Summary.-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 / human chorionic gonadotrophin (/-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.

Summary.-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.PATIENTS with breast cancer frequently develop abnormalities in their calcium metabolism, which are usually associated with osteolytic bone metastases (Galasko and Burn, 1971) and are caused by excessive mobilization of calcium from the skeleton. This raises the possibility that the mechanism for this skeletal calcium mobilization, and the ability of tumour deposits to develop into destructive bone deposits, may both depend on the ability of tumour cells to produce osteolytic substances.To investigate this hypothesis, we have used an in vitro organ culture system of neonatal mouse bone which releases calcium in response to known osteolytic substances. We have found that some, but not all, human breast carcinomata when added to the organ culture caused increased calcium release from the bones and that this osteolysis could be inhibited by aspirin.To test whether these in vitro observations were relevant to the in vivo behaviour of tumours, it was necessary to develop a suitable animal tumour model. For this, we chose the intra-aortic injection of Walker carcinosarcoma cells into the rat, because this tumour has been reported to cause hypercalcaemia (Raue et al., 1972) These were cultured for a further 3 days under the same conditions as for the preliminary period, during which time calcium passed from the bone into the medium. At the end of the culture period the 45Ca in the medium, and remaining in the bone, was estimated using a Packard scintillation counter. The

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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. Images Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 5 Fig. 6 Fig. 7 Fig. 8

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.

Summary.-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 nonprostaglandin 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.

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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.