Spontaneous Primary Hepatomas in Mice of Strain C3H IV. A Study of Intracytoplasmic Inclusion Bodies and Mitochondria Edward L. Burns, M.D., and John R. Schenken, M.D. (From the Department of Pathology and Bacteriology, Louisiana State University School o[ Medicine, New Orleans, La.) (Received for publication May 24, 1943) In describing the histology of spontaneous hepatomas as to dosage and time of injection have been given~ in of strain C3H mice, we pointed out (2) that the a previous report (6). cytoplasm of the tumor cells contained two types of Forty-nine of the mice did not have liver tumors. inclusion bodies. One of these was a large, homo- Of these 16 were untreated controls, of which 7 were geneous, or finely granular, hyaline body that often breeding and 6 nonbreeding males and 1 was a breed- pushed the nucleus to one side; the other a rounded, ing and 2 were nonbreeding females. Thirty-three were or sometimes indented, pink-staining body that varied treated animals. Three nonbreeding males and 1 non- greatly in size and sometimes showed a doubly refrac- breeding female were treated with a-estradiol benzoate; tile ring at the periphery. 10 nonbreeding males and 1 nonbreeding female were Other investigators also have found cell inclusions treated with ketohydroxyestrin; 9 nonbreeding males in hepatomas. Edwards and Dalton (4) described and 9 nonbreeding females were treated with testos- globular inclusions in the cytoplasm of cells of a pri- terone propionate. mary liver neoplasm of a strain C3H mouse, as well Most of the animals were killed with chloroform as in the first generation transplant of this tumor. when they appeared ill. The remainder died spon- Edwards and White (5) described cytoplasmic inclu- taneously and were examined as soon as possible after sions in hepatomas produced in rats by feeding death. Pieces of liver were fixed in Helly's fluid and p-dimethylaminoazobenzene. These inclusions varied in 10 per cent formalin. Microscopic sections, stained in size, the larger ones being somewhat larger than with hematoxylin and eosin, and with Mallory's phos- the average nucleus of the hepatoma cells. They were photungstic acid-hematoxylin stain for mitochondria, not acid-fast and usually were acidophilic. When were prepared on hepatoma and liver tissue from all stained with Mallory's aniline blue stain they showed animals. In addition, sections of many, but not all, a central blue core surrounded by an unstained zone hepatomas and livers were stained with Mallory's outside of which was a blue marginal rim. The in- aniline blue stain for connective tissue, Van Gieson's clusions were sometimes found free in spaces that stain for connective tissue, Mallory's phloxine and were possibly lymph vessels. methylene blue, Mallory's stain for alcoholic hyalin, The purpose of this paper is to report further Ziehl-Neelsen's carbolfuchsin, methyl violet, acidulated studies on the various types of inclusion bodies found potassium ferrocyanide, and sudan IV. in the cells of hepatomas arising in strain C3H mice. RESULTS MATERIALS AND METHODS Macroscopic studies.--The gross appearance of the The 88 mice used in these experiments were of the tumors was the same as that described in a previous C3H strain. The source of the animals and the report (2). In general the neoplasms were solid, methods of breeding and caring for them have been rounded, well localized, but not encapsulated masses. outlined in a previous report (2). When located within the liver, they were gray; the Thirty-nine of the mice had hepatomas. Eighteen rarer pedunculated tumors were sometimes hemor- were untreated control mice; of these 16 were breed- rhagic. Metastases were not observed. ing males, 1 was a nonbreeding male, and 1 a non- Microscopic studies.--On the whole the tumor cells breeding female. Twenty-one were treated animals; closely resembled normal liver cells and arranged them- of these 15 were nonbreeding males treated with selves in cords. Accurate reproduction of liver lobules a-estradiol benzoate; 5 were nonbreeding males treated did not occur and, although bile canaliculi were ob- with ketohydroxyestrin, and 1 was a nonbreeding served regularly, bile ducts were rarely, if ever, present. female treated with testosterone propionate. Details The cytoplasm and nuclei of the liver and tumor 4 697 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1943 American Association for Cancer Research. 698 Cancer Research cells contained various bodies that were made the cytoplasm usually varied from 1 to 50; occasionally subject of special study. Some were mitochondria. there were more. The cells containing these inclu- The others were cellular inclusions, of which we were sions often appeared little altered but in some cases, able to identify two types: (a) intracytoplasmic especially when the inclusion bodies were numerous hyaline bodies, and (b) intracytoplasmic lipoprotein or large, the cell nucleus was pyknotic or absent and bodies. the cell membrane ruptured. Mitochondria.--The presence of numerous granules The bodies varied from 3 to 13 microns in diameter, of varying size, shape, and staining qualities in the the smaller ones being the most numerous (Fig. 2). neoplastic and nonneoplastic liver cells makes it neces- The majority appeared to be spheroidal, but many sary to define our interpretation of mitochondria. were ovoid and others appeared as indented spheroids With Mallory's phosphotungstic acid-hematoxylin or crescent-shaped bodies. Most of the smaller bodies stain for mitochondria after fixation in Helly's fluid, appeared to be homogeneous. Many of the medium only the small coccoid or rod-like, sharply defined sized and larger ones, however, contained pale, rounded bodies that stained deep blue were accepted as mito- or ovoid masses that in rare instances presented pro- chondria (Fig. 1). trusions resembling those seen in budding yeast cells In the livers of untreated control animals, mito- (Fig. 3). Other bodies contained either a brownish chondria were constantly present. The cells contain- bur-like mass or scattered brownish granules. ing them were irregularly distributed in small to large In sections stained with hematoxylin and eosin most patches. In the livers of animals treated with testos- of the lipoprotein bodies were strong!y eosinophilic. terone propionate the mitochondria were not reduced Others showed unstained or pale green doubly re- in numbers, but in those that received estrogens the fractile peripheral rims and a central eosinophilic mitochondria were either definitely reduced in num- granular or nongranular mass or masses (Fig. 3). In bers or completely absent. In the hepatomas the mito- some tumors many of the bodies were unstained; others chondria were usually sparse or absent. Occasionally were pale pink or light green. they occurred in very large numbers throughout the With the phosphotungstic acid-hematoxylin stain tumor. One necrotic tumor contained numerous (Fig. 4) most of the bodies stained ldeep blue or mitochondria. reddish blue though a few had a reddish brown color lntracytoplasmic hyaline inclusion bodies.--These or a distinct greenish tint. Among the medium sized consisted of homogeneous or very finely granular and larger bodies some had a deep blue peripheral rim masses that measured 10 to 15 microns in diameter and a central mass, or masses, that stained a paler blue. and were confined to the cytoplasm. They were present With phloxine-methylene blue most of the bodies in the majority of the hepatomas. The only non- stained deep red or purplish red, although some of neoplastic liver tissue that contained these bodies was them were stained pale pink and others were mottled that at the periphery of one tumor. With hematoxylin with red and blue stain. and eosin these bodies stained pink and with Mallory's With Mallory's aniline blue connective tissue stain phosphotungstic acid-hematoxylin, pale blue. A few the bodies stained orange or reddish orange, with the contained small, rounded, pale staining bodies that peripheral rim often staining more deeply than the resembled lipoprotein bodies. central portion. Intracytoplasmic lipoprotein inclusion bodies.- With Van Gieson's connective tissue stain they These bodies were found exclusively in the cytoplasm stained yellow, with the peripheral rims sometimes of the neoplastic cells and were noted in every tumor. colored brownish yellow. The total number in each tumor varied from a few Mallory's phloxine stain for alcoholic hyalin showed to innumerable bodies. The size of the growth and that the central portions of a few bodies stained pink. the presence of necrosis bore no demonstrable rela- tionship to the number present. In the tumors that Ziehl-Neelsen's carbolfuchsin stain showed that many had undergone complete coagulation necrosis the of the bodies, both large and small, were acid-fast. number of visible bodies was diminished, and faint, The peripheral rim was stained more deeply in some rounded "ghost-like" bodies suggested that some had cases. undergone degeneration. The distribution within the Frozen sections of formalin-fixed tumor tissue, neoplasms was highly irregular. The location of the stained with sudan IV, showed that almost all the bodies within the tumor bore no relationship either bodies stained intensely red. In some of the bodies to the central or peripheral portions of the growth, the peripheral rims stained more deeply. In tissues or to the blood vessels. No one portion of the cyto- that had been dehydrated with alcohol, infiltrated with plasm was more frequently the site of these inclusions chloroform, embedded in paraffin, and treated with than any other. The number contained within a single xylol, many of the bodies had a reddish orange color Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1943 American Association for Cancer Research. Burns and Schenken--Spontaneous Primary Hepatomas. IV 699 FiG. 1.--Mit()chondria in liver cells of an untreated mah' Fro.