Proc. Natl. Acad. Sci. USAVol. 77, No. 8, pp. 4948-4952, August 1980Medical Sciences

Liver tumors distinguished by immunofluorescence microscopy withantibodies to proteins of intermediate-sized filaments

(tumor pathology/angiosarcomas/cytoskeleton/vimentin/keratin)

PETER BANNASCH*, HEIDE ZERBAN*, ERIKA SCHMIDt, AND WERNER W. FRANKEt*Division of Cytopathology and tDivision of Membrane Biology and Biochemistry, Institute of Experimental Pathology, German Cancer Research Center,D-6900 Heidelberg, Federal Republic of Germany

Communicated by Hans Popper, April 28, 1980

ABSTRACT Antibodies against constitutive proteins ofdifferent types of intermediate-sized filaments were used inimmunofluorescence microscopy on frozen sections of normalrat liver and various rat liver tumors induced by treatment withnitrosamines. Antibodies to tonofilament prekeratin stained bileduct epithelia and hepatocytes of normal liver and hepatocel-lular carcinoma cells and ductal cells of cholangiofibromas.These cells were not significantly stained by antibodies tovimentin. By contrast, antibodies to vimentin stained mesen-chymal cells of normal liver and cells of early and advancedangiosarcomas and of undifferentiated spindle cell sarcoma.These mesenchymal tumor cells were not stained with anti-bodies to prekeratin. The presence of intermediate-sized fila-ments in these tumors, often in large whorl-like aggregates, wasalso demonstrated by electron microscopy. The results show thatimmunofluorescence microscopy with antibodies to cytoskeletalproteins is a powerful tool for the classification and differentialdiagnosis of mesenchymal and epithelial liver tumors. Wepropose that staining with antibodies to proteins of differenttypes of intermediate filaments can be used to improve theidentification of tumors of other organs, including metastases,as well as non-neoplastic proliferative lesions.

Classification and diagnosis of tumors and their precursor stagesin biopsies and autopsies is primarily based on descriptivemorphology. The use of cytochemical markers in studies ofcarcinogenesis, especially hepatocarcinogenesis, has indicatedthe potential of such tools in the identification and classificationof preneoplastic and early neoplastic disorders of epithelialtissues (1-3). Reliable cytochemical markers for the identifi-cation and differential diagnosis of mesenchymal neoplasiashave not been available. Recently, it has been shown by im-munofluorescence microscopy that antibodies to the proteinsof different types of intermediate-sized (8-11 nm) filamentsallow epithelial, mesenchymal, myogenic, and neuronal cellsto be distinguished in diverse tissues (4-10). The intermediatefilaments, which are the predominating, if not exclusivelypresent, ones in epithelial cells, are chemically and immu-nologically related to epidermal prekeratin ("cytokeratins";ref. 11) and are different from the structurally similar filamentspresent in mesenchymal cells, which contain a constitutiveprotein called vimentin (5). The finding that the expression offilaments containing cytokeratin is maintained in transformedepithelial cells growing in culture (5, 6, 8, 11-13) has stimulatedour interest in the possible maintenance of such molecularmarkers in tumors grown in the body.

As a first example we have used the liver (in which differenttypes of tumors, such as adenomas, hepatocellular carcinomas,cholangiofibromas, cholangiocarcinomas, angiosarcomas, andfibrosarcomas can occur) to determine whether epithelial andmesenchymal tumors could be distinguished by the use of an-tibodies to different filament proteins. The search for a mo-

lecular marker for identification of mesenchymal tumors ofliver was especially desirable because mesenchymally derivedliver tumors are important in human (14) and experimentalanimal (15) pathology. Reports of a high incidence of angio-sarcomas in workers exposed to inorganic arsenic and gaseousvinyl chloride and in patients treated with Thorotrast or certainsteroid hormones have increased the interest in this tumor (14,16). The structure and morphogenesis of vascular liver tumorsin humans and experimental animals appear to be largelyidentical (15, 17). In both humans and animals, differentialdiagnosis of epithelial and mesenchymal liver tumors is some-times difficult. A clear distinction, however, would be importantnot only for prognostic appraisals in human pathology but alsofor evaluation of animal experiments such as dose-responsestudies of carcinogenesis induced by chemical compounds (18,19).

In this study we show that immunofluorescence microscopywith antibodies to two molecular markers, prekeratin andvimentin, allows the character and origin of epithelial andmesenchymal cells to be distinguished in neoplastic liver cellsand thus facilitates the differential diagnosis of liver tumors.

MATERIALS AND METHODSAnimals. Liver tumors were induced in male Sprague-

Dawley rats (n-200 g body weight at the beginning of treat-ment) by oral treatment with nitrosamines (for methods,see ref. 20). Seven hepatocellular carcinomas, 3 cholangio-fibromas, 10 angiosarcomas, and 1 undifferentiated spindle cellsarcoma were examined. Four of the hepatocellular carcinomashad developed in animals treated with 50 mg of N-nitroso-morpholine (in a total of 100 ml of their drinking water) for 3weeks and killed between weeks 52 and 77 after withdrawalof the carcinogen. (Three of these carcinomas occurred in as-sociation with benign cystic cholangiomas.) The other threecarcinomas had been induced by application for 66 weeks ofboth dimethylnitrosamine (0.3 mg/kg body weight) and N-nitrosopyrrolidine (0.5 mg/kg body weight) or of nitrosopyr-rolidine (same dose) alone; tumors were found between weeks12 and 14 after withdrawal of the drugs. Cholangiofibromaswere induced as described (21) and taken from animals killedbetween weeks 60 and 64 after withdrawal of the drug. Fiveangiosarcomas were induced by application of both di-methylnitrosamine and nitrosopyrrolidine (doses as above), andanimals were killed on day 87 after withdrawal. The five otherangiosarcomas and the spindle cell sarcoma were found in an-imals treated with 50 mg of nitrosomorpholine (in 100 ml ofdrinking water) for 3 weeks and killed between weeks 60 and87 after withdrawal.

Antibodies. The following antibodies were used: (i) guineapig antisera against total reconstituted bovine hoof prekeratinand monospecific antibodies prepared therefrom by affinitychromatography (5, 11, 22); (ii) guinea pig antisera againstreconstituted prekeratin of desmosome-attached tonofilaments

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FIG. 1. Immunofluorescence microscopy on frozen sections ofliver of a rat (untreated control animal) stained with antibodies toprekeratin from desmosomal tonofilaments (a and b) and to vimentin(c). Antibodies to this type ofprekeratin show intense staining in bileduct (BD) epitheliumn and moderate staining in hepatocytes, espe-cially in the hepatocyte periphery (shown at higher magnification inb); prekeratin antibodies do not stain mesenchymal cells and bloodvessel walls (A, artery; V, vein). By contrast, vimentin antibodies stainmesenchymal cells only (c), including those of blood vessels (26). (Bars= 30,om.)

from bovine muzzle (23); (iii) guinea pig antisera against mu-rine vimentin as well as IgG fractions and monospecific anti-bodies made therefrom (5, 13, 24); and (iv) rabbit antibodiesto chicken gizzard actin (22, 25). Controls included preimmunesera and incubations at elevated ionic strength (phosphate-buffered saline containing 100 mM MgCl2; ref. 22).

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FIG. 2. Electron micrographs, showing rat liver tumor cells withhigh densities, of intermediate filaments. a, Angios~arcoma; b, ductularcells of cholangiofibroma (D, desmosome). (Bars, = 0.5 um.)

Immunofluorescence Microscopy. Small pieces of liverwere frozen in isopentane cooled with liquid nitrogen, andair-dried cryostat sections (4 /.Lm thick) were fixed in acetoneat -20°C and processed for indirect immunofluorescencemicroscopy as described (26). Specimens were photographedwith a Zeiss photomicroscope equipped with epifluorescenceillumination (25).

Electron Microscopy. Tissue samples were fixed and pro-cessed for electron microscopy as described (27).

RESULTSNormal Liver. The distribution of intermediate-sized fila-

ments in different cell types present in mammalian liver hasbeen studied in detail by electron microscopy and by immu-nofluorescence microscopy with antibodies to prekeratin andvimentin (26). Strong staining with antibodies to bovine hoofprekeratin was noted-in bile duct epithelial cells but not inhepatocytes (5, 8, 26), except for toxically damaged hepatocytescontaining Mallory bodies (28). We have prepared guinea pigantibodies against the prekeratin from desmosome-attachedtonofilaments that reacted specifically, in immunoreplica tests(for methods see refs. 13 and 24), with polypeptide componentsI, III, IV, V, and VI of bovine muzzle prekeratin (11). On var-ious mammalian epithelia, these antibodies gave results iden-tical to those described previously with antibodies to total epi-

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FIG. 3. Immunofluorescence microscopy of frozen sections of different rat liver tumors (a and b, hepatocellular carcinoma; c and d, cho-langiofibroma; e and f, angiosarcoma) stained with antibodies to prekeratin (a, c, and e) and vimentir (b, d, and f). Note staining with prekeratinantibodies in the epithelial tumors (a and c) in contrast to the specific staining with vimentin antibodies in the angiosarcoma (f) and themesenchymal cells of stroma (b and d) and isolated intraductular cells of the cholangiofibroma (d). In e, we demonstrate the absence of prekeratinin the angiosarcoma cells (lower part) in contrast to the positive reaction of prekeratin antibodies in the hepatocytes of adjacent normal livertissue (upper part). (Bars = 30 um.)

Proc. Natl. Acad. Sci. USA 77 (1980)

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Proc. Natl. Acad. Sci. USA 77 (1980) 4951

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FIG. 5. Immunofluorescence microscopy showing the intensestaining of cells of a spindle cell sarcoma in rat liver with antibodiesto vimentin. (Bar = 30 ,m.)

FIG. 4. Immunofluorescence microscopy showing the stainingof vimentin-positive cells in an early stage of an angiosarcoma in ratliver (S, sinusoidal spaces). (Bar = 30,gm.)

dermal prekeratin of human (6, 8-10) and bovine (11) originbut, in addition, they also stained parenchymal cells of glandssuch as pancreas acinar cells (not shown) and hepatocytes (Fig.1 a and b). Staining in hepatocytes was prominent along thelateral cell walls, especially in the pericanalicular region, whichprobably reflects the local density of desmosome-attached to-nofilaments (26, 29), but fine meshwork arrays of fibrilsthroughout the cytoplasm were also stained (Fig. lb; ref. 30).By contrast, vimentin antibodies showed the typical stainingin mesenchymal cells, most prominently in Kupffer cells, en-

dothelial cells (Fig. lc), and cells of blood vessel walls (5,26).

Liver Tumors. Intermediate-sized filaments have been ob-served by electron microscopy in diverse types of liver tumors(31-36). In our study they were especially abundant in thecytoplasm of cells of angiosarcomas (Fig. 2a) and cholangio-cellular tumors (e.g., Fig. 2b). In cholangiocellular tumors (Fig.2b) and in hepatocellular carcinomas, association of such fila-ments with desmosome was often observed. Although the ul-trastructure of the filaments present in different cells and tu-mors was practically indistinguishable (4, 5, 9, 12, 26), immu-nofluorescence showed that different types of filaments werepresent in different types of tumors.

Hepatocellular carcinomas. Like normal hepatocytes,hepatocellular carcinoma cells contained intermediate-sizedfilaments, some of them connected with typical desmosomesand hence identified as tonofilaments. In immunofluorescencemicroscopy with antibodies against prekeratin, fluorescencewas observed at plasma membrane regions opposite the sinus-

oidal side and along the lateral plasma membrane (Fig. Sa).Immunofluorescence was weak or absent on the sinusoidal cellsurface. Bright immunofluorescence was seen at luminal plasmamembranes of tumor cells forming tubular structures. Bycontrast, antibodies against vimentin did not significantly stainstructures in the hepatocarcinoma cells but intensely stainedmesenchymal cells of the stroma (Fig. 3b).

Cholangiofibromas. Intermediate filaments were abundantin ductular cells of cholangiofibromas induced by nitroso-morpholine (Fig. 2b; ref. 36). In immunofluorescence micros-copy, most neoplastic ductular cells reacted strongly withprekeratin antibodies (Fig. Sc). Mesenchymal cells again gavea negative reaction with prekeratin antibodies (Fig. Sc) but apositive reaction with antibodies against vimentin (Fig. 3d).Some neoplastic ductular cells were also weakly stained by thevimentin antibodies, but when compared with controls, this wasnot significant. Polymorphonuclear or mononuclear cells, whichwere frequently found within the duct lumina, reacted stronglywith vimentin antibodies (Fig. 3d). Mucous depositions andnecrotic cells, which were often found in the lumina, showeda nonspecific staining.

Angiosarcomas. All angiosarcomas contained many cells richin dense aggregates of intermediate filaments (Fig. 2a), oftenarranged in bundles. Such extended whorl aggregates of fila-ments (up to 5 ,m in diameter) were readily visible in the lightmicroscope in hematoxylin- and eosin-stained sections as denseand finely striated acidophilic cytoplasmic inclusions. Des-mosomes were not found in angiosarcoma cells. Immunofluo-rescence microscopy showed that these masses of intermediatefilaments were strongly stained by antibodies to vimentin (Fig.Sf) but not by antibodies to prekeratin (Fig. Se) or to actin (notshown). At higher resolution the fibrillar nature of the fluo-rescent material was resolved. The difference between pre-keratin-negative angiosarcoma cells and prekeratin-positivehepatocytes or bile duct epithelial cells was seen particularlywell at the border between neoplastic and normal tissue (Fig.Se). Moreover, early proliferative changes during the devel-opment of angiosarcomas were easily detected by immu-nofluorescence microscopy with antibodies against vimentin.

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Under these conditions the hepatocyte trabeculae were linedby a nearly continuous layer of atypical, vimentin-richmesenchymal cells (Fig. 4).

Spindle cell sarcoma. Like angiosarcoma cells, the cells ofthe undifferentiated spindle cell sarcoma contained abundantintermediate filaments, often in association with typical "densebodies" similar to those described in smooth muscle and variouscultured cells (37). Immunofluorescence microscopy with an-tibodies to vimentin resulted in a strong staining of fibrillarmaterial present in these cells (Fig. 5); by contrast, antibodiesto prekeratin gave negative results (not shown).

DISCUSSIONOur results show that epithelial and mesenchymal tumor cellsof liver differ in the type of intermediate filaments present intheir cytoplasm. Whereas neoplastic cells of hepatocellularcarcinoma and cholangiofibroma contain filaments of theprekeratin type, sarcoma cells are characterized by the presenceof vimentin filaments, often in excessive amounts. These ob-servations on tumors grown in the body correspond to recentfindings in cultured cells. The presence of filaments of theprekeratin type has been described in various cultured hepa-toma cells (refs. 13 and 38; for the special cases of the HTC andH4 cell lines, see refs. 8 and 13). The widespread, apparentlyubiquitous occurrence of filaments of the vimentin type intransformed mesenchymal cells in culture has also been dem-onstrated (5, 9, 13, 39). From our study we conclude that vim-entin filaments are also present in mesenchymal tumors incontrast to cytokeratin filaments. We further have shown thatat least two epithelial tumors grown in the body, hepatocellularcarcinoma and cholangiofibroma, maintain the specific epi-thelial type of intermediate filaments (i.e., the prekeratin type)and do not have detectable amounts of vimentin filaments.Therefore, these tumor cells appear to maintain the specifictype of cytoskeletal components characteristic of the cell fromwhich they are derived. Our data, however, do not allow us todecide whether carcinoma cells growing as primary tumors oras metastases will not begin to form vimentin in later stages; theadvent of filaments of the vimentin type has been demonstratedin normal and neoplastic epithelial cells, including hepatomacells, growing in nitro (5, 9, 12, 13, 26, 38-40). The maintainedformation of cytokeratin filaments in neoplastic epithelial cells(see also ref. 41) and of vimentin-type filaments in neoplasticmesenchymal cells provides an excellent additional criterionfor the classification and differential diagnosis of tumors, in-cluding metastases. We propose that antibodies to the differentintermediate filament proteins known so far (9) can be used inimmunofluorescence microscopy to improve the identificationof neoplastic or preneoplastic cell populations as well as non-neoplastic proliferative lesions.

We thank H.-J. Hacker and B. Stiles for the excellent cryostatpreparations and our colleagues from the Institute of Toxicology andChemotherapy (German Cancer Research Center) for providingtumor-bearing animals.

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