Histogenesis of Ewing's sarcoma. An evaluation of intermediate filaments and endothelial cell markers

Four cases of Ewing's sarcoma, three in bone and one from an extraskeletal site, were studied immunohistologically using monospecific antibodies against intermediate filament proteins of keratin, vimentin, desmin and neurofilament types. All cases were also evaluated for the presence of Factor VIII-related antigen (FVIIIR:Ag) and for the binding of Ulex europaeus I lectin (UEA I), both of which are endothelial markers. In all cases the tumor cells contained vimentin but not keratin, desmin or neurofilaments. The tumor cells could not be decorated with either anti-FVIIIR:Ag or UEA I, whereas the vascular endothelium was positive for both markers. The vimentin-positivity indicates a mesenchymal derivation of Ewing's sarcoma, while the lack of endothelial markers argues against the proposed endothelial origin of this tumor.     

Cytology of fine needle aspiration biopsy of primary breast rhabdomyosarcoma in an adolescent girl

A bilateral breast tumor in an 11-year-old girl was diagnosed as a malignant mesenchymal neoplasm by fine needle aspiration biopsy, and its muscular origin was suggested. An immunoperoxidase stain for the demonstration of intracellular myoglobin in destained cytologic aspirates exhibited a positive reaction of variable intensity in some of the undifferentiated round cells and in the more mature bipolar sarcomatous elements. These findings were confirmed in the histologic sections of the excised tissue. Needle aspiration is helpful in these rare cases of primary breast rhabdomyosarcoma, which often present with a solid, undifferentiated histologic pattern in this age group. In addition to the cytologic characteristics, the possibility of detecting muscle antigens as markers for these embryonal small cells, even in previously stained slides, provides a successful method for defining the specific type of sarcoma.     

Chemically induced rhabdomyosarcomas in rats. Ultrastructural, immunohistochemical, biochemical features and expression of alpha-actin isoforms

A series of 14 primary and two metastatic rat rhabdomyosarcomas (RMS) induced with nickel sulfide was studied by light microscopy, transmission electron microscopy, indirect immunofluorescence, avidin-biotin-peroxidase immunohistochemistry and two-dimensional gel electrophoresis. Monoclonal or affinity-purified polyclonal antibodies were used for the immunohistochemical demonstration of vimentin, desmin, alpha-smooth muscle (alpha-sm) actin and alpha-sarcomeric (alpha-sr) actin. By histological and ultrastructural studies, four categories of RMS were diagnosed on the basis of the neoplastic cell types. These were: (1) well-differentiated RMS (n = 2), (2) pleomorphic RMS (n = 8), (3) embryonal RMS (n = 4), and (4) embryonal myosarcomas (n = 2). Immunohistochemically, all these neoplasms expressed desmin and alpha-sr actin, reflecting their rhabdomyoblastic origin. Two dimensional gel electrophoresis performed on five neoplasms demonstrated alpha, beta and gamma actins spots in all cases. This study demonstrates that the alpha-sr actin antibody represents a good marker for rhabdomyoblastic differentiation is useful in the diagnosis of RMS since it was present in all morphologically confirmed RMS and in two ultrastructurally undifferentiated sarcomas positive for desmin. Neoplastic cells positive for alpha-sm actin were noted in 11 confirmed RMS. Double indirect immunofluorescence showed that all alpha-sm and alpha-sr positive cells also contained desmin. Co-expression of alpha-sr and alpha-sm actins was studied in serial sections of formalin-fixed, paraffin-embedded tumor tissue. Both alpha-sm and alpha-sr actins were localized in some rhabdomyoblasts. This study confirms our previous observations in human tumors and shows, for the first time, that alpha-sr and alpha-sm actins can be present in the same neoplastic cell in vivo.     

Vimentin-typing in diagnostic surgical pathology: a comparative study using four antibodies after different fixations

Vimentin-typing was carried out on various normal and neoplastic tissues using four anti-vimentin antibodies in order to evaluate the effect of different fixation treatments on tissue reactivity in comparison to the results obtained on frozen sections. All antisera were reactive on frozen material; on paraffin embedded material staining of tissues depended on the type of fixation method applied (formalin, methacarn or absolute alcohol) and each antibody behaved differently in relation to the fixative used. Only mesenchymal normal structures were revealed on frozen material whilst on paraffin embedded material three of the four antibodies reacted also with non-mesenchymal normal structures (epithelia, central and peripheral nervous system cells). All four antibodies decorated, regardless of treatment, neoplastic cells of mesenchymal and non-mesenchymal derivation, but not germ cells or germ cell tumors. The reactivity of vimentin to its specific antibodies depends on the fixative used: therefore, in routine pathology more than one antiserum should be available for testing. Furthermore, given the variety of non-mesenchymal structures stained by the anti-vimentin antibodies, the differential diagnosis of undifferentiated tumors must not be based on vimentin positivity alone. The expression of vimentin by non-mesenchymal neoplastic cells seems to parallel that of normal tissues during embryogenesis; therefore, this intermediate filament appears to be not only a marker of mesenchymal cells but also of many immature elements.     

Antibodies to intermediate filament proteins in the immunohistochemical identification of human tumours: an overview

Intermediate-sized filament proteins (IFP) are tissue specific in that antibodies to keratin, vimentin, desmin, glial fibrillary acidic protein (GFAP) and the neurofilament proteins can distinguish between cells of epithelial and mesenchymal origin as well as of myogenic and neural origin respectively. Malignant cells retain their tissue-specific IFP, which makes it possible to use these antibodies in tumour diagnosis. Carcinomas are exclusively detected by antibodies to keratin. Monoclonal antibodies to keratin have allowed the differentiation between subgroups of epithelial tumours until now between adenocarcinomas and squamous cell carcinomas. Lymphomas, melanomas and several soft tissue tumours are distinctly recognized by antibodies to vimentin. On the other hand, rhabdomyosarcomas and leiomyosarcomas are positive for desmin, while astrocytomas give a strong reaction with GFAP antibodies. Thus, antibodies to IFP are useful tools for differential diagnosis in surgical pathology.     

The distribution of keratin type intermediate filaments in human breast cancer. An immunohistological study

Antibodies to different intermediate filament proteins can be used to distinguish cells of epithelial, mesenchymal, muscle, glial and neuronal origin. Antibodies to prekeratin which characterize cells of epithelial origin, and antibodies to vimentin which recognize cells of mesenchymal origin have been used to study twenty cases of breast carcinoma (sixteen infiltrating ductal carcinomas and four infiltrating intraductal carcinomas), two cases of cystic breast disease, two fibroadenomas and one case of benign cystosarcoma phylloides. The prekeratin and vimentin were detected using specific antibodies to these proteins by immunofluorescence microscopy using alcohol fixed paraffin-embedded tissues. In eighteen out of the twenty carcinomas the tumor cells were strongly and specifically stained by antibodies to prekeratin. DIfferent tumors gave different patterns of prekeratin staining. In contrast, when the same specimens were tested with the vimentin antibody, the tumor cells were unstained, and instead only the usual strong staining to fibroblasts and blood vessels in the stroma was observed. In cystic breast disease, fibroadenomas, and benign cystosarcoma phylloides, cells of epithelial origin were strongly stained by the prekeratin but not by the vimentin antibody.     

Diagnostic significance of coexpression of intermediate filaments in fine needle aspirates of human tumors

A study was undertaken of the diagnostic significance of the coexpression of intermediate filaments in fine needle aspirates of human tumors. Three types of coexpression were found: (1) true coexpression, in which tumor cells simultaneously express more than one intermediate filament protein; (2) pseudocoexpression, in which various tumor cell types from histogenetically different parts of a complex tumor show different results; and (3) false coexpression, in which tumor cells with one or two types of intermediate filaments are present together with benign cells expressing a different filament type. True coexpression of vimentin and keratin was documented in renal cell carcinomas, endometrial carcinomas, certain thyroid carcinomas and Hürthle cell adenomas. Coexpression of keratin and neurofilaments was seen in Merkel cell carcinomas, and coexpression of desmin and vimentin was found in leiomyosarcomas. Keratin, vimentin and neurofilament expression was seen in medullary thyroid carcinomas, and keratin, vimentin and glial fibrillary acidic protein expression was observed in pleomorphic adenomas of the salivary gland. Pseudocoexpression was noted in synovial sarcoma, epithelioid sarcoma, benign cystosarcoma phyllodes of the breast, teratocarcinoma, malignant granular cell tumor, progonoma, Wilms' tumor and triton tumor. Sources of false coexpression are also discussed.     

Distinction of nephroblastomas from other childhood tumors using antibodies to intermediate filaments

Ten nephroblastomas were investigated by antibodies to intermediate filaments. In seven cases, which in light microscopy were characterized by the presence of blastema and tubules, immunofluorescence microscopy with IF-specific antibodies reveals expression of cytokeratin and vimentin in blastema cells, while tubules were only labelled by the cytokeratin antibodies. This result was independent of whether the conventional cytokeratin antibody or monoclonal antibodies specific for cytokeratin 18 were used. Stroma cells were vimentin-positive. In two cases nephroblastomas were undifferentiated and also lacked tubuli formation. In both these tumors blastema cells were vimentin-positive and cytokeratin-negative. Finally one case of clear cell sarcoma of the kidney could only be labelled by the vimentin antibody. Thus antibodies to intermediate filaments seem to be useful tools to distinguish nephroblastomas from neuroblastomas or rhabdomyosarcomas, especially in cases of metastasis.     

Balance between activities of Rho kinase and type 1 protein phosphatase modulates turnover of phosphorylation and dynamics of desmin/vimentin filaments

To analyze the cell cycle-dependent desmin phosphorylation by Rho kinase, we developed antibodies specifically recognizing the kinase-dependent phosphorylation of desmin at Thr-16, Thr-75, and Thr-76. With these antibodies, phosphorylation of desmin was observed specifically at the cleavage furrow in late mitotic Saos-2 cells. We then found that treatment of the interphase cells with calyculin A revealed phosphorylation at all the three sites of desmin. We also found that an antibody, which specifically recognizes vimentin phosphorylated at Ser-71 by Rho kinase, became immunoreactive after calyculin A treatment. This calyculin A-induced interphase phosphorylation of vimentin at Ser-71 was blocked by Rho kinase inhibitor or by expression of the dominant-negative Rho kinase. Taken together, our results indicate that Rho kinase is activated not only in mitotic cells but also interphase ones, and phosphorylates intermediate filament proteins, although the apparent phosphorylation level is diminished to an undetectable level due to the constitutive action of type 1 protein phosphatase. The balance between intermediate filament protein phosphorylation by Rho kinase and dephosphorylation by type 1 protein phosphatase may affect the continuous exchange of intermediate filament subunits between a soluble pool and polymerized intermediate filaments.     

Differential location of different types of intermediate-sized filaments in various tissues of the chicken embryo.

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11--20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to--and specific for--epithelial cells; vimentin filaments are seen--at this stage of embryogenesis--only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structurees provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta

The presence of intermediate filament proteins in vascular tissue cells has been examined by immunofluorescence microscopy on frozen sections of the aortic wall of diverse vertebrates (rat, cow, human and chicken) and by gel electrophoresis of cytoskeletal proteins from whole aortic tissue or from stripped tunica media of cow and man. Most cells of the aortic wall in these species contain vimentin filaments, including smoooth muscle cells of the tunica media. In addition, we have observed aortic cells that are positively stained by antibodies to desmin. The presence of desmin in aortic tissue has also been demonstrated by gel electrophoresis for rat, cow and chicken. In aortic tissue some smooth muscle cells contain both types of intermediate filament proteins, vimentin and desmin. Bovine aorta contains, besides cells in which vimentin and desmin seem to co-exist, distinct bundles of smooth muscle cells, located in outer regions of the tunica media, which contain only desmin. The results suggest that (i) intermediate-sized filaments of both kinds, desmin and vimentin, can occur in vascular smooth muscle in situ and (ii) smooth muscle cells of the vascular system are heterogeneous and can be distinguished by their intermediate filament proteins. The finding of different vascular smooth muscle cells is discussed in relation to development and differentiation of the vascular system.     

Monoclonal antibodies to desmin, the muscle-specific intermediate filament protein

A set of monoclonal antibodies to desmin has been isolated from a fusion of mouse myeloma cells with spleen cells from mice immunized with purified porcine desmin. Eleven group I antibodies recognized desmin in the immune blot, and using defined desmin fragments the epitope has been tentatively assigned as lying between residues 325 and 372. When cell lines were tested in immunofluorescence only the human line RD and hamster BHK-21 were positive. When tissue sections were used, skeletal, cardiac, visceral and some vascular smooth muscle cells were positive. Thus, the group I antibodies appear specific for desmin and do not recognize other intermediate filament proteins. Group II monoclonals recognized not only desmin in the immune blot but also other polypeptides. The epitope of this class is located between residues 70 and 280. In immunofluorescence on cell lines and tissues, the staining patterns of group II antibodies were more complicated and demonstrate that not only other intermediate filament proteins but also additional antigenic determinants are being recognized. The group I antibodies stain, as expected from their desmin specificity, rat and human rhabdomyosarcomas and thus appear to be useful reagents in pathology.     

Various sympathetic derived human tumors differ in neurofilament expression. Use in diagnosis of neuroblastoma, ganglioneuroblastoma and pheochromocytoma

We have extended our analysis of human tumors using antibodies specific for each of the five types of intermediate filaments to neuroblastoma, ganglioneuroblastoma, pheochromocytoma, ependymoblastoma, and alveolar soft part sarcoma. Tumor cells in the three cases of neuroblastoma, as well as in the single case of alveolar soft part sarcoma, did not react positively with sera directed against any of the five intermediate filament types. We suppose, therefore, that neuroblastoma at least may be derived from a cell type - possibly present in peripheral neurones - which in vivo has very few or no intermediate filaments. In ganglioneuroblastoma and in pheochromocytoma the tumor cells were positive when tested with antibodies directed against neurofilaments and negative with those directed against other intermediate filament types. The ependymoblastoma was positive when tested with antibodies directed against glial fibrillary acidic protein (GFA) and negative when tested with antibodies against other intermediate filament types. Use of antibodies to the different intermediate filament types appears to be a valid way in which to classify tumors, and so far the data presented here and elsewhere support the hypothesis that tumor cells retain the intermediate filament type typical of their cell of origin. Wider use of these sera would seem particularly useful in cases such as neuroblastoma, rhabdomyosarcoma or lymphoma where diagnosis is currently difficult using conventional histological stains.     

Intermediate filament protein profiles of human testicular non-seminomatous germ cell tumors: correlation of cytokeratin synthesis to cell differentiation

The patterns of cytoskeletal differentiation were studied in 20 testicular non-seminomatous germ cell tumors by immunohistochemistry, using diverse monoclonal antibodies specific for different intermediate filament (IF) proteins and for desmoplakin. Immunofluorescence and immunoperoxidase methods on both formalin-fixed and frozen tissues were applied, in some cases together with a gel electrophoretic analysis of IF proteins. The tumors examined included embryonal carcinoma (EC), endodermal sinus tumor (EST), choriocarcinoma and teratoma. Nine of the tumors were composed of only one histological type, the others showed mixed components. Cytokeratins 8 and 18 were identified in all these neoplasms, but their immunostaining was weak in ECs. Cytokeratin 19 was absent or very scarce in ECs, but strongly expressed in ESTs, choriocarcinomas and teratomas, thus allowing the identification of small EST and choriocarcinoma elements in ECs even when they were morphologically not obvious. Occasionally, some cells in ECs and ESTs also stained for cytokeratins 4 and/or 17, indicating potential for epithelial stratification. The majority of the germ cell tumors showed varied amounts of vimentin, often in co-existence with cytokeratins. Neurofilaments were demonstrated in scattered tumor cells in a single case of EST. In the teratomas studied, each type of tissue component present showed the expected IF protein. However, in many germ cell tumors some stromal cells and blood vessels contained, in addition to vimentin and desmin, also cytokeratins 8 and 18. This heterogeneity of the cytoskeletal profile of germ cell tumors is indicative of the varied differentiation potential inherent in these neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Location of Different Types of Intermediate-Sized Filaments in Various Tissues of the Chicken Embryo

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11–20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to – and specific for – epithelial cells; vimentin filaments are seen – at this stage of embryogenesis – only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structures provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

Vimentin and desmin of a cartilaginous fish, the shark Scyliorhinus stellaris: sequence, expression patterns and in vitro assembly

In the shark Scyliorhinus stellaris we have biochemically identified and cDNA-cloned orthologs of human vimentin and desmin, SstV and SstD, as deduced from immunoblotting and sequence alignment with teleost, frog and human vimentin and desmin, respectively. This allowed us to further clarify the relationship of previously identified lower vertebrate intermediate filament proteins to mammalian vimentin and desmin. Immunofluorescence microscopy with antibodies H5 and VIM13.2 showed vimentin expression in shark eye and brain and absence in epithelia, which resembles the situation in higher vertebrates. In addition, SstV is expressed in many mesenchymal cell types which corresponds to the case in terrestrial vertebrates but strongly differs from teleosts. Surprisingly, shark interstitial cells, including fibroblasts, express neither SstV nor keratins but other as yet unidentified intermediate filament proteins as deduced from their reactivity with antibody IFA. In vitro assembly studies of recombinant SstV revealed a temperature optimum for uncompromised filament assembly of 15 degrees C. At 18 degrees C, but more pronounced at 21 degrees C and 24 degrees C, which is notably above the animal's inherent preferred environmental temperature, both, SstV and SstD assemble into thick and inflexible fibers. Thus, environmental temperature apparently is, as a general principle, a driving force for the fine tuning of protein primary structure and eventually 3D structure.     

Pediatric sarcomas display a variable EpCAM expression in a histology-dependent manner

EpCAM is a transmembrane glycoprotein typically overexpressed in cancer of epithelial origin and mainly involved in the epithelial-to–mesenchymal transition (EMT) of tumor cells that spread and disseminate. Strategies for the targeting and capture of EpCAM-expressing tumor cells are showing promise in cancers prone to metastatize, both as diagnostic tools and potential therapies. Sarcomas are among the most aggressive tumors in children, with a common mesenchymal origin that comprises both soft tissue sarcomas (STS) and bone sarcomas. The aim of this study was to assess EpCAM expression in pediatric sarcomas and correlate its expression with disease progression. To do so, we analyzed a set of cell lines and primary tumor tissues from rhabdomyosarcoma (RMS), Ewing sarcoma (ES), synovial sarcoma (SS) and desmoplastic small round cell tumor (DSRCT) STS, or osteosarcoma (OS) bone cancer. We demonstrated that EpCAM was variably expressed in pediatric sarcomas, with DSRCT, a rare, aggressive and almost fatal tumor type, characterized by the highest EpCAM expression levels. Interestingly, although EpCAM expression was lower in RMS tumors, high levels at diagnosis correlated with reduced patients' overall survival (p < 0.05). Indeed, membrane-bound EpCAM was detected in circulating sarcoma tumor cells, revealing its potential to be used as dissemination biomarker in this type of childhood cancers. This reinforces the concept that pediatric sarcomas do express both epithelial and mesenchymal markers and reside in an intermediate condition that most likely contributes to their aggressive phenotype and low survival rate.     

Localization of vimentin and desmin in BHK21/C13 cells and in baby hamster kidney

Specific antibodies against the intermediate filament protein subunits, desmin and vimentin, were used to characterize the fibroblastic tissue culture cell line BHK21/C13 and the cells comprising baby hamster kidney (BHK). The BHK21/C13 cells have previously been shown to contain desmin and vimentin by biochemical techniques. The results from double immunofluorescence analysis show that both immunologically distinct intermediate filament subunit proteins are expressed simultaneously within the same BHK21/C13 cell, and that the filamentous patterns are very similar, if not superimposable even in cells treated with colchicine. There are some cells that may contain vimentin only. Double immunofluorescence on cryostat sections of BHKs and preparations of dissociated kidney cells demonstrate that the cells, most likely smooth muscle, comprising the blood vessel walls contain vimentin and desmin simultaneously. The simultaneous expression of vimentin and desmin is not a phenomenon which is restricted to tissue culture cells. Thus, the simultaneous presence of these two intermediate filament proteins within the BHK21/C13 cell may not be the result of growth in tissue culture.     

Immunomorphological study of 1,2-dimethylhydrazine-induced carcinomas of the large intestine in rats using monoclonal antibodies to intermediate filament proteins

Cryostatic sections of rat large bowel tumors induced by 1,2-dimethylhydrazine were stained with monoclonal antibodies against different proteins of intermediate filaments: (a) against prekeratin (mol. mass 49 000, PK49) found in many epithelial cells and (b) against vimentin, a constituent of intermediate filaments of mesenchymal cells. Immunofluorescence study showed that large bowel tumor cells as well as normal cells of this organ contain PK49 but not vimentin. High sensitivity of the method allowed one to clearly identify small invasive nodules and groups of tumor cells not visible in usual histologic preparations. Moreover, in some cases single atypical tumor cells were identified in tumor stroma and in the submucosal layer underlying the tumor, that were indistinguishable from normal mesenchymal cells at the light microscopy level.     

Changes in the cellular phenotype and extracellular matrix during progression of estrogen-induced mesenchymal kidney tumors in Syrian hamsters.

The cellular origin of estrogen-induced kidney tumors in male Syrian hamsters has been repeatedly the subject of controversy. Several authors have proposed that the tumors arise from proximal tubules, from a combination of tubular and interstitial stromal cells, or solely from interstitial cells. Because of the model character of this tumor for hormone-associated cancer, it was further investigated in this study with respect to morphology, enzyme and intermediate filament pattern, the expression of alpha-smooth muscle actin and the extracellular matrix proteins fibronectin and tenascin. These analyses were carried out with early and late tumors as well as metastases to determine possible changes in expression of biochemical parameters during the development and progression of this neoplasm. The enzyme histochemical and intermediate filament patterns were usually the same as those described previously for proliferative foci and early tumors, i.e. highly elevated activities of glucose-6-phosphate dehydrogenase, adenylate cyclase and alkaline phosphatase, a lack of glucose-6-phosphatase and gamma-glutamyltransferase and coexpression of vimentin and desmin, alpha-smooth muscle actin could not be detected in early lesions. In five of 24 advanced tumors inclusions of kidney tubules were found which showed various degrees of alteration in their morphology and enzyme histochemical pattern, but were often directly connected with tubular segments of normal appearance outside the tumor. Like the normal tubules, the enclosed tubular segments were strongly positive for cytokeratin but never expressed vimentin or desmin. Among the 24 tumors studied, two contained cysts which expressed cytokeratin and sometimes also vimentin but not desmin. The enzyme histochemistry of the cells lining the cysts was similar to that of the surrounding tumor mass, except adenylate cyclase was lacking and alkaline phosphatase was not uniformly distributed. In tumors containing cytokeratin-positive cysts, there often were cytokeratin-positive, vimentin-negative and desmin-negative tumor formations in close contact to these cysts. With the exception of cyst formation, the pattern of metastases were identical to that of the primary tumors. All large tumors and the main component of the metastases expressed vimentin, desmin and fibronectin. Mesothelia surrounding metastatic tumor complexes were positive for vimentin, desmin, alpha-smooth muscle actin, fibronectin, cytokeratin and tenascin. It was concluded from these and previous observations on early stages of tumor development that the estrogen-induced hamster kidney tumor originates from mesenchymal interstitial cells (probably pericytes) which may rarely acquire an epithelial phenotype by metaplastic transformation during tumor progression.