[CANCER RESEARCH 29, 223—229,January 1969]

MATERIALS AND METHODS

A spontaneous tumor was discovered in the dorsal musclesof the left proximal portion of the tail of a 2.5-year-old axolotl. The lumbosacral region of the animal, together with theproximal portion of the tail, was fixed in toto in a modffication of Zenker's fixative (A. L. Roque, manuscript in preparation). Superficial incisions were made in order to facilitatepenetration of the fixative. A couple of hours later, when thetissues were hardened, the tumor and the surrounding tissueswere cut transversely into four pieces for gross examination.

After each piece of tumor had been photographed, it wasdecalcified, dehydrated, cleared, and embedded in paraffin intoto. Sections at 6@ were made from both surfaces of eachblock and were stained with hematoxylin and eosin for generalhistologic study and photomicrography. For demonstratingmuscular, connective, and neural tissues, a trichrome stain (9)and the Puchtler phosphotungstic acid hematoxylin stain (8)were used. Periodic acid-Schiff (PAS) and mucicarmine wereemployed for demonstrating glycogen and other polysaccharides. A modification of the May-Grünwald-Giemsa technicwas used for demonstrating basophiic and acidophiic cytoplasmic materials. Photomicrographs were made at X 1 5, 40,100, 200, and 610. Special attention was given to the preparation of one compound photograph consisting of many separatephotomicrographs under very low magnification (X 1 5), inorder to provide a general picture of an almost complete crosssection of the tumor.

INTRODUCTION RESULTS

Amphibian and human neoplasms show remarkable similarities in cellular structure (4). Almost all human tumors havetheir analogs in other animals, but most studies have dealt withmammals or birds, and only a few with reptiles, amphibians, orfishes, mostly because of the assumption that tumors in coldblooded vertebrates are rare and hence difficult to obtain forstudy (6). Luck@ and Schlumberger (7) showed otherwise infishes and amphibians. Even so, tumors appear to be less frequent in urodeles than in anurans (1).

The Mexican axolotl (Siredon mexicanum) is the neoteniclarval form of a salamander, which nevertheless becomes capable of reproduction (2). Axoloth have developed several typesof tumors, melanoma (5, 10, 1 1, 13, 14, 16), adenocarcinoma(13), neuroepitheioma (3), and possibly epitheioma (12), butsuch spontaneous tumors are rare. According to Stevens (1 5),the number of species in which teratomas have been observedis small. Teratomas have never been noted in dogs, rats, rabbits, or amphibians.

While the animal was still alive, the tumor measured 19.7 x19.8 x 8.2 mm (Fig. 1). It bulged under the skin, which wascompressed by it. The tumor extended deep into the subcutaneous tissue and penetrated some distance into the dorsalmuscles, which were partially destroyed, compressed, and displaced (Figs. 2, 3). It had a loculated appearance, being composed of cavities separated by thin septa of connective tissue.Some of these cavities were cystic and contained a mucoidmaterial. Others contained a solid material of epithelialappearance.

Histologic examination showed that the tumor consisted of awide variety of tissues, foreign to the surrounding muscle (Fig.7) and mostly atypical for a normal organism. The most unportant tissue was epitheium, partly organized in the form ofglan d-like structures (Fig. 4). These neoplastic glands werecomposed of cuboidal or cylindrical cells arranged in variouslayers. They rested on a basement membrane well demonstrated by the PAS and reticulum-trichrome stains. Some

JANUARY 1969 223

A Spontaneous Teratoma in an Axoloti (Siredon mexicanuin)

Victor V. Brunst and Augustine L. Roque

Departments ofExperimentai Biology and Pathology, Rosweli Park Memoriailnstitute, Buffalo, New York 14203

SUMMARY

A spontaneous tumor originated in the dorsal muscles of theproximal portion of the tail of a 2.5-year-old axoloti (Siredon

mexicanum). Histologically, the tumor consisted of tissues derived from ectoderm, mesoderm, and endoderm. Its most significant cells were epithelial, partly organized in the form ofgland-like structures. The neoplastic glandular epithelium wasarranged mostly in solid sheets or cords resting on a thin basement membrane; this tissue did not resemble any of the welldeveloped or differentiated epitheium or glandular tissueknown to occur in the axolotl. The cells contained scantycytoplasm with abundant basophilic material. Their nucleiwere large and hyperchromatic and had one or two prominentnucleoli. Only these cells underwent mitosis, which was extensive in some areas. A second common tissue most likely resulted from differentiation and gradual degeneration of theepithelial tumor tissue, producing rounded pieces of compacttissues with a comparatively small number of nuclei and gradu

ally increasing amounts of intercellular material. The nucleibecame pycnotic and fewer in number. The tumor also contamed loose, compact, and embryonic connective tissues. Insepta composed of connective tissue, isolated striated musclewas found. In some areas there were neuroepithelial structuresand abnormal cartilage. There were few blood vessels, manyblood sinuses, and many blood cells in various stages ofdevelopment.

This tumor was evidently a spontaneous teratoma.

on April 8, 2020. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Victor V. Brunst and Augustine L. Roque

expected to occur, there were only blood cells (Fig. 25); somepieces of cartilage contained large blood sinuses. In otherplaces, however, there were small clusters of epithelial cellswhich, because of their form, structure, and arrangement, canbe regarded as neuroepithelium (Fig. 27).

Only a few (mostly small) true blood vessels were found inthe tumor (Figs. 7, 9, 10, 12, 23); all had blood vessel walls(Fig. 23). More often the blood supply of the tumor was provided by large blood sinuses (Fig. 7). These sinuses characteristically lacked blood vessel walls. Some were surrounded bysimple epitheium, others partly by epitheium and partly onlyby a thin basement membrane (Fig. 22), and still others onlyby a cartilaginous walL In many places, the blood sinuses hadno definite borders, and blood cells in large numbers penetrated into the degenerate differentiated tissue that predominated in the tumor (Fig. 7). It is noteworthy that largenumbers of erythrocytes and leukocytes, in various stages of

development, accumulated in the blood sinuses.

DISCUSSION

A single tumor, composed of tissues derived from ectoderm,mesoderm, and endoderm, was observed in one animal; nometastasis was found. It seems to have originated in skeletalmuscle, but neither its gland-like epithelial tissue nor the tissueprobably resulting from differentiation of that epithelial tissueresembled any of the well-developed epitheium, glandular tissues, or other tissues of the normal adult axolotl. Thus thetumor satisfies Willis' definition of a teratoma as “atrue tumoror neoplasm composed of multiple tissues foreign to the partin which it arises―(17). At least one gross feature of thetumor, cystic cavities in which solid components were growing, was also characteristic of a teratoma.

REFERENCES

1. Balls, M. Spontaneous Neoplasms in Amphibia: A Review and Dcscription ofSix New Cases. Cancer Res., 22: 1142—1154, 1962.

2. Brunst, V. V. The Axolotl (Siredon mexicanum). I. As Material forScientific Research. Lab. Invest., 4: 45—64, 1955.

3. Brunst, V. V., and Roque, A. L. Tumors in Amphibians. 1. Histology of a Neuroepithelioma in Siredon mexicanum. J. Natl. CancerInst., 38: 193—204, 1967.

4. Duryee, W. R., Long, M. E., Taylor, H. C., Jr., McKelway, W. P.,and Ehrmann, R. L. Human and Amphibian Neoplasms Compared.Science, 131: 276—280, 1960.

5. Krontovsky, A. Comparative and Experimental Pathology of Tumors. In Russian. Kiev: Bacteriological Institute, 1916.

6. Luckd, B., and Schlumberger, H. G. Common Neoplasms in Fish,Amphibians and Reptiles. J. Tech. Meth. Bull. mt. Assoc. Med.,22: 4—17,1942.

7. Luck@, B., and Schlumberger, H. G. Neoplasia in Cold-BloodedVertebrates. PhysioL Rev., 29: 91—126,1949.

8. Puchtler, H., Sweat, F., and Doss, N. 0. A One-Hour Phosphotungstic Acid-Hematoxylin Stain. Am. J. Clin. Pathol., 40: 334—337,1963.

9. Roque, A. L. Chromotrope Aniline Blue Method of Staining Mallory Bodies of Laennec's Dirrhosis. Lab. Invest., 2: 15—21, 1953.

10. Sheremetieva, E. A. An Investigation of Spontaneous Tumours inAxolotls (Siredon pisciformis): Pigmentary Tumours. Rept. Inst.Zool Acad. Sri. Ukr. S.S.R., 12: 37—61,1938 (in Russian).

giands had a wide, empty lumen; in others, the lumen wasfilled with a mucoid material that reacted positively to mucicarmine and PAS and was visible in tumor cytoplasm. Accu

mulation of this material seemed to have brought about thedistension and cystic degeneration of the neoplastic gland-likestructures; nuclei of tumor cells were embedded in it (Fig. 28).

Some of this material was still recognizable as debris from thecytoplasm of tumor cells.

The neoplastic glandular cells were arranged mostly in solidsheets or cords resting on a thin basement membrane (Figs.5—8,10, 11, 15). These tumor cells contained scanty cytoplasmwith abundant basophilic material; their nuclei were large andhyperchromatic, and had one or two prominent nucleoli. Anoccasional giant cell, probably triploid (Fig. 16), was observed.

Only these glandular neoplastic cells underwent mitosis, whichwas extensive in some areas (Figs. 16—21).An apparent minormodification was found in sheets of epitheium, consisting

mostly of one layer of cylindrical cells (Figs. 7—9, 12). Thisepithelium sometimes formed the walls of large cavities (Figs.7—9) or penetrated between other compact tissues (Fig. 12).

Most likely it was differentiation and gradual degenerationof the glandular neoplastic tissue that resulted in the development of the second most significant tissue. This consisted ofrounded pieces of compact tissue with a relatively small number of nuclei and a gradually increasing amount of intercellularmaterial. These pieces of tissue had definite borders and werestructurally similar to cartilage (Fig. 7A). The intercellularmaterial, presumably representing the beginning of the development of this tissue, was almost completely homogeneous(Fig. 11), but the process of differentiation resulted in theformation of numerous fibers (Figs. 5, 1 2, 14, 1 5). This tissue

occurred mostly in large pieces (Fig. 7) but was sometimessubdivided into many small, rounded pieces (Figs. 7, 13); mitosis was never observed. Evidently the tissue had graduallydegenerated, the nuclei decreasing greatly in number and becoming pycnotic. Giant degenerate nuclei were observed insome cases (Fig. 14). Such tissue is never present in a normalaxoloti.

The tumor also contained small amounts of other types oftissues (Fig. 7), particularly the connective tissue of the septa

separating the lobules of the tumor. In some places, there wasembryonic fibrous tissue, easily distinguished from adult connective tissue with its bands of collagen fibers (Figs. 5, 23, 24,28), being composed of fibroblasts laying down fibers of collagen, as shown by trichrome stain. The trichrome and thePuchtler phosphotungstic acid hematoxylin stains revealed fibroglia in the cytoplasm of these fibroblasts.

In the septa composed of connective tissue, isolated piecesof striated muscle were found (Figs. 10, 26, 28). These pieceswere independent of the striated muscles surrounding the tumor, just as the loose connective tissue and the epitheium ofthe tumor were independent of the subcutaneous loose connective tissue and the skin epitheium in the vicinity of thetumor (Fig. 7). A few normal pigment cells (melanocytes)were also present, chiefly in connective tissue (Fig. 10).

Small pieces of abnormal cartilaginous tissue were found insome portions of the tumor. This tissue contained intercellularmaterial typical of cartilage but completely lacked cartilaginous cells. In empty spaces where cartilaginous cells might be

224 CANCER RESEARCH VOL.29

on April 8, 2020. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

A Spontaneous Axoloti Teratoma

1 1. Sheremetieva, E. A. Spontaneous Melanoma in Regenerating TailsofAxolotls. J. Exptl. Zool., 158: 101—122, 1965.

12. Sheremetieva-Brunst, E. A. An Epithelioma in the Axolotl. Proc.Am. Assoc. Cancer Rca., 1: 51, 1953.

13. Sheremetieva-Brunst, E. A. Melanoma and Adenocarcinoma in theRegenerating Organs of the Axolotl. In: W. R. Duryee and L.Warner (eds.), Transcript of Proceedings, Frog Kidney Adenocarcinoma Conference, Natl. Cancer Inst. Bethesda, Md., pp.115—123, 1961.

14. Sheremetieva-Brunst, E. A., and Brunst, V. V. Origin and Trans

plantation of a Melanotic Tumor in the Axolotl. Spec. Pub. NewYork Acad. Sci. 4: 269—287,1948.

15. Stevens, L. C. The Biology of Teratomas. In: M. Abercrombie andJ. Brachet (eds.), Advances in Morphogenesis, Vol. 6, pp. 1—32.New York: Academic Press, 1967.

16. Teutschlander, 0. Beitrage zur vergleichenden onkologie mitBerucksichtigung der Identitätsfrage. Z. Krebsforsch, 17: 285—407,1920.

17. Willis, R. A. Teratomas. In: Atlas of Tumor Pathology, Sec. III,Fasc. 9. Washington, D. C. : Armed Forces Institute of Pathology,1951.

Fig. 1. Axolotl, 2.5 years old, with spontaneous tumor (T) on proximal dorsal portion of tail. Centimeter rule is included for comparison.Fig. 2. Cross section of whole animal shown in Fig. 1, rostral to tumor; normal situation of tissues. M, muscle; S, spinal cord; V, vertebral

column. Centimeter rule is included for comparison.Fig. 3. Two cross sections of animal shown in Fig. 1. M, muscle; S, spinal cord; T, tumor; V, vertebral column. Centimeter rule is included for

comparison.Fig. 4. Tumor cross section. C-C, glandular cell tissue. In many places, a glandular tube with an empty lumen (L) was formed. X 40.Fig. 5. Tumor cross section. CT, loose connective tissue; DT, differentiated tissue derived from glandular tissue (CC). X 40.Fig. 6. Tumor cross section. This portion of the tumor consisted only of glandular cells, and its lobules were separated by septa (SE) composed

of connective tissue. There was little lumen (L). x 40.Fig. 7. Compound photomicrograph (prepared from many separate photomicrographs) of almost complete cross section of tumor. A,

differentiated tissue, resulting from transformation of glandular cells (D); B, tissue consisting of sheets of epithelium; BS, blood sinuses; BV, bloodvessels; C, tissues consisting of limited numbers of glandular cells and large empty lumens; CT, loose connective tissue; E, skin epithelium; M,muscle (muscle inside the tumor, as well as normal muscle outside); MT, tumor margin. x 15.

Fig. 8. Tumor cross section. BC, blood cells in blood sinus; DT, differentiated tissue; E, sheets of epithelium; CC, glandular cells; L, lumen. X40.

Fig. 9. Epithelial part of tumor. By, blood vessel; E, sheet of epitheium; MM, mucoid material in the lumen. x 100.Fig. 10. Connective tissue septa (CT) between glandular lobules (CC). BV, blood vessels; M, muscle; PC, pigment cells. x 200.Fig. 11. Glandular (GC) and differentiated (Dfl portions oftumor. x 100.Fig. 12. Epithelial layer (E) between differentiated portions (Dl') of tumor. BV blood vessels. x 100.Fig. 13. Differentiated portion of tumor, consisting mostly of pycnotic cells. x 200.Fig. 14. Degenerate portion of tumor, with pycnotic nuclei (P) or degenerate giant nuclei (C). x 200.Fig. 1 5. Portion of tumor, with final differentiation (DT). CC, glandular cells. x 200.Figs. 16—21. Glandular cells oftumor. C, giant cell; Mt, mitotic figures. x 610.Fig. 22. Blood sinus in tumor, lined with epithelium (E) and thin basement membrane (M). BC, blood cells. X 200.Fig. 23. Portion of tumor, consisting of glandular cells (CC) and loose connective tissue (CT) surrounded by thin simple squamous epithelium

(E). B V, large blood vessel; PC, pigment cells. x 200.Fig. 24. Connective tissue between tumor lobules. CTC, connective tissue cell; F, collagen fibers. x 610.Fig. 25. Abnormal cartilage in tumor. BC, blood cells in the spaces (S) in the intercellular substance (CR). X 610.Fig. 26. Striated muscle (M) in connective tissue (CT). x 610.Fig. 27. Neuropithelial cells (N). CT, loose connective tissue. x 610.Fig. 28. Portion of tumor, consisting of glandular tissue (CC) and interstitial connective tissue (CT), with striated muscle (M). L, lumen;MM,

mucoid material with nuclei. x 200.

JANUARY 1969 225

on April 8, 2020. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

M

2@ V

II

s•.@.@. .:@, DI

‘ .- .

(,,,I. @,

t@ ,a .@‘@ ---

,._@=•‘@

@.@ ..@, -

... ‘ @:@ •@t'. .a@@\)@'-@

.‘@ - ‘@ - @:@‘@ • .•@ ,. 4,

. --@@@ :@:@ ‘@@ :

@: ‘.: “-.‘-@!:‘@ 4, . - ,. ..,- .,-@. 4

@-

b'—@ %:l@ @. @.

@,@ \ S •

4,@ , @‘

£@__

@pi'•__ -w.q ó.@i$',@

,,@p •

@,

226CANCER RESEARCH VOL.29

Victor V. Brunst and Augustine L. Roque

:@@.1/@) @i -

on April 8, 2020. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

.@

c-;'@.@_;. .y,@@

-‘i,: . , -,, •

‘I.@ : :@ , @.@ ‘c..

.,.. ,: @..@@

, ‘ ,@ .- . I • . •@

@. _‘%,@ .@. . (, . . - . . .@ .@ :@f .:

JANUARY 1969 227

on April 8, 2020. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

ci. @.@.

@k

-@-1@M@. .— @:

I

S•

A

‘I,

, 4.

a

S

0

GC

. •4@@—@ -@.‘.

S

S Dr

%, ,. .5

0 t' .,.‘I. 4g@I:•.;,\a.@S @‘• \@ @_.*@‘. •(l@1@,

-p

•1

•@@

.5@ I.

0@ .@,

â€p̃S•1

141

‘44.

S

__@‘.ii__..@-@-, -@ JIL

@\O‘‘4j-@ ‘ :@;“‘

.- -@ - /_\ .@ •:-‘@ ‘@‘@?/:@

@ . p :.@ @-@@@@@ “@

\@ )‘@..‘ • , >‘ 1

@ ‘.@ @-‘. @:k@@ - I@

•DT,!@;@, :‘

@ :@@ •@ @,@@

*‘@ @.J .—--@ ?@@—%-.-- ‘ @;. L__...@@ ..,@.,@

‘I@ :@.O@ •@@ @,,@

5'@'iS@ @:@)\:@?@

—.@—-@\

..@#.

..

-I

‘.

@@1

13@,

228 CANCER RESEARCH VOL.29

/1@4@,ff,

@e/,,ift .@

on April 8, 2020. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

.@

1'•

CT

idi“.4

II. I'

I

27

\@,

... CT@

JANUARY 1969 229

@A\b@

, . - I

\@\@ . .@.@. @\‘@

on April 8, 2020. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1969;29:223-229. Cancer Res   Victor V. Brunst and Augustine L. Roque 

)Siredon mexicanumA Spontaneous Teratoma in an Axolotl (

  Updated version

  http://cancerres.aacrjournals.org/content/29/1/223

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/29/1/223To request permission to re-use all or part of this article, use this link

on April 8, 2020. © 1969 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from