morphological studies on transmissible feline fibrosarcoma

[CANCER RESEARCH 30, 1658â€”1667,June 1970]

SUMMARY

Inoculations s.c. of cell-free Millipore filtrates of the S-Tfeline sarcoma virus resulted in the rapid induction offibrosarcomas in kittens. Metastatic tumors, seen in a largepercentage of the inoculated kittens, were of two distincttypes. The first was a solid tumor, similar to the tumorslocated in the subcutaneous tissue. The second type, seenprimarily in the liver and brain, was formed by numerous,cystic, blood-filled cavities surrounded by a thin shell ofneoplastic cells. Ultrastructural studies confirmed light microscopic findings of three cell types in all solid tumors:fibroblasts, macrophage-like cells, and mast cells. C-type viralparticles seen in tumor tissue of the original source cat withspontaneous fibrosarcomas and from all inoculated experimental kittens were uniform in morphology and apparentmode of production and were similar to those of felineleukemia. The findings help to substantiate the viral etiologyof this feline fibrosarcoma.

INTRODUCTION

Lymphosarcoma is one of the most common spontaneousneoplasms of cats (3 1). Recently, the association of thisneoplasm with a virus, which is evidently causative, has beenshown by several groups of research workers (16, 19, 28, 35)whose findings have stimulated wider interest in felinetumors.

Spontaneously occurring feline fibrosarcomas are foundmuch less commonly than lymphosarcomas and occur mainlyin adult or aged animals (23). They are usually found assolitary masses and often recur following surgical excision(15). Dueto infrequencyof theiroccurrenceandto thelackof particular interest in them, little morphological or biological data are available for review. The paucity of knowledgeconcerning spontaneous fibrosarcomas in cats is reflected insurveys of feline neoplasms (7, 25, 31).

Kasza et a!. (17) demonstrated the presence of a cytopathogenic agent from an unclassified cat sarcoma in an

1Supported in part by United States Drug Administration Grant529-15.1, General Research Support Grant 68-124, Cancer ResearchFunds of the University of California, and USPHSContract 43-65-609in the Special Viral Cancer Program.

2Postdoctoral Fellow, USPHSGrant 2T1-GM537.Received September 4, 1969; accepted January 29, 1970.

established canine melanoma cell line. They suggested thatthe virus was similar to a myxovirus. Fairly large doses oftissue culture fluid were ineffective in inducing tumors inkittens within the experimental period of 5 months following inoculation.

Recently, we reported the 1st successful transmission offibrosarcomas in cats (32). Puppies and rabbits also developed fibrosarcomas following s.c. inoculation of either cellular homogenates or cell-free Millipore filtrates of the felinefibrosarcoma material. Associated with the original andinduced tumors are large numbers of C-type viral particleswhich were indistinguishable morphologically from felineleukemia virus (18). The relationship of the S-T felinesarcoma virus to the K-T feline leukemia virus is beinginvestigated, and it may prove similar to the relationships ofthe avian sarcoma-leukosis (1 3) and the murine sarcomaleukemia systems (9). The present communication is anaccount of the morphological characteristics of experimentally induced sarcomas in cats.

MATERIALS AND METHODS

Experimental Animals. In this experiment, 4 litters ofdomestic thorthaired kittens were used, I for each of 4successive serial passages. Adult females were fed commercialcat food supplemented with pasteurized bovine milk, andtheir kittens were inoculated s.c. in the dorsal thoracicregion when they were 1 to 6 days old.

Inoculum. Tumor tissue which was frozen at â€”70Â°from 2weeks to 6 months was used as the source of inoculum. Thetumor tissue was collected aseptically from kittens underhalothane anesthesia or after barbiturate euthanasia andexsanguination. Samples of the frozen tumors were thawedin a 25Â°water bath, minced with scissors, and ground inTenBroeck tissue grinders. Sterile 0.9% NaC1 solution wasadded to give a final dilution of I : 1 tissue to 0.9% NaClsolution (w/v). The homogenate was then centrifuged in arefrigerated centrifuge at 2000 rpm for 15 min. The supernatant fluid was removed and passed through 450-mj.t Milliporefilters. The filters were checked and found to be imperviousto a suspension of Escherichia coli. Additional 0.9% NaClsolution was added to restore the filtrate to the volume ofthe original homogenate. One-ml aliquots of the filtrate wereinoculated into each kitten.

Light Microscopy. For histopathology, tissues were fixed ineither formalin or Zenker's fluid and embedded in paraffin.Sections were cut at 4 or 6 p and routinely stained with

1658 CANCER RESEARCH VOL.30

Morphological Studies on Transmissible Feline Fibrosarcoma'

Stanley P. Snyder,2 Gordon H. Theilen, and W. P. C. Richards

Departments of Pathology JS.P.S., W.P.C.R.Jand ainical Sciences [G.H.T.J, School of Veterinary Medicine, University of California, Davis,California 95616

Research. on February 16, 2018. © 1970 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

PassageAnimalNo.SexAge

wheninoculated

(days)Latentperiod(days)â€•Age

whenkilled

(days)Comment1FS-1F22028All

tumors insubcutis1FS-2F22141Alltumors insubcutis1FS-3F23141Alltumors insubcutis1FS-4F23756Widespread

metastases1FS-5M26165Palpabletumorregression2FS-6F11523All

tumors insubcutis2FS-7F15bDiedS dayspostinoculation2FS-8M11023Palpable

tumorregression3FS-29MCC35Nolesions3FS-30F21035All

tumors insubcutis3FS-31M21442Widespreadmetastases3FS-32M21

235Widespreadmetastases4FS-47F51334Widespreadmetastases4FS-48M5834Widespread

metastases4FS-49F5834Widespreadmetastases4FS-50M51034Widespreadmetastases4FS-51FC34No

lesions

Transmissible Feline Fibrosarcoma

hematoxylin and eosin. Phosphotungstic acid-hematoxylin,Mallory's trichrome, Gordon and Sweet's reticulum, Alcianblue-periodic acid-Schiff, and Giemsa stains were also used.Impression smears of tumors were routinely stained withGiemsa stain.

Electron Microscopy. Tumor tissue was sliced into 1-mmcubes, fixed in 1 .5% glutaraldehyde in 0.06 M phosphatebuffer (300 to 310 milliosmols) for 3 hr (30), washed severaltimes with phosphate buffer, postfixed in 1% 0s04 in phosphate buffer, pH 7.4 (21), and embedded in Epon 812 (20).Ultrathin sections on grids were stained with uranyl acetate(36) and lead citrate (27). Specimens were examined with aPhilips EM 200 electron microscope at 80 kV.

RESULTS

Transmission Studies. Results of serial transmissions in 4litters of kittens are summarized in Table I.

Gross Pathology. Tumors in all kittens arose as nonencapsulated, multiple or multinodular, firm, white to pinknodules in the subcutaneous tissue (Fig. 1). Many tumorswere firmly anchored to the dermis; others were freelymovable in the subcutaneous tissue. Some tumor massescontained red focal areas of hemorrhage or brown areas ofnecrosis. The 1st tumor nodules arose at the site of s.c.inoculation; but within 10 days, many smaller ones could bepalpated surrounding the original mass and often at aconsiderable distance from it. Gross evidence of invasion ofsurrounding soft tissues was often readily appreciated at thetime of biopsy or necropsy. In some kittens, the combinedweight of the tumors comprised more than 10% of theanimal's body weight. Metastatic tumors were widely distributed in the various organs and were found in skeletal muscle,

liver, central nervous system, eyes, tongue, omentum, spleen,pleura, kidneys, tonsils, heart, and lymph nodes. The metastases were of 2 basic types. The most commonly encountered type was a solid tumor nodule similar to those foundin the subcutis. These occurred in the skeletal musculatureof all kittens with metastases, and were also occasionallyfound in internal organs. The 2nd type was characterized bysoft to firm red foci from which fresh or clotted bloodcould be readily expressed. These lesions were most frequently seen in the 3rd and 4th serial passages. They rangedfrom a few small red blebs in liver, brain parenchyma, ormeninges to patterns in which the organ was riddled withnumerous cystic, blood-filled cavities (Figs. 2 and 3).

Histopathology. All tumors produced were fibrosarcomas(33) with some individual variation. The majority of the cellswere fusiform with abundant eosinophiic cytoplasm andround, oval, or cigar-shaped nuclei containing clumps ofchromatin material. The basic histological pattern was produced by bundles of parallel cells traversing the tumor tissue invarious planes (Fig. 4). Cells in mitosis were numerous andoften displayed abnormal mitotic spindles. Hemorrhage andnecrosis were a common finding, especially in the morerapidly enlarging tumors (Fig. 5). Some tumors contained nogiant cells; others had numerous giant cells with single largeor multiple smaller centrally clustered nuclei.

Metastatic tumors were either solid (Fig. 6) or cavernous.The solid metastases had structures like that described above.The metastases with cavities, found most often in the liverand central nervous system, consisted of free or clottedblood surrounded by a thin layer of neoplastic cells (Fig. 7).Histologically, these tumors were similar to hemangiosarcomas. The amount of collagen in the tumors was variablebut generally sparse. The neoplasms were well vascularized.

Table 1

Serial transmissions of S-T feline sarcoma virus in kittens

aj@tent period refers to time after inoculation to 1st palpable evidence of tumor formation.b}4jstologjt@aJevidence of early tumor formation at inoculation site.C@pjj@oc@ted contact controls.

1659JUNE 1970



S. P. Snyder, G. H. Theilen, and W. P. C Richards

The solid tumor masses were characterized by 3 basic celltypes: fibroblasts, macrophage-like cells, and mast cells. Themacrophage-like cells were pleomorphic neoplastic cellswhich grade into the fusiform fibroblastic cells and theircharacteristics were seen best in impression smears andelectron micrographs. In impression smears, the macrophagelike cells had vacuolated or foamy cytoplasm, round or ovalnuclei, and often contained phagocytized debris (Fig. 8). Themast cells, evidently nonneoplastic, were most numerousalong small blood vessels, but were also scattered throughoutthe neoplastic tissue (Fig. 9). Impression smears made fromthe cut surface of tumors revealed that the fibroblasts werethe most numerous; macrophage-like cells and mast cellswere respectively fewer. We were unable to determine ifthese 3 cell types also formed the cystic tumors, since theselesions were very difficult to examine.

Lymphocytes were present in variable numbers at theperiphery of tumor masses from animals that had palpableevidence of spontaneous tumor regression. In tumors of 1kitten (FS.8), lymphocytes were found infiltrating the entireneoplasms. Neutrophils were often seen in or near necroticfoci in tumors.

Electron Microscopy. During the earlier stages of development the tumors tended to have more intercellular spacethan in later stages, when they were characterized byincreased compactness and collagen fibrils in small packetsbetween cells. The amount of collagen increased with the ageof the tumors, but it was never extensive.

The three cell types seen with light microscopy were notedin electron micrographs. When sectioned longitudinally,fibroblasts were readily recognized by their elongated nucleiwith infoldings of the nuclear membranes (Fig. 10). Theirnuclear chromatin was coarsely granular and was distributedhomogenously with isolated areas of heterochromatin.Commonly, several nucleoli were seen per fibroblast nucleus,with well-developed nucleolonema surrounding pools of parsamorpha. As in normal fibroblasts (5), rough-surface endoplasmic reticulum was organized and mitochondria and theGolgi apparatus had no special features. Ribosomes andpolysomes were numerous. Small bundles of fine filamentswere sometimes seen within the cytoplasm, and tended to beperipherally located in the cell.

The macrophage-like cells had large round, oval, or indented nuclei with coarsely granular nuclear chromatin andusually a single nucleolus. Some clumping and marginationof chromatin was commonly found. The Golgi apparatus andmitochondria were poorly developed. Small amounts oforganized ergastoplasm were seen but ribosomes were scattered randomly through the ground substance. Characteristicfeatures of these cells were their numerous cytoplasmicprocesses and dilated cytoplasmic vacuoles (Fig. 11).Lysosomes and myelin figures were commonly seen in thesecells. The cells frequently contained phagocytized erythrocytes and cellular debris in areas of hemorrhage and necrosis.An occasional cell contained lipid droplets.

Typical mast cells were scattered throughout all solidtumors; their cytoplasm was packed with electron-densegranules (Fig. 12). Their round or indented nuclei weresmaller than those of the other cells and usually contained asingle nucleolus (Fig. 13).

Virus particles were not found in either the subcutaneoustissues or Achilles tendons (examined as a different site offibrous tissue) of the littermates that served as uninoculatedcontact controls. Typical C-type virions, with outer diameterof approximately 115 mhz, were found budding from cytoplasmic membranes and into membrane-bound vacuoleswithin the cytoplasm of both the fibroblasts and the macrophage-like cells in tumors of all inoculated kittens. Buddingand immature [or enveloped A (9)] forms had inner concentric shells of about 90 and 65 nip (Figs. 14 to 16). Maturevirions contained electron-dense nucleoids measuring approximately 90 mi in diameter (Fig. 17). Budding and immatureparticles were found much more frequently than were themature forms. Intracisternal virus particles were also occasionally observed. C type virions were found in all stages oftumor development and were also seen in tumors undergoingspontaneous regression.

DISCUSSION

Many similarities exist between this transmissible cat sarcoma and those of the avian and murine species. The sarcomasin all 3 species are rapidly induced, have many morphological similarities, sometimes regress spontaneously, and areassociated with C-type virus.

Examination of kittens with induced fibrosarcomas revealedthat tumors were invariably multiple and metastases werefrequent. Metastatic tumors were either solid or cystic,closely resembling those described for both avian (10, 29)and murine (14) sarcomas.

Histologically, the subcutaneous neoplasms and the solidtype metastatic tumors were typical fibrosarcomas (33).Their basic pattern was formed by interwoven bundles ofparallel cells. The tumors were well vascularized and fre.quently showed invasion of surrounding soft tissues. As withRous sarcomas, 3 basic cell types were observed by light andelectron microscopy (I 3). Fibroblasts were the most numerous cells and were morphologically similar to normal fibroblasts (5, 24), except for the presence of replicating C-typevirus particles. Additionally, tumor tissue contained a relativelack of collagen in contrast to normal fibrous connectivetissue. The macrophage-like cells were also quite numerous.These cells, because of their tendency to form cytoplasmicpseudopodia, their paucity of organized endoplasmic reticulum, and their ability to phagocytize extravasated erythrocytes, were considered to be less differentiated than thefibroblastic cells. Other cells, morphologically intermediatebetween fibroblasts and macrophage-like cells, helped tosubstantiate further that the macrophage-like cells wereundifferentiated neoplastic fibroblasts. Mast cells in thetumors were not thought to be neoplastic and were indistinguishable from normal mast cells. Although the role of themast cells in the neoplastic process is not understood, it maybe that they are involved in the formation of hyaluronic acidground substance in a manner similar to that reported forRous sarcomas (1).

Lymphocytes that were present in the few regressingtumors were often adjacent to tumor cells in various stagesof degeneration and necrosis. The role of the lymphocytes inspontaneous tumor regression is also poorly understood andis being investigated.





The morphogenesis of the cystic blood-filled cavities seenin the liver, brain, and occasionally in other organs may besimilar to that suggested by Duran-Reynals for avian sarcomas (1 0). He thought that the cavities may have resultedfrom the action of virus on endothelial cells. In our series,the tumors often closely resembled hemangiosarcomas andprogression in size of the more discrete spherical cavities wasobvious.

Measurements of the C-type virus particles seen in electronmicrographs of sarcomas of the original source cat and of allexperimental kittens show that these virions were morphologically indistinguishable from those associated with catleukemia as reported by us (18, 34).

Additional pathogenesis studies involving continued serialtransmission, different routes of inoculation, age effects, andspecies susceptibility are now being investigated in this andother laboratories. In addition, serological and tissue culturestudies are in progress to test the hypothesis that a felinesarcoma-leukemia system exists in a manner similar to theavian and murine systems.

ACKNOWLEDGMENTS

We thank Dr. D. R. Cordy, Dr. T. G. Kawakami, Mr. R. J. Munn, MrJ_B. Harrold,and Mi. S. H. Clemmensen for their advice and assistance.

REFERENCES

1. Asboe-Hansen, G., Levi, H., and Wegeius, 0. Mast Cells andIntercellular Mucopolysaccharides in Rous Fowl Sarcoma I.CancerRes.,17:792â€”793,1957.

2. Bernhard, W. Electron Microscopy of Tumor Cells and TumorViruses: A Review. Cancer Res., 18: 491â€”509, 1958.

3. Bernhard, W. The Detection and Study of Tumor Viruses withthe Electron Microscope. Cancer Res., 20: 712â€”727, 1960.

4. Bernhard, W., Oberling, C., and Vigier, P. L'Ultrastructure deVirus dans le Sarcome de Rous, leur Rapport avec le Cytoplasmede CellulesTumorales. Bull. Cancer, 43: 407â€”422,1956.

5. Chapman, J. A. Fibroblasts and Collagen. Brit. Med. Bull., 18:233â€”237,1962.

6. Clause, A., Porter, K. R., and Pickels, E. G. Electron MicroscopeStudy of Chicken Tumor Cells. Cancer Res., 7: 421â€”430,1947.

7. Cotchin, E. Neoplasia in the Cat. Vet. Record, 69: 425â€”434, 1957.8. Dalton, A. J. An Electron Microscopic Study of a Virus-induced

Murine Sarcoma (Moloney). NatL Cancer Inst. Monograph, 22:143â€”168,1966.

9. Dc Harven, E. Morphology of Murine Leukemia Viruses. In: M.A. Rich (ed.), Experimental Leukemia, pp. 97â€”129.New York:Appleton-Century.Crofts, 1968.

10. Duran-Reynals, F. A Hemorrhagic Disease Occurring in ChicksInoculated with the Rous and Fuginami Viruses. Yale J. Biol.Med., 13: 77â€”98,1940.

11. Gaylord, W. H. Virus-like Particles Associated with the RousSarcoma as Seen in Sections of the Tumor. Cancer Res., 15:80â€”83,1955.

12. Haguenau, F. Significance of Ultrastructure in Virus-inducedTumors. Natl. Cancer Inst. Monograph,4: 211â€”247,1960.

13. Haguenau, F., and Beard, J. W. The Avian Sarcoma-LeukosisComplex; Its Biology and Ultrastructure. In: A. J. Dalton and F.Haguenau (eds.), Tumors Induced by Viruses: UltrastructuralStudies, pp. 1â€”59.New York: Academic Press. Inc., 1962.

14. Harvey, J. J. An Unidentified Virus Which Causes the RapidProduction of Tumors in Mice. Nature, 204: 1104â€”1105, 1964.

15. Head, K. W. Skin Diseases. Neoplastic Diseases. Vet. Record, 65:926â€”929,1953.

16. Jarrett, W. F. H., Crawford, E. M., Martin, W. B., and Davis,F.Leukemia in the Cat. A Virus-like Particle Associated withLeukemia (Lymphosarcoma). Nature, 202: 566â€”568,1964.

17. Kasza, L., Hayward, A. H. S., and Betts, A. 0. Isolation of aVirus from a Cat Sarcoma in an Established Canine MelanomaCell Line. Res. Vet. Sd., 10: 216â€”218, 1969.

18. Kawakami, T. G., Theilen, G. H., and Munn, R. J. ElectronMicroscopy of California Isolate of Cat Leukemia Virus, Proceedings Electron Microscopic Society of America Twenty-sixthAnnual Meeting, p. 24. Baton Rouge, Louisiana: Claitors PublishingCo., 1968.

19. Laud, H. M., Jarrett, 0., Qighton, G. W., and Jarrett, W. F. H.An Electron Microscopic Study of Virus Particles in SpontaneousLeukemia in the Cat. J. NatL Cancer Inst., 41: 867â€”878, 1968.

20. Lockwood, W. R. A Reliable and Easily Sectioned EpoxyEmbedding Medium. Anat. Record, 150: 129â€”140,1964.

21. Millonig, G. J. Advantages of a Phosphate Buffer for 0s04Solution in Fixation, (Abstract). J. AppL Phys., 32: 1637, 1961.

22. Moloney, J. B. A Virus-induced Rhabdomyosarcoma of Mice.Natl. Cancer Inst. Monograph, 22: 139â€”142,1966.

23. Moulton, J. E. Fibrosarcoma. In: Tumors in Domestic Animals,pp. 24â€”26.Berkeley, Calif.: University of California Press, 1961.

24. Movat, H. F., and Fernando, N. V. P. The Fine Structure ofConnective Tissue. I. The Fibroblast. Exptl. MoL Pathol., 1:509â€”534,1962.

25. Mulligan, R. M. Spontaneous Cat Tumors. Cancer Res., 11: 271,1951.

26. Perk, K., and Moloney, J. B. Pathogenesis of a Virus-inducedRhabdomyosarcoma in Mice. J. Nail. Cancer Inst., 37: 581â€”599,1966.

27. Reynolds, E. S. The Use of Lead Citrate at High pH as anElectron-opaque Stain in Electron Microscopy. J. Cell BioL, 17:208â€”212, 1963.

28. Rickaxd, C. G., Post, J. E., Noronha, F., and Barr, L. M. ATransmissible Virus-induced Lymphocytic Leukemia of the Cat.J_NatL Cancer Inst., 42: 987â€”1014, 1969.

29. Rous, P., and Murphy, J. B. Variations in Chicken Sarcoma Causedby a Filterable Agent. J. Exptl. Med., 17: 219â€”231,1913.

30. Sabatini, D. D., Bensch, K., and Barrnett, R. J. Cytochemistryand Electron Microscopy. The Preservation of Cellular Ultrastructure and Enzymatic Activity by Aldehyde Fixation. J. CellBioL, 17: 19â€”58,1963.

31. Schmidt, R. E., and Langhaxn,R. F. A Survey of Cat Neoplasms.J. Am. Vet. Med. Assoc., 151: 1325â€”1328, 1967.

32. Snyder, S. P., and Theilen, G. H. TransmissibleFeline Fibrosarcoma.Nature,221:1074â€”1075,1969.

33. Stout, A. P. Fibrosarcoma, the Malignant Tumor of Fibroblasts.Cancer, 1: 30â€”63,1948.

34. Theilen, G. H., Dungworth, D. L., Kawakami, T. G., Munn, R. J.,Ward, J. M., and Harrold, J. B. Experimental Induction ofLymphosarcoma in the Cat with â€œCâ€•-typeVirus. Cancer Res.,30: 401â€”408,1970.

35. Theilen, G. H., Kawakami,T. G., Dungworth,D. L., Switzer, J.W., Munn, R. J., and Harrold, J. B. Current Status of TransmissibleAgents in Feline Leukemia. J. Am. Vet. Med. Assoc., 153:1864â€”1872,1968.

36. Watson, M. L. Staining of Tissue Sections for Electron Microscopy with Heavy Metals. J. Biophys. Biochem. CytoL, 4:475â€”478,1958.

1661JUNE 1970




Fig. 1. Experimental Kitten FS-31, 4 weeks postinoculation, showinga large multinodular tumor over the shoulders. X 0.4

Fig. 2. Brain of Experimental Kitten FS-32 showinga few cavernousmetastases on the meningeal surface. X 1.8.

Fig. 3. Liver of Experimental Kitten FS.49 showing extensivereplacement of hepatic parenchyma by dark, cystic structures. X 1.3.

Fig. 4. Subcutaneous tumor showing the basic fibrosarcomatous histological pattern. H & E, X 220.Fig. 5. Subcutaneous tumor containing foci of hemorrhage and necrosis. H & E, X 220.Fig. 6. Solid.type metastatic lesion in the iris and ciliary body of the eye. Lens is at left. H & E, X 80.Fig. 7. Cavernous type of metastatic lesion in the liver. H & E, X 220.Fig. 8. Impression smear of a tumor showing gradation from a foamy macrophage-like cell (left) to a fusiform fibroblastic cell (right).

Giemsa, X 1500.Fig. 9. Tumor containing several mast cells (arrows). Giemsa, X 550.Fig. 10. Fibroblastic cell in a tumor. Adjacent tumor cells show extensive interdigitations of cell membranes. X 12,900.Fig. 11. Macrophage-like cell in a tumor showing extensive cytoplasmic processes. X 12,900.Fig. 12. Mast cell in a tumor. X 16,500.Fig. 13. Tumor, showing relationship of a mast cell to the neoplastic cells. X 12,900.Figs. 14 to 17. C-type viral particles, showing apparent mode of production and maturation in the sequence. X 80,500.

1662 CANCER RESEARCH VOL. 30




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JUNE 1970



1970;30:1658-1667. Cancer Res Stanley P. Snyder, Gordon H. Theilen and W. P. C. Richards Morphological Studies on Transmissible Feline Fibrosarcoma

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