TransmissibilityandSome Histopathology of a Spontaneously Originated Visceral Tumor … · gaster which had received tumor supernatant 74 days previously and two normal newts, each

Several characteristics of a spontaneous visceraltumor found in the Japanese newt, Trituru. pyrrhogaster, have already been reported by one of theauthors (5, 6). During the last 9 years about 30successive generations of inoculations have beencarried out beginning with the original tumor. Themethod of successful transfer of tumor was at firstimplantation of pieces into the belly; then injection of the supernatant from freshly homogenizedand centrifuged tumorous liver, spleen, and kidney; and, more recently, injection of the centrifuged supernatant of homogenized tumor afterrepeated freezing in a mixture of dry ice and acetone and then thawing in water at about 38°C.Other points of interest about the tumor are thatit has the features of a lymphosarcoma;@ thatascites cells can be collected in large amount fromthe peritoneal cavity of tumorous animals; andthat the supernatant of the centrifuged ascitesfluid also induces the tumor.

The present report describes successful inocu

S Supported in part by grants from the Amerinan Cancer

Society and the National Institutes of Health.t On leave from the Institute of Endocrinology,Schoolof

Medicine, Gunma University, Maebazhi, Japan.

Received for publication June 17, 1968. • .

lation in related species and various cytologicalfeatures of the neogrowth. It also describes tumorformation in the regenerating and nonregeneratingforelimb tissues of Triturus ptjrrhogaster after inoculation with the supernatant of the tumorhomogenate.

MATERIALS AND METHODS

Supernatants of homogenates and frozen-driedsamples of the visceral tumor of the newt, Trituruspyrrhoga@ter, were transferred to the urodeles,Hyno&ius lichena@us, Triturus virideacens, and Amblystoma maculatum.

Homogenates of the tissue were prepared in avolume of physiological saline solution about 10times that of the tumor tissue. In some instances,the tissue was frozen (see below) and then thawedto about 38°C., a small amount of saline wasadded, and the tissue was ground into a paste;then saline was added to about 10 times the origin.al volume of the tissue and the suspension furtherhomogenized. The homogenate was centrifuged at92500-4500 r.p.m. for 5—10minutes, and then thesupernatant was drawn off for intraperitoneal injection.

In the case of frozen-dried specimens, the tissue

1679

Transmissibility and Some Histopathology of a SpontaneouslyOriginated Visceral Tumor in the Newt,

Triturus pyrrhogaste@r*

SAKAE INOUEt AND MARCUS SINGER

(Dspartm.n@of Anatomy, SChOOLof Medicineand DeVØiOpnW,UaZBiolo@@yCen@sr,We@IernReserveUniveraily,Clsveland@Ohio)

SUMMARY

The characteristics of the spontaneous tumor of the liver found in Triturus p@jrrhogaster are described in the present work, and it is shown that it can be transferred successfully to other urodeles, including T. viridescens, Hynobius lichenatus, and Anthlyatoma maculatum. The transfer can be made by direct implantation of a piece of fresh tumor into the peritoneal cavity. Frozen-dried specimens also induce a growth, as doesthe supernatant of a homogenized and centrifuged sample after repeated freezings andthawings. The supernatant can also induce an invasive and destructive growth wheninjected into the limb. Moreover, such growths metastasize to the liver.

In addition to involving the liver and spleen primarily, the tumor also is found in thekidney, gonad, and along the intestines. Ascites cells and fluid are also a sign of the neogrowth. The nature of the agent of tumor transmission has not been identified. However, the ensemble of information suggests that it is a microorganism.

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Cancer Research Vol. @3,November 19631680

in a test tube was frozen by dipping the tube intoa mixture of dry ice and acetone and then dried inthe frozen state by vacuum distillation. Dryingwas continued in a desiccator, after which procedure the sample was stored in the open for anumber of days before it was homogenized insaline solution and injected into the peritonealcavity of the host species.

After 492—175days the host animals were sacrificed and autopsied for evidence of the tumor.Tissues were fixed in Bouin's solution, cut at 8 @i,then stained with Delafield's hematoxylin andeosin or with Mallory's trichrome stain. For cytoplasmic basophilia other samples were fixed informol-acetic acid, cut at 6@ and stained with 0.1per cent toluidine blue solution after the methodof Lison (9) ; control sections were treated with1/5000 aqueous solution of crystalline ribonucleasefor 1 hour at 60°C. Some tissues were fixed inZenker's or Helly's fluid, cut at 6 @,and stainedwith Heidenhain's iron hematoxylin for mitochondna—after mordanting with iron alum or beingstained with Schiff's reagent for the Feulgen reaction or the periodic acid-Schiff reagent after themethod of McManus.

Ascites cells were observed under the phasecontrast microscope ; or dried smears were madewhich were stained with the Giemsa method. Forelectron microscopy small pieces of tumor tissueswere fixed with 1 per cent osmic acid solution inphosphate buffer at pH 7.5 for 1.5 hours at 5@C.;and after dehydration in ethanol they were embedded in a butyl methyl methacrylate mixture inthe proportion of 7:3.

For radioautography, three Triturtss pyrrhogaster which had received tumor supernatant 74days previously and two normal newts, eachweighing about 3 gm., were given injections of920 sc. Di,.methionine-S35 in 0.5 ml. saline solution.Six hours later their livers were fixed in Bouin'ssolution. Paraffin sections were made at 6@ andmounted on slides, deparaffinized, and stainedwith hematoxylin and eosin, covered with a membranous photographic film, and dried and storedfor 60 days in the refrigerator. After development,the slides including the film were dehydrated inalcohol and placed in xylene. The photographicmembranes, heavily labeled over the cells, werestripped from the slides and then mounted separately on a slide and compared with the stainedsections.

@ RESULTS

Morphological observation.i.—Grossly, the tumorappears as irregular nodules particularly involvingthe dorsal surface of the liver (Fig.@ 192). The en

largements distend the capsule of the liver andinfiltrate into the parenchyma. The nodules arefrequently many but vary greatly in size. In addition to the liver the spleen is often involved as welland, sometimes, the kidneys and other visceralorgans. The growths have a milky-white color andoften appear fluffy. In advanced growths there aresigns of hemorrhage and extensive tissue destruction ; yet, there are large areas of the organ whichappear normal. Samples of tissue for histologicalstudy were mainly of liver, since it was primarilyinvolved and included normal as well as tumortissue so that the two could be compared histologically.

Histologically, the tumor is composed mainly ofmononuclear cells and some scattered pigmentedcells. Histological sections show that the capsularregion of the liver and other organs is involvedfirst, and the tumor then infiltrates deeply into theparenchyma. The nuclei of tumor cells after staining with Feulgen's reagent appear rather irregularand notched (Fig. 1). The nuclear chromatin issparse and lightly stained in some, and dense anddeeply colored in others. The latter are fewer innumber and generally seen in smaller cells, although an occasional large cell may have considerable deeply staining chromatin. Feulgen stainingin the heavily colored nuclei of tumor cells isstronger than that for nuclei of liver cells but resembles that for the mononuclear cells found inthe cortical part of the normal liver. There is arather strong periodic acid-Schiff reaction (PAS)(Fig. 92) which appears to exceed that for cellsfound in the cortical part of the normal liver. ThePAS-positive material is present either as largeaccumulations or as scattered granules, sometimesconcentrated near the nucleus. The amount ofstaining of the cytoplasm varies among the tumorcells and is different from those of the cortical cellsin the normal liver.

After toluidine blue staining, cytoplasmic basophilia of the tumor cells is often greater than thatof the normal cortical cells. The stainable materialis in the form of small scattered patches, and sometimes tiny vacuolar structures are associated withthe formations. Staining is abolished by pretreatment of the sections with ribonuclease.

The mitochondria of pigmented and unpigmented tumor cells stain with iron hematoxylin(Fig. 3). They vary in number and are generallylarger than those of the normal cortical cells; theyalso show various shapes. Some of them are straightrods; others are crooked or spherical.

Various types of tumor cells are seen in theelectron microscope. The reticulum is rather sparsein all but varies in amount and kind from one cell

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INOUE AND SINGER—SpOntaneOus and Transmi@sibk Tumor in Newt 1681

to the next. Sometimes there is a rough-surfacedlamellar endoplasmic reticulum (Fig. 4). In most,there are scattered vesicles. In some free ribonucleoprotein granules are abundant (Fig. 4), inothers sparse. The nuclei are granulated in general,and sometimes the two limiting membranes of thenucleus are widely separated to form lumens (Figs.4 and 5). The Golgi apparatus may be hypertrophied and the membranous structures spreadout widely in the cytoplasm (Fig. 5). Inclusionswhich might be interpreted as microorganismswere searched for but could not be identified withcertainty. Many mitochondria show apparent degenerative changes, including signs of swellings,loss of internal structure, vacuolation, and assimilation of lamellated vesicles and osmiophilicinclusions (Fig. 5). Sometimes there are shrunkenmitochondria with increased osmiophilia showingloss of cristae. Peculiar darkly stained inclusionbodies are frequently observed ; they are oftenclustered, and each is often surrounded by a haloof unknown substance.

Observations on ascites cells in tumorous animals.—The intraperitoneal fluid of tumorous newts oflong standing is sticky and contains small whitemasses which can be detected with the unaided eyeand which, under the phase-contrast microscope,consist of adherent tumor cells (Fig. 6). The cellsare mostly mononuclear, but some are binuclearand a few multinuclear; the nuclei are round oroval. Most of the ascites cells, stained with theGiemsa stain, show a light blue cytoplasm with avariable number of deeply staining granules.

Radioautographic studies.—In the normal liver,strongest incorporation of DL-methionine-S35 is observed in the parenchyma cells, and the weakestin the capsular region. In the tumorous liver, thereaction of the tumor area is weak as comparedwith that of the parenchyma cells. The resultssuggest a relatively low rate of protein synthesiswhich may be attributed, among other possibilities, to the characteristic slow growth of thistumor.

Implantation of tumor tissues in Triturus yindescens.—Pieces of liver tumor, taken from T.pyrrhogaster 107 days after the injection of tumorsupernatant, were implanted into the body cavityof T. viridescens. Forty-two days later seventeenanimals were examined macroscopically. Most ofthe implant and parts of visceral organs appearedmilky-white, a sign of tumor growth. Scatteredtumor nodules were found clinging to the liver,intestine, gonad, and fat body and clustered in thearea neighboring the implant. Figure 7 shows inhistological section an implant attached to theventral surface of the host liver. The central part

shows widespread degeneration and histolysis, sunrounded by clusters of pigmented cells. The periphery of the implant is composed of a mass oftumor cells and dissociated capsular and corticaltissue of the liver. Tumor cells of the implant haveapparently invaded the surface of the host liverand induced a tumor response there so that theboundary between host and implant is obscuredin places and constitutes a region of transition between the normal liver and implanted tumortissue. The host tumor tissue involves the cortexof the liver and is composed mainly of mononuclear cells; the normal cortex contains manymultinuclear and polymorphonuclear cells the cytoplasm of which is often granular. Farther inwardthe host tumor cells are seen in some places to haveinvaded the liver parenchyma in cords so thatthere is a mixture of normal and tumor cells. Atother places the boundary is rather sharp andbroad, with areas of dissociated cells and scatteredfibrous structures. At the boundaries the arrangement is so complicated that the origin of the transition cellular types from host or implant tissuecould not be distinguished. Figure 8 shows an induced tumor nodule of the spleen of T. viridescens,beginning to invade the normal tissue.

Injection of tumor supernatant into Tniturus viridescens.—Tumorous livers, spleens, and kidneysinduced in T. pyrrhogaster 107 days previously byinjection of tumor supernatant intraperitoneallywere removed and alternately homogenized, frozen,and thawed 5 times. Then 0.1 ml. of the supernatant of the homogenate was injected intraperitoneally; the animals were killed 4@ days (threeanimals) and 1@8days (four animals) after injection. In all animals tumor nodules were found,especially on the dorsal surface of the liver andspleen. Most of the tumor nodules of the liver andspleen were brown in color and consisted, in histological section, of mononuclear cells, the charactenistic component of this tumor (Fig. 9). Whenthe tumorous liver of T. viridescens was homogenized and the supernatant injected into T. pyrrhogaster, tumor growth was induced.

Injection of tumor supernatant into Amblystomamaculatum.—Tumor supernatant, prepared in thesame way as above from three 140-day-old tumorsof T. pyrrhogaster, was injected intraperitoneallyin ten adult salamanders, A. maculatum, 0.5 ml!animal. Five animals survived an adequate period.Two of these, examined 58 days after injection,showed advanced tumor development, especiallyin the liver, the dorsal surfaces of which were a!most completely covered with tumor nodules. Histological sections showed deep invasion of theparenchyma. Indeed, the response of Amblystoma,

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Cancer Research Vol. @8,November 1963l68@

in terms of the speed, extent, and degree of invasiveness of the growth, appeared to be greater eventhan in T. pyrrhogaater, the original source of thetumor. The spleen ordinarily in T. pyrrhogaster ismore refractory than the liver, but in the case ofAmblystoma the spleen was heavily involved.There were many tumor nodules inside the organas well as on the surface, and vast areas were replaced by tumor cells (Fig. 10). The pancreas, intestine, and kidney were also involved. The response of Amblystoma is also peculiar in that themorphology of tumor cells was different from thatseen in other species. They were rich in cytoplasmcontaining a number of mitochondnia and variousamounts of pigment granules, and usually thenucleus was located eccentrically (Fig. 11). Hereand there colloidal accumulations were found inthe intercellular spaces. Another interesting phenomenon, observed at autopsy, was a greatly expanded belly, and a reddish ascites fluid, smearsof which revealed intermingled blood and tumorcells. The remaining three animals, kept muchlonger, showed enormous swelling of the belly.They died within 3 months but were not autopsied.

Injection of tumor supernatant into Hynobius!ichenatus.—About 0.5 ml. of centrifuged tumorsupernatant taken from fresh homogenates of livertumor of T. pyrrhogaster 63 days after tumor initiation was injected intraperitoneally into eightadult newts, H. lichenatus. Forty-three, 70, and175 days later the animals were sacrificed. Macroscopic signs of tumor formation were not found infive animals after 43—70days, but were obvious inone individual of 175 days. In the latter case,there were large, white nodules on the dorsal sunface of the liver and spleen and many minute onesscattered •elsewhere over the liver, spleen, and

. kidney. In histological sections, the tumor nodules

of the liver appeared, as in preceding cases, toarise from the cortical part of the liver and toconsist principally of mononuclear cells having thesame morphological characteristics of the originaltumor. Tumor provocation was also positive whenthe homogenate of H. lichenatus tumor was transferred back to T pyrrhogaster.

Injection of frozen and dried tumor samples.—When frozen and dried pieces of tumor of T.pyrrhogaster were homogenized in saline solution,centrifuged, and the supernatant injected intoother newts of the same species (five animals) orinto T. viridescens (eighteen animals), tumor formation occurred regularly (Fig. 1@). The natureof the tumors and their location, particularly inthe liver, was the same as described above. Onedifference, noted already for tumor implants in T.

t@iridescens,was the greater frequency of pigmentedcells.

Evidence for a tumor-inducing factor in animalsthat failed to develop a tumor.—In the case ofHynobius lichenatus (see above) some recipientanimals did not show development of macroscopically visible tumor nodules after the injection ofsupernatant. Pieces of liver of such animals taken43, 70, and 175 days after the injection of tumorsupernatant, when removed and transplanted intraperitoneally or subcutaneously to twelve T.pyrrhogaster, induced tumors in each instance.Moreover, if homogenates instead of implantswere injected back into T. pyrrhogaster, tumorswere also invariably induced.

Control experiments.—Two kinds of control experiments were performed with T. pyrrhogaster. Inthe first, supernatant drawn from the homogenateof normal liver and spleen was injected intraperitoneally into six animals. No tumor was observedin the viscera of recipient animals 167 days afterinjection. In the second, five normal newts andfive tumorous ones which had received belly injections of tumor supernatant %5Odays in advancewere kept together for 138 days in a small aquarium tank containing about 4 liters of water plussome plant material. The water was changedmonthly. On autopsy no signs of tumor were recognized in the normal newts, although the tumorgrowth in the others was by this time severe. Theresults show that within the period of about 4months, under the conditions we employed, thetumor was not transmitted from one animal tothe next. However, it is possible that a substantially longer period and even more crowded conditions are needed.

Growth of tunwr in the forelimb.—The supernatant of tumor homogenate taken from the liverof T. pyrrhogaster, which had themselves receiveda similar supernatant intraperitoneally 146 dayspreviously, was injected into the soft tissues of theleft upper arm of@ newts of the same species.Enough fluid was injected to distend temporarilythe arm to about 1.3—1.5times its usual diameter.Signs of tumor growth in the limb appeared about36 days later; the upper arm was swollen in somecases more than in others, but in all instances itwas always greater in diameter than the oppositecontrol limb. In some instances there was ulceration of the skin with intermittent hemorrhage fromthe surface. Healing was never complete, and thesurface appeared ragged, denuded, and inflamed(Fig. 13). At this time the limb was amputatedthrough the swollen tumorous region in fifteencases and the amputated part fixed for histological

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INOUE AND Sncom@—Spontaneous and Transmi@ible Tumor in Newt 1683

study; the remaining seven served as nonamputated controls. The limbs of newts can regeneratereadily after amputation, and amputations wereperformed to ascertain whether processes of renewalof the amputatedlimb byregeneration wouldaffect the growth of the tumor.

Histological study of the amputated tumorouslimbs showed a heavily infiltrating tumor widelyspread throughout tissues of the upper arm. Inits growth the tumor had gradually destroyed andreplaced the muscle and connective tissue until thenormal internal structures of the limb, except forthe bone, were no longer recognizable. Autopsyshowed metastasis from the limb to the viscerawith small tumors studding the liver, spleen, andother parts.

In the case of the amputated limbs, epitheliumattempted to move over the surface to close thewound as it ordinarily does. In some cases closurewas incomplete, or the closed epithelium was seeondarily interrupted and ulcerated by the tumor.In these instances, there was also intermittenthemorrhage. The epithelium tended to persist onlyover nontumorous tissue. Regeneration of the limbdid not occur in limbs with advanced ulceratingtumors, and the wound surface failed to heal( Fig. 14) . In instances where there was an area of

normal tissue at the wound, including muscle andskin, a small regenerate was formed (Fig. 15) ; insome instances the growth was aborted as thetumor spread through the underlying normal tissue and undercut the new growth.

DISCUSSIONRelatively few cases of spontaneous tumor have

been reported among the urodeles (15). The rarityof spontaneous tumors in this order has given riseto the view that normal tissues of the urodele exerta stronger organizing effect, causing them to differentiate into normal cells rather than to growuncontrolled (15). Indeed, it has been reported byRose and Wallingford (14) that, when the renaladenocarcinoma of the frog, Rana pipiens, is implanted into the regenerating limb of T. viridescens, muscle, cartilage, and fibrous connectivetissue differentiate from the graft. There is evidence that tissues of the unodele are not as resistant to carcinogenesis as previously thought,because tumor formations can be induced in thelimb of salamanders by methylcholanthrene (@)and by a mixture of 3,4-benzpyrene and bloodalbumin (7) ; and tumors of the notochord of larvalAmblystoma punctatum have been evoked by thecrystalline Lathyrus factor (8). Moreover, thepresent work and previous studies by one of us

(5 and 6) report a spontaneous tumor in the liverof T. pyrrhogastsr which can also grow in otherviscera and in somatic tissue as well—e.g., thelimb. It is probable that amphibians are not asimmune to invasive neogrowths as previouslythought and that other tumors will be found inthis group of animals. Indeed, the tumor may bereadily transferred from one species to another asthe present work shows. Furthermore, the tumoris capable of metastasis as shown in the limb experiments and also in the formation of ascites cells inthe peritoneal fluid.

The newt tumor can be successfully transferredto individuals of the same and related species byway of frozen and dried specimens. Moreover, thesupernatant of a centrifuged homogenate of frozendried or fresh tumor induces a neogrowth. Exceptfor tumors in Amblystoma, the nature of whichare still under investigation, the tumor induced inother species always has the same essential grossand histological features. These experiments showthat whole cells of the original tumor were nottransplanted and did not provide the genetic lineof the new tumor. Some factor on some fragmentextracted from the tissues or cells was the causative agent. The nature of the agent of tumor induction has as yet not been ascertained. A possible interpretation is that the tumor is caused bya microorganism, since the results, at the moment,are commensurate with those of studies of tumorsknown or strongly suspected to be caused by amicroorganism.

The adenocarcinoma of the frog (10) can alsobe transplanted from one animal to another of thesame species (1@). It will grow in related speciesas well (1@), and has been transplanted to the limbof the urodele T. viridescens (14 and others) wherethe tumor agent has been reported (13) to causetransformation in the cells of the young salamanden or in regenerating cells of the adult. Moreover,the tumor may be transferred after it is frozen,desiccated, and glycerinated (11). Fawcett (4) reported virus-like bodies in the nucleus, cytoplasm,and intercellular spaces by electron microscopy.

In animals other than the Amphibia, tumorscan provoke neogrowths in related species—e.g.,Rous sarcoma in the pheasant (1) and duckling(3) ; and the infectious papillomatosis found incottontail rabbit is readily transmissible to thedomestic rabbit (16).

REFERENCES1. ANDREWEB,C. H. The Transmission of Fowl Tumours to

Pheasants. J. PathoL Bacteriol., 35:407-18, 1932.2. BREamS,C. TransplantableSarcomaof the Salamander

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1684 Cancer Research VoL @3,November 1963

Induced by Methylcholanthrene. Cancer Res., 11:239,1951.

3. Dusu@N-RE@ALs,F. The Reciprocal Infection of Ducksand Chickens with Tumor-inducing Viruses. Cancer lies.,2:843—69, 1942.

4. FAWCIcrr,D. W. Electron Microscope Observations onIntracellular Virus-like Particles Associated with the Cellsof the LuckéRenal Adenocarcinoma. J. Biophys. Biochem.CytoL, 2:725—49, 1956.

5. INOUE,S. On the Transplantable Spontaneous VisceralTumor in the Newt, Triturus pyrrhogasier. Sci. Rep. TohokMUniv., 20:226—86,1954.

6. . Experiments on the Potency of the Tumor Supernatant and the Peritoneal Exudate of the Newt (Trituru.pyrrhogasier) Bearing Spontaneously Originated VisceralTumor. Gunma J. Med. Sci., 3:269—79, 1954.

7. LEONE, V. Richerche e considerazioni sulla concerizzazionenegli anfibi. Tumori, 39:420—42, 1953.

8. Luvy, B. M., and GODMAN,G. C. Tumors of the Notochordof the Salamander, Amblyatomapunctaium, Produced by

Crystalline Lathyrus Factor. Cancer Res., 15:184—87,1955.

9. LisoN, L. Histochimie et cytochimie animales. Paris:Guatier-Villars, 1958.

10. Luc@g,B. A Neoplastic Disease of the Kidney of the FrogRana pipiens. Am. J. Cancer, 20:352-79, 1934.

11. . Carcinoma in the Leopard Frog: Its ProbableCausation by a Virus. J. Exp. Med., 68:457-68, 1988.

12. Lucx@,B., and SCHLUMBERGER,H. Heterotransplantationof Frog Carcinoma; Character of Growth in the Eyes ofAlien Species.J. Exp. Med., 72:311—20,1940.

13. Rosz, S. M., and Ross, F. C. Tumor Agent Transformation in Amphibia. Cancer lies., 12: 1—12,1952.

14. Rosa, S. M., and WALLINOFORD,H. M. Transformation ofRenal Tumors of Frogs to Normal Tissues in RegeneratingLimbs of Salamanders. Science, 107:457, 1948.

15. SCHLUMBERGER, H. G., and Lucx@, B. Tumors of Fishes,

Amphibians and Reptiles. Cancer lies., 8:657—753, 1948.16. SIIOPE,R. E. Infectious Papillomatosis of Rabbits with a

Note on the Histopathology by E. W. Hurst. J. Exp. Med.,58:607—24,1938.

Fio 1.—Feulgen-stained section of Zenker's fixed tumorous with homogenate supernatant. The cell mass on the left ofliver of T. pyrrhogaster 98 days after inoculation with tumor the photograph is a tumor nodule. Compare the strong PASsupernatant intraperitoneally showing intermingled tumor and reaction of tumor cytoplasm with that of normal cortical tissueliver parenchyma cells. Note the variation in nuclear shape on the right (bottom) of the figure. X450.and density. X240. Fm. 3.—Mitochondria (arrow) of liver tumor cells after

Fia. 2.—Periodic acid-Schiff reaction in tumor cells of Zenker's fixation and staining in iron hematoxylin, 87 daysZenker's fixed liver of T. pyrrhogasisr 87 days after inoculation after injection of tumor supernatant. X1750.

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FIG. 4.—Electron micrograph of liver tumor cells showing,in the upper one, some rough-surfaced ergastoplasmic lamellaeand vesicles. Note abundance of free granules. X26,000.

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FIG. 5.—Electron micrographs of liver tumor cells showing

irregular nucleus with separation of the limiting membrane.Endoplasmic reticulum is sparse and vesicular. Note the degenerating mitochondria and dark round inclusion bodies witha surrounding light halo. X26,000.

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FIG. 6.—Phase-contrast photomicrograph of ascites tumor

cells of T. pyrrhogo.ster. Most of the nuclei are mononucleated,some of them binucleated or multinucleated. Some pigmentedcells are also present. X1100.

FIG. 7.—Implant of liver tumor tissue of T. pyrrhogaster to

T. viride8censattached to the ventral surface (left) of the hostliver. Growing area can be seen at the periphery of the implant involving implant and host cells invading the parenchyma. X100.

FIG. 8.—Discrete tumor nodule growing on the dorsal sur

face (left) of the spleen of the same animal shown in Figure 12.The tumor nodule consists mainly of mononucleated cellsX270.

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FIG. 9.—Tumor nodules developed in the dorsal cortical

part (left) of the liver of T. viridescens after injection of thesupernatant of a frozen-dried tumor homogenate taken fromT. pyrrhogo4er. There are a number of slightly pigmentedcells in the tumor. Hematoxylin-eosin stain. X 120.

FIG. 10.—Section of a tumorous spleen of Amblystoma 58

days after intraperitoneal injection of tumor supernatant froma frozen-thawed specimen taken from the tumorous viscera

of T. pyrrhogaster. Extensive tumor growth is seen at thedorsal side (left) of spleen, and vast areas of the parenchymaare replaced by tumor cells. Hematoxylin-eosin stain. X 105.

FIG. 11.—Tumor cells of Amblystoma liver induced by intraperitoneal injection of the supernatant of T. pyrrhogaster'stumor, stained with iron hematoxylin after Helly's fixation.Some cells at center of the photograph are loaded with mitochondria. The cytoplasm is voluminous and the nucleus usually eccentric. X 1455.

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FIG. 12.—Tumors Oil the dorsal side of a liver of T. pyrrhoga4er induced by injection of supernatant of homogenatesprepared from frozen and dried tumorous viscera of T. pyrrhogaster. Note the many nodules that dot the surface of the liver.X8.

FIG. 18.—A ragged upper arm (left) of T. pyrrhogaster 162days after injection of tumor supernatant. Vast areas of skinand muscle were eaten away, and the humerus was partlyexposed. Fingers and part of the hand decayed and weresloughed. Swelling of the limb preceded ulceration and sloughing. Right limb served as normal control. X3.

FIG. 14.—Histological section of nonregenerating tumorouslimb which was amputated 36 days after tumor supernatantinjection and fixed 70 days later, at which time the normal

control side showed an advanced regrowth of a new limb. Endof humerus marks original amputation line. Note limited cartilage regeneration at end of bone and incomplete epidermalclosure of wound. Upper part of figure also shows normalproximal muscle. Lower part of figure shows absence of epithelium and a mass of tumor tissue. The bone marrow wasalso invaded by tumor cells. Fixed with Bouin's fluid, stainedaccording to Bodian's silver proteinate and Masson's trichromemethods. X35.

FIG. 15.—Tiny regenerate in the center of the cut surfaceof the limb in which tumor supernatant was introduced 106days before amputation. Fixed 70 days after amputation ofthe limb. Remainder of amputation surface was ulcerated andunderlaid by concentrated masses of tumor that had replacedthe normal tissue. The regenerate was packed with blastemacells, but intermingled here and there among them were somesmall masses of tumor. Original amputation line marked byend of humerus on right. Bouin's fluid fixation and silver impregnation, counterstained with Masson's trichrome stain.X53.

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1963;23:1679-1684. Cancer Res   Sakae Inoue and Marcus Singer 

Triturus pyrrhogasterOriginated Visceral Tumor in the Newt, Transmissibility and Some Histopathology of a Spontaneously

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