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.

Time-lapse filming of cellular interactions within living tissues. III. The role of cell shape.

By a further development of the technique for time-lapse filming of organ cultures, it has become possible to observe the internal structure of the living tissues and to study the dynamics of intercellular contacts within the tissue. This method has been applied to a study of the interaction between the tissues of the chick chorioallantoic membrane and non-malignant fibroblasts, polyoma virus-transformed fibroblasts, and Harding-Passey melanoma cells. The behaviour of these cells in the absence and in the presence of colcemid has been investigated. From these studies it is concluded that disturbances in cell shape brought about by colcemid do not affect the invasive properties whereas the surface polypodial activity shown by the malignant cells is a major factor in tumour invasion.

Measurement of the ability of cells to infiltrate normal tissues in vitro.

Organ cultures of chorioallantoic membranes of hen eggs have been used to establish a quantitative method of measuring the infiltrative ability of a variety of normal and tumour cells. Normal fibroblasts, mouse peritoneal cells and cells of low tumorigenicity infiltrated poorly and slowly whereas most tumours infiltrated rapidly. Some cells of the more invasive tumours achieved minimum rates of migration through the normal tissue of 2-3 µm/h. One tumour line which tended to form aggregates on the chorioallantoic membrane elicited a pronounced rejection response from the ectoderm of the membrane. Colcemid, which inhibits the formation of cell processes and the directional migration of cells, and dibutyryl cyclic AMP, which restores certain aspects of normal behaviour to tumour cells in vitro, had little effect on the invasion of the membrane by tumour cells.

Selective culture of epithelioid cells from a human squamous carcinoma using a monoclonal antibody to kill fibroblasts.

Monoclonal antibody LICR LON/FIB86 is cytotoxic for human fibroblasts from at least some sources, with rabbit complement. Explants of human squamous carcinomas of the head and neck were put into culture and fibroblasts and epithelioid cells began to grow out. The cultures were treated regularly with antibody plus complement: the fibroblasts were killed consistently, as they emerged, leaving sheets of epithelioid cells. We conclude that this is a practical method for controlling human fibroblast growth in culture.

Human breast tumour-induced osteolysis and prostaglandins.

Osteolysis effected in vitro by breast carcinomas can be inhibited by aspirin. Some prostaglandins stimulate in vitro bone resorption. Our results indicate that whilst osteolytically active PGE and PGF are released by the carcinomas in most cases, some other osteolytic principle is released as well.

Breast-cancer osteolysis, bone metastases, and anti-osteolytic effect of aspirin.

23 (60%) of 38 human breast carcinomas had significant in-vitro osteolytic activity. All patients presenting with bone metastases or hypercalcaemia had active tumours. Over a subsequent three-year follow-up period, bone metastases did not develop in any of the 15 patients with inactive tumours, and metastases at other sites developed in only 2. Of the 23 patients with active tumours, 7 either had, or have since developed bone metastases; in 4 of these hypercalcaemia also developed. 14 tumours, chosen at random, were tested for in-vitro osteolytic activity in the presence and absence of aspirin, which inhibits prostaglandin synthetase. The activity of 8 of the 9 osteolytically active tumours was significantly, though not completely inhibited by aspirin. Although the number of patients is limited, these results indicate that the in-vitro osteolysis assay may detect substances, perhaps including prostaglandins, produced by breast tumours which affect prognosis and contribute to the subsequent formation of bone metastases.

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.

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.

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.

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.

The inhibition by aspirin and indomethacin of osteolytic tumor deposits and hypercalcaemia in rats with Walker tumour, and its possible application to human breast cancer.

Walker carcinosarcoma cells cause in vitro osteolysis which may be inhibited by aspirin. In the rat, this tumour produces osteolytic bone deposits and hypercalcaemia, both of which can be prevented by aspirin and indomethacin, whereas soft tissue tumour deposits are unaffected by these drugs. Some human breast tumours cause in vitro osteolysis which may be inhibited by aspirin.

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.