Immunohistochemical characterization of extracellular matrix components of yolk sac tumors.

Proteoglycans (PGs) were isolated from yolk sac tumor and chondroitin sulfate large PG (core molecule with a molecular weight congruent to 200,000) and small PG (core molecule with a molecular weight congruent to 50,000) were detected. Immunohistochemical localization of PGs in three yolk sac tumors was investigated using monoclonal antibodies raised against both small and large PGs, which were purified from human ovarian fibroma capsule and a yolk sac tumor, respectively. The localization of large PG was observed to be distinct from that of small PG. A markedly positive reaction for antibody against large PG was observed in myxomatous areas, perivascular and perivesicular portions; hyaline globules were the most intensely reactive. In the areas showing a polyvesicular vitelline tumor pattern, the compact connective tissue stroma consisted of small PGs. It is conceivable that large PGs are synthesized by immature mesenchymal cells and also by epithelial-like cells as a basement membrane component, whereas small PGs are synthesized by mature fibroblastic cells synthesizing collagen. Immunohistochemical localization of other extracellular matrix components (laminin, fibronectin, type I-IV collagen) was also studied in relation to PG localization.

Immunohistochemical localization of proteoglycans in interstitial elements of human pancreas and biliary system.

The immunohistochemical localization of large proteoglycan and small proteoglycan was observed, using antibodies 2B1 and 6B6 (Sobue et al., 1988, 1989a), in fetal and adult pancreas and biliary system as well as in tumour tissues, obtained from 11 autopsies and 74 biopsies. The distribution of chondroitin 4- and 6-sulphate side chains, type I and IV collagen and elastin were also studied. In adult pancreas and all the biliary tracts examined, periductal fibrous tissues consisted mainly of dermatan sulphate small proteoglycan with networks of fibrous elements, which were composed of large proteoglycan, elastin, type I collagen and type IV collagen. In the interstitial components of cystadenoma of pancreas and biliary duct carcinoma, similar small proteoglycan-rich components were relatively abundant, although large proteoglycan was present in much larger amounts than that in non-neoplastic adult tissues. In some cholangiomas, the extra- and intracellular hyaline globules formed by the carcinoma cells were found to contain chondroitin sulphate large proteoglycan, laminin and fibronectin. The distribution of proteoglycans was observed to be different in the arterial walls of the interlobular tissues of the adult and the fetal pancreas. The biological significance of large and small proteoglycans in the interstitial connective tissues was discussed.

Effects of beta-D-xylosides on proliferation and matrix formation of adherent fibroblastic cells in mouse bone marrow culture.

Mouse bone marrow cells were seeded on small pieces of cover glasses placed in culture dishes, and after 3 days, the pieces of glass to which only small spindle cells adhered were transferred to another dish containing fresh medium. The adherent small spindle cells proliferated and some of them became large polygonal cells with abundant cytoplasm. When phenyl beta-D-thioxyloside (0.5 mM), an artificial initiator of chondroitin sulfate chain synthesis, was added to the culture medium, the cells showed a marked increase in number as compared to controls, with conversion of about 35% of the cells to large size cells. Immunohistochemical staining with monoclonal antibodies showed that the intercellular matrix of adherent cells consists mainly of a large proteglylcan with chondroitin 6-sulfate side chains. By histochemical analysis, the amount of chondroitin sulfate was shown to be greater in the intercellular matrices of xyloside-treated groups than those of control cultures. The amount of chondroitin sulfate in the growth medium of the adherent cells, as measured by uronic acid analysis, was also significantly increased by treatment with phenyl beta-D-thioxyloside compared with controls.

Production and immunohistochemical characterization of a monoclonal antibody raised to proteoglycan purified from a human yolk sac tumour.

A large proteoglycan with chondroitin sulphate and dermatan sulphate side chains has been isolated and purified from a yolk sac tumour of the left ovary from a 23-year-old female. A monoclonal antibody, designated 2B1, was produced which reacted specifically with the intact molecule of the large proteoglycan and the chondroitinase ABC-treated core molecule. The localization of substances showing cross-reactivity to this antibody was studied in a variety of human tissues by means of indirect immunohistochemistry. The interstitial elements of nearly all tissues of a 5-month-old foetus were intensely reactive with the antibody, but in adult tissues structures that gave positive reactions were limited; only the perivascular and perimuscular fibrous elements were reactive, except for the aorta, which reacted extensively. In contrast, the interstitial elements of the carcinoma tissues tested were intensely reactive. Thus antibody 2B1 can be regarded as a useful tool for studies on the immunohistochemical localization of large proteoglycan in various human tissues.

Establishment of a cell line producing basement membrane components from an adenoid cystic carcinoma of the human salivary gland.

A new cell line has been established from an adenoid cystic carcinoma arising in the submandibular gland of a 63-year-old woman. The cultured epithelial-like cells grew vigorously and adhered together to form a sheet. Immunohistochemical stainings for type IV collagen, laminin and fibronectin were clearly positive in the intercellular matrix and on the surface of the culture cells. Chondroitin 6-sulfate proteoglycan and heparan sulfate were also detected. Ultrastructural studies showed that the cells had abundant rough endoplasmic reticulum and a well-developed Golgi apparatus. Rough endoplasmic reticulum near the cell surface was markedly dilated, and contained material of low electron density. This cell line would be useful for biological and biochemical studies on the mechanisms by which the stromal component is formed.

Immunohistochemical techniques for detection of dermatan sulfate proteoglycan in tissue sections.

Dermatan sulfate proteoglycan chains were detected in tissue sections treated with chondroitin B-lyase (0.01 units/ml) in 20 mM Tris-HCl (pH 8.0) for 1 hr, followed by staining with antibody 9A2 specific for unsaturated uronic acid coupled to N-acetylgalactosamine-4 sulfate. In contrast, after treatment with chondroitin B-lyase, no positive staining was observed with antibodies 3B3 and 1B5 which react to the unsaturated uronic acid coupled to N-acetylgalactosamine 6-sulfate and unsaturated uronic acid coupled to N-acetylgalactosamine, respectively. The distribution of dermatan sulfate thus revealed was confirmed by comparison with that found by monoclonal antibody 6B6 which reacts with small proteoglycans carrying dermatan sulfate side chains. The localization of positive staining in fibrous connective tissues was almost identical with these two procedures.

Characteristics of "embryoid body" in human gonadal germ cell tumors.

The characteristics of so-called "embryoid body" appearing in gonadal germ cell tumors were studied histologically and immunohistochemically on serial sections of three cases (one ovary and two testes). The embryoid bodies were usually observed to be contiguous with immature or mature intestinal ducts, hepatic nests, or epidermal cell nests on serial sections, though they appeared to be isolated in one section. The "amniotic cavity"-like structure of embryoid body was continuous with intestinal duct, and rarely with squamous cell nests, while the "yolk sac" was continuous with hepatic tissue. In these immature or mature structures, differentiation was always found independently of "disc," and portions of "ectoderm" and "endoderm" remained less differentiated in comparison with others. These findings were in contrast with a normal embryo in which immature and/or mature structures are derived from the embryonic disc. The amniotic cavity connected frequently with yolk sac. From the present results, the embryoid body is not considered to be a real or teratomatous embryo, but only a product during a divergent differentiation into intestine and liver from the plastic epithelium, which seems to be derived from an embryonic gut.

Production and characterization of monoclonal antibody to dermatan sulfate proteoglycan.

We purified dermatan sulfate proteoglycan (PG) from the capsule of human ovarian fibroma for use as an immunogen. A monoclonal antibody, designated 6B6, was produced which reacts to the intact molecule of dermatan sulfate PG and the chondroitinase AC-treated core molecule on Western-blotted nitrocellulose membrane. Localization of materials showing crossreactivity to this antibody was studied in human tissues by indirect immunohistochemistry. The interstitial elements of almost all tissues examined were positive for the antibody: dermis, submucosal layer of digestive tract, perichondral layer, perivascular connective tissue, perineurium, adventitia of aorta, vessel wall of vein, pleura, and fibrous capsule of kidney and liver. Positive staining was also observed in fibrous elements at post-necrotic foci of cardiac muscle and pancreas, and at atherosclerotic lesions of aorta. The distribution of the antigen, core protein of the dermatan sulfate PG, revealed with 6B6 was compared to that of the dermatan sulfate side chain, which was demonstrated with antibody 9A-2 (Couchman et al.: Nature 307:650, 1984) after treatment with chondroitin sulfate B-lyase. The distribution of both antigens, core protein, and dermatan sulfate side chains showed the same pattern, with minor exceptions. The antibody 6B6 will be a useful tool to study the immunohistochemical localization of dermatan sulfate PG.

Immunohistochemical studies of DMBA-induced rat mammary tumors.

In order to clarify the biological characteristics of rat mammary tumors induced by 7,12-dimethylbenz-[a]-anthracene (DMBA), histochemical and immunohistochemical studies were performed. Two types of luminal spaces were observed within the tumor. In one type, the lumen was surrounded by eosinophilic columnar cells which were strongly reactive for soybean agglutinin (SBA) but weakly stained with keratin antibodies. In the luminal spaces, substances positive for PAS, dialyzed iron ferrocyanide or alcian blue and resistant to mucopolysaccharidase were occasionally observed. Ultrastructurally, the luminal surface was characterized by the presence of microvilli and tight junctions. In the other type, the lumen was often found in highly cellular foci and surrounded by pale, polygonal or elongated cells which were weakly stained with keratin antibodies but not SBA. The luminal spaces presented a peculiar structure filled mainly with mucoid substances sensitive to hyaluronidase, chondroitinase ABC and heparitinase, and the inner surface of the spaces was surrounded by basement membrane components: laminin, fibronectin and type IV collagen. The results of the present study therefore showed that DMBA-induced mammary tumor consists, partly, of a structure resembling human adenoid cystic carcinoma.

Extracellular matrix of cultivated, poorly differentiated human gastric adenocarcinoma cells promotes attachment and spreading of mesenchymal cells.

To clarify interactions between carcinoma and mesenchymal cells, we examined the extracellular matrix-substance remaining on culture dishes after confluent growths of gastric carcinoma cells were removed with EDTA. The matrix synthesized by poorly differentiated adenocarcinoma cells (cell lines KATO-III and MKN-45) cultivated in serum-free medium has a fibroblast (cell line WI38)-attachment activity, which is not detected in the matrix synthesized by a well differentiated adenocarcinoma (cell line MKN-28). This activity was not observed in KATO-III-matrix extracted with 6 M urea, but could be detected in a 1% SDS extract from the remaining matrix on the culture dishes after 6 M urea extraction. The activity was abolished by treatment with pronase (16 micrograms/ml), trypsin (0.005%) or alkali, but was unaffected by collagenase (80 micrograms/ml, 4 h) or chondroitinase ABC (1 U ml, 1 h). It is conceivable that the fibroblast-attachment activity of the matrix produced by poorly differentiated adenocarcinoma cells is related to the proliferation of interstitial connective tissue in vivo.

Immunohistochemical localization of chondroitin sulphate and dermatan sulphate proteoglycans in tumour tissues.

Immunohistochemical localization of chondroitin sulphate and dermatan sulphate proteoglycans (PGs) was observed in 70 tumour tissues, using monoclonal antibodies 9A-2 and 3B-3 raised against core molecules obtained from chondroitin sulphate PG by chondroitinase ABC-treatment. They recognize a stub of delta Di-4S and delta Di-6S binding to core protein via a linkage tetrasaccharide, respectively. The antibody 6B6 raised against dermatan sulphate PG obtained from an ovarian fibroma capsule in our laboratory was also used. The interstitial fibrous elements, so-called 'specific stroma' within the cancer cell nests contained chondroitin 4-sulphate PG as revealed with 9A-2, whereas the surrounding connective tissue and the preexisting fibrous connective tissue involved in the tumour growth consisted of dermatan sulphate PG with a considerable amount of chondroitin 4-sulphate PG. Chondroitin 6-sulphate PG as revealed with 3B-3 was located in the connective tissue proliferating from blood vessels and muscle tissue in association with the invasive growth of tumour cells. Chondroitin 6-sulphate PG was also observed in the basement membrane components of some tumours. In non-epithelial tumours (fibrogenic, chondrogenic, osteogenic and neurogenic tumours), chondroitin 4-sulphate was in fibrous portions. When collagenization and hyalinization progressed, dermatan sulphate PG was observed to increase in quantity.

The frequency and histology of hepatic tissue in germ cell tumors.

The frequency of hepatic tissue and its histological characteristics were examined in 516 germ cell tumors. Hepatic tissue was observed in 48 cases (9.3%). The incidence of hepatic tissue was low in tumors of the ovary (5%), high in both retroperitoneal (27%) and sacro-coccygeal (24%) tumors, and low in both mature (0.3%) and immature teratomas (11%). It was usually encountered in infancy, and the frequency was high in both yolk sac tumors (48%) and mixed germ cell tumors (52%). The hepatic tissue found mainly in mature or immature grade 1 teratomas was similar to adult normal human liver tissue (Ha-type). Tissue in areas consisting of some immature somatic elements of a mixed germ cell tumor was similar to embryonic or fetal liver tissue (Hf-type). Many hepatic nests found in a polyembryoma were of both Ha- and Hf-types. The hepatic tissue found in close relation to yolk sac elements showed predominantly hepatocellular carcinoma-like features (HCLS). Immunohistochemically, the cytoplasm of adult liver-type cells was positive for alpha-1-antitrypsin (AAT), human albumin (ALB), and the third (C3) and fourth (C4) components of the complement system. The cytoplasm of fetal liver-type cells showed the same positivity; in addition, these cells were positive for alpha-fetoprotein (AFP) in 25% of the cases. The cytoplasm of hepatic cells of HCLS was positive for AFP, AAT, ALB, C3, and C4. A weakly positive reaction for CEA and CA19-9 was observed in bile duct-like structure in some Hf-type cases.

beta-D-xylosides and their analogues as artificial initiators of glycosaminoglycan chain synthesis. Aglycone-related variation in their effectiveness in vitro and in ovo.

A series of aryl and alkyl O-beta-D-xylosides and their analogues with S, NH or CH2 in the glycosidic linkage were prepared and examined for their ability to act as artificial chain initiators of chondroitin (dermatan) sulphate synthesis in embryonic chick cartilage, foetal rat skin and 6-week-old-rat aorta under conditions where normal protein-core synthesis was inhibited by cycloheximide. For all these tissues in culture, phenyl O-beta-D-xyloside and phenyl beta-D-thioxyloside were clearly more effective than the corresponding N-xyloside and homo-C-xyloside. Introduction of a carboxy group to the para position of their aglycone yielded derivatives with far lower initiator activity. In a concentration range lower than 0.1 mM, the effectiveness of alkyl beta-D-thioxyloside was greatly influenced by the carbon number (n) of the alkyl group and was at a maximum at n = 7 or 8 for the cartilage, at n = 5 for the skin and at n = 4 for the aorta. In the beta-xyloside-treated cartilages, the average length of newly formed chondroitin sulphate chains reflected the chain-initiator activity of added xyloside, i.e. the higher the initiator activity, the shorter the average chain length. In the skin and aorta, none of the drugs could relieve the inhibition of heparan sulphate synthesis caused by cycloheximide. Fertilized hens' eggs were each injected on day 9 with 9.2 mumol of beta-xyloside and the skeletal systems of embryos were examined a week later. The embryos treated with beta-xylosides of relatively high initiator activity showed a 30-40% decrease in the overall growth rate of skeletons, whereas those treated with beta-xylosides of low initiator activity showed little or no decrease in the growth rate. The results are consistent with the notion that the observed change in skeletal morphology results mainly, if not completely, from beta-xyloside-induced synthesis of core-protein-free chondroitin sulphate, and further suggest that a procedure employing a series of beta-xyloside homologues with various initiator activities will furnish an easily applied criterion on which to test the specificity of xyloside action on biological processes.

Isolation and characterization of proteoglycans from human nonepithelial tumors.

Proteoglycans (PGs) and glycosaminoglycans (GAGs) were identified in myogenic and fibrogenic tumors. More PGs containing mainly chondroitin sulfate could be detected in malignant tumors (leiomyosarcomas) than in benign tumors (leiomyomas and fibromas). Two groups of PGs were detected in the malignant tumors by ion-exchange chromatography and gel chromatography. One group was a large molecule with chondroitin sulfate side chains, seemingly composed of two or more subpopulations that were eluted from Sepharose CL-4B with a kav of 0.45. After removal of GAG side chains from the PG by chondroitinase AC digestion, core molecules with molecular weights greater than 200,000 were obtained. Another PG detected was a fraction of small PG eluted from Sepharose CL-4B with a kav of 0.45. It consisted of a core molecule with a molecular weight approximately equal to 48,000 and GAG side chains containing chondroitin sulfate-dermatan sulfate hybrids. The mixed sequence of L-iduronic acid with D-glucuronic acid in the same GAG chain was demonstrated by the formation of a small proportion of tetrasaccharide after chondroitinase AC digestion. In the benign tumors, the large PG was found only in very small amounts, and PG detected was composed mainly of the small one eluted from Sepharose CL-4B with a kav of 0.45. Its core protein had a molecular weight of approximately equal to 46,000, which was similar to that of small PG obtained from leiomyosarcomas, but its GAG side chains were composed mainly of dermatan sulfate containing small amounts of glucuronic acid. The results suggest that the core molecules of small PGs from both benign and malignant tumors are the products of the same gene but that they are processed in a different manner to form proteoglycans with different types of GAG chains.

Histochemical studies on pseudocysts in adenoid cystic carcinoma of the human salivary gland.

Pseudocysts are unique structures found in adenoid cystic carcinomata of human salivary glands. They were studied in 13 such cases by histochemical and immunohistochemical means. The pseudocysts contained an abundance of mucoid materials which reacted strongly with both Alcian Blue and dialysed iron ferrocyanide. The mucoid material was digested with chondroitinase ABC and heparitinase, but was resistant to Streptomyces hyaluronidase. The inner surfaces of the pseudocysts were strongly reactive for laminin, whereas the interface between the tumour cell nests and the outer stromal area was intensely reactive for fibronectin. Numerous fibronectin-reactive fibrils and blood coagulation factor XIII (F-XIII)-positive cells were distributed extensively in the outer stromal area. The F-XIII-positive cells were also found within some pseudocysts. The results obtained in the present study have shown that the pseudocysts represent a peculiar structure consisting of basement membrane components; laminin, fibronectin, heparan sulphate and chondroitin sulphate.

A cell line from pleomorphic adenoma of the rat salivary gland.

Pleomorphic adenoma of rat salivary gland is extremely rare, and culture of cells obtained from rat salivary gland tumors has not been reported. We have established a long-term cell line from a pleomorphic adenoma of a Sprague-Dawley rat submaxillary gland. The pleomorphic adenoma was composed of oval or spindle-shaped cells occasionally forming a small duct. Alcian blue-positive intercellular matrices, consisting mainly of glycosaminoglycans, were abundant. The cultured cells showed characteristics similar to those of the original tumor. This cell line should be useful for biological and biochemical studies of glycosaminoglycan-synthesis of pleomorphic adenoma cells.

Histochemical AMD electron microscopic studies of intercellular matrices of papilloma induced by painting DMBA on the skin of mice.

The localization and distribution of glycosaminoglycans (GAG) in papilloma tissue which was introduced by painting DMBA on the back skin of ICR mice were observed by light and electron microscopy. GAG consisting mainly of hyaluronic acid could be detected histochemically on the cell surface and extracellular matrices of the basal layer of the papilloma, but it could not be observed in the non-neoplastic epidermis. After incubation of tissue segments in a medium containing 35SO4, an autoradiograph was made, and 35S-radioactivity was observed in the interface between the papilloma epidermis and dermis. Ultrastructurally, the cell surface of keratinocytes in the basal layer of the papilloma was stained intensely with ruthenium red. A cell line was obtained from the papilloma, and GAG could be demonstrated on the cell surface and in the intercellular matrix of the cultivated keratinocytes by treatment with hyaluronidase. The significance of GAG in the proliferating squamous cells was discussed.