Biological studies of ten human squamous carcinoma cell lines: an overview.

Ten cell lines established from surgical specimens of human squamous carcinomas of the tongue and larynx have been investigated with respect to their motility, ultrastructure, karyotypes, certain biochemical features, interaction with normal epithelial and stromal elements and capacity to infiltrate three-dimensional organoid systems. All the cell lines have maintained several morphological and biochemical characteristics indicating a common origin, although the extent to which each line displays this heritage is variable. The phenotypes of each of the individual cell lines are, however, notably stable. Data are provided for epithelial surface markers (including epidermal growth factor, EGF) and for the synthesis and release of prostaglandins and proteases which may be involved in invasive mechanisms. Encounters between the cell lines and organoid substrata (embryonic chick heart spheroids, human amnion, chick chorioallantoic membrane) are described: the results indicate a scale of invasiveness ranging from lack of penetration to full-thickness infiltration by cells showing various distinctive growth patterns. Correlation between in vitro and in vivo findings is discussed, and it is suggested that the biological heterogeneity of the lines may reflect inherent properties of the original carcinoma cell populations which are more distinctly expressed in vitro.

Interactions between normal epithelial and squamous carcinoma cells in monolayer culture.

Confrontations of rings of adult human oral mucosa epithelial cells enclosing islands of similar normal epithelium, fibroblasts, and cells of three established lines of human squamous carcinoma in monolayer culture were investigated by phase and reflection microscopy and by time lapse cinematography. Measurements of the dimensions of the rings and islands of cells revealed that, while normal epithelial rings confronted with normal epithelium or fibroblasts migrated continuously inwards, similar rings confronting islands of the carcinomas retreated progressively outwards from the tumor islands. The persistence of substantial cell-free space between the epithelium and tumor cells indicated that the outwards migration of the epithelial rings was not solely due to proliferation of the tumor cells. The tumor-induced migration of normal epithelium in monolayer culture may reflect the response of normal epithelium to carcinoma cells in certain in vivo situations.

Five human tumour cell lines derived from a primary squamous carcinoma of the tongue, two subsequent local recurrences and two nodal metastases.

Five tumour cell lines have been derived from a primary squamous carcinoma of the tongue, from 2 subsequent local recurrences, and from 2 lymph-node metastases--all from the same patient. While the cell lines shared many morphological and biochemical characteristics, those derived from recurrences and metastases appeared to be less differentiated, were less well organized in culture, and displayed fewer desmosomes and tonofilaments than cells in the primary tumour line. A recurrent line showing greatest morphological divergence from the primary tumour line also demonstrated the greatest differences at the ultrastructural level, in increased production of plasminogen activator and in the composition of cell-surface glycoproteins.

Ten human carcinoma cell lines derived from squamous carcinomas of the head and neck.

Ten cell lines of human squamous carcinomas of the tongue and larynx have been established from surgical specimens removed from 36 unselected patients, in order to provide systems for investigating the invasive and tissue-destructive capacity of squamous carcinomas of the head and neck. The morphology, ultrastructure and growth characteristics of the 10 lines are described. Detailed cytogenetic analysis of the first 4 lines indicates that each is karyotypically unique, with no evidence of cross-contamination. Nine of the 10 cell lines secrete immunoreactive beta human chorionic gonadotrophin (beta-hCG) in the culture medium. No correlation was demonstrated between the ability of the cell lines to secrete plasminogen activator and their capacity to grow in soft agar or as xenografts in immune-deficient mice.

Establishment and properties of human germ cell tumors in tissue culture.

Monolayer cultures of nine human testicular germ cell tumors have been attempted, and five have been established as proliferating long-term cultures. All five grew in suspension in nutrient agar, but three proliferated in monolayer culture only when in contact with stromal cells derived from the tumors. Two have been established as "pure" lines, and one of these has been cloned. All contain elevated levels of alkaline phosphatase, approximately one-half of which displays placental isoenzymic characteristics. Three tumor cultures formed differentiated tissues and synthesized significant quantities of beta-chorionic gonadotrophin or carcinoembryonic antigen for a brief period after a 5-month culture. These properties were subsequently lost and could not be reinduced.

Some mechanisms of local bone destruction by squamous carcinomas of the head and neck.

An in vitro osteolysis assay with 45Ca-labelled mouse calvaria has been used to investigate mechanisms of direct bone invasion by squamous carcinomas of the head and neck. Short-term (3-day) organ cultures of 8 fresh squamous carcinomas showed varying degrees of in vitro bone-resorbing activity which was blocked by indomethacin, an inhibitor of prostaglandin synthesis. Supernatant media from 6 established cell lines also induced bone resorption in vitro and evoked an osteoclastic response in the cultured calvaria. Osteolysis by supernatant media was not blocked by indomethacin in all the tumour-cell lines, and the production of non-prostaglandin osteolysins by the indomethacin-resistant lines is postulated. The two principal findings that emerge are: (1) Stimulants for osteoclastic activity are derived from both squamous-carcinoma cells and from host cells in the tumour stroma. (2) These stimulants are diverse. Indomethacin-sensitive agents, presumed to be prostaglandins, are most convincingly demonstrated in the fresh tumours. Indomethacin-resistant agents, presumably not prostaglandins, are more characteristic of the carcinoma cell lines.

Asymmetrical distribution of epithelial membrane antigen on the plasma membranes of human breast cell lines in culture.

A technique for localizing antigens by immunoperoxidase staining on the surfaces of unfixed culture cells is described. The cells can subsequently be studied by light or electron microscopy. These methods have been used to localize epithelial membrane antigen (EMA) on human mammary carcinoma (MCF7) cells in culture. This antigen was expressed on the plasma membrane, being localized on the interface with the culture medium and absent from the other external cell surfaces. This heterogeneous distribution reflects the distribution observed in mammary epithelial cells in vivo.

Preparation and identification of human breast epithelial cells in culture.

The routine preparation of gram quantities of lobuloalveolar and ductal structures from human reduction mammaplasties by treatment with collagenase is described. When cultured on plastic or glass surfaces these structures give rise, initially, to epithelial sheets consisting of cells which retain many morphological characteristics of myoepithelial cells and do not stain with an antiserum which reacts with the surfaces of the lining epithelium of breast ducts and lobulo-alveoli. Subsequently, cells which resemble the lining epithelial cells migrate from these structures and react strongly with the antiserum. Cell proliferation in these cultures is minimal. Explants of major ducts dissected from mastectomies produce vigorously proliferating epithelial sheets which contain morphologically similar cells not identifiable as either lining epithelium or myoepithelium, although nearly all the cells in direct contact with the medium react with the lining epithelium specific antiserum.

Polycyclic hydrocarbon activation and metabolism in epithelial cell aggregates prepared from human mammary tissue.

The metabolism of benz(a)anthracene (BA), 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BP) by human mammary epithelial cell aggregates in culture has been investigated using non-neoplastic tissues obtained from eight patients undergoing reduction mammoplasty. All three hydrocarbons were metabolized to water-soluble and organic solvent-soluble products and the latter included both K-region and non-K-region dihydrodiols. The major dihydrodiols detected as metabolites of the parent hydrocarbons were the 8,9-dihydrodiols of BA and DMBA and the 9,10-dihydrodiol of BP. The 1,2-dihydrodiols of BA and DMBA and the 11,12-dihydrodiol of BP were not detected. The hydrocarbons also became bound to the proteins and DNA of the epithelial cells but there were wide differences in the extents of binding occurring with the different hydrocarbons and in the extents of metabolism and binding occurring with tissue preparations from different patients. Some of the hydrocarbon-deoxyribonucleoside adducts formed from DMBA and BP appeared to have arisen through reactions of "bay-region" diol-epoxides with DNA, but only very low levels of reaction with DNA were detected in tissue preparations treated with BA.

Preparation of highly purified cytotrophoblast from human placenta with subsequent modulation to form syncytiotrophoblast in monolayer cultures.

The preparation of highly purified monolayer cultures of human cytotrophoblast cells essentially free of stromal and syncytial cells is described. Such cells subsequently form multinucleate syncytial cells in vitro. This is accompanied by the synthesis of heat-stable alkaline phosphatase and beta-HCG. A significant proportion of the multinucleate cells that form in monolayer cultures of early placentae arise as a result of an amitosis. It is suggested that this in vitro behavior may reflect an in vivo process.

An experimental model of cachexia induced by a xenografted human tumor.

A hypernephroma removed from a male patient who had lost 30 kg in weight in the 2 months preceding surgery was established in immunosuppressed CBA/Lac mice as a nonmetastasizing transplantable xenograft. The xenografted tumors, although comprising less than 5% of the total body weight of the mice, produced considerable weight loss (greater than 25%). A slight reduction in food intake of tumor-bearing mice was noted, but some animals bearing mouse or human tumors not inducing cachexia had equally low food intake without accompanying weight losses. No obvious defects in gastrointestinal histology or absorption were observed. The precise mechanism(s) producing the severe cachexia remains to be established.

A new experimental model of human cachexia.

A hypernephroma removed from a male patient who had lost 30 kg in weight in the two months preceding surgery was grown as a non-metastasizing transplantable xenograft in immune-suppressed mice. The tumour produced a considerable weight loss (greater than 25 per cent) in the mice at a stage when it comprised less than 5 per cent of the total body weight. A slight fall in food intake of the tumour-bearing mice was noted, but animals bearing other non-cachectic mouse and human tumours had much lower food intakes without accompanying weight loss. No obvious defects in gastrointestinal absorption were detected nor was any gross increase in basal metabolic rate observed. The precise mechanism producing the severe cachexia remains to be established, but elaboration of humoral factors by the tumour seems probable. This model of cachexia bears a closer relation to the clinical situation than do other experimental animal tumour models currently available.