zeigel, R. (1966). Suggestions for the classification of oncogenic RNA viruses. Journal of the jVationaE Cancer Institute, 37, 395-397. Dalton, A. J., and Potter, M. (1968). Electron microscopic study of the mammary tumor agent in plasma cell tumors. Journal of the National Cancer Institute, 40, 1375-1377. Dmochowski, L., Seman, G., and Gallager, H. S. (1969). Viruses as possible etiologic factors in human breast cancer. Cancer, 24, 1241-1249. Feldman, D. G. (1963). Origin and distribution of virus-like particles associated with mammary tumor in DBA strain mice. II. Virus-like particles in blood and organs. Journal of the National Cancer Institute, 30, 503-515. Feldman, D. G., and Gross, L. (1971). Electron microscopic study of spontaneous mammary carcinomas in cats and dogs: Virus-like particles in cat mammary carcinomas. Cancer Research, 31, 1261-1267. Haguenau, F., and Arnoult, J. (1959). Le cancer du sein chez la femme. Etude comparative au microscope electronique et au microscope optique. Bulletin de l’dssociation Franfais pour l’kude du Cancer, 46, 117-2 11. Hamperl, H. (1939). Uber die Myoepithelien (myoepitheliale Elemente) der Brustdrtise. Virchows Archiv, Pathologische Anatomie, 305, 17 l-2 15. Misdorp, W. (1964). Malignant Mammary Tumours in the Dog and the Cat compared with the same in the Woman. Druckerei G. van Dijk N.V., Breukelen. Murad, T. M. (1971). Cytologic differentiation of carcinoma of the breast by electron microscopy. Acta Cytologica, 15, 400-409. Murad, T. M., and Scarpelli, D. G. (1967). The ultrastructure of medullary and scirrhous mammary duct carcinoma. American Journal of Pathology, 59, 335-360. Pulley, L. T. (1973). Ultrastructural and histochemical demonstration of myoepithelium in mixed tumors of the canine mammary gland. American Journal of Veterinary Research, 34, 15 13-l 522. Schafer, A. (1967) _ Zur Feinstruktur des nichtinfiltrierenden Milchgangkarzinoms. ~entralblatt fgr Allgemeine Pathologie und Pathologische Anatomic, 111, (1968), 473-474, Herbsttagung Nord- und Westdeutscher Pathologen, 1967. Seman, G., Guillon, J. C., Proenca, M., and M&y, A. M. (1968). Enterovirus observes au microscope Clectronique dans des biopsies de tumeurs malignes chez le chien. Revue Franfaise d’Etudes Cliniques Biologiques, 13, 1006- 1008. Thomssen, R., Bandlow, G., and StankoviC, P. (1972). Virus-ahnliche Partikel im Mammagewebe bei Mammakarzinom und Mastopathia chronica cystica. Deutsche medizinische Wochenzeitschrift, 97, 2 19-22 1. Weijer, K., Head, K. W., Misdorp, W., and Hampe, J. F. (1972). Feline malignant mammary tumors. I. Morphology and biology: Some comparisons with human and canine mammary carcinomas. Journal of the National Cancer Institute, 49, 1697-1704. Wellings, S. R., and Roberts, P. (1963). Electron microscopy of sclerosing adenosis and infiltrating duct carcinoma of the human mammary gland. Journal of the JVational Cancer Institute, 30, 269-287. [Receivedfor publication,

February 11 th, 19741