A panel of eight conditionally immortal lines derived by infection of human breast epithelial cells with an amphotropic retrovirus transducing a ts mutant of SV40 large T-antigen was analyzed with respect to individual retroviral integration patterns. Each line contained multiple integration sites which were clonal and stable over extended passage. Similar integration patterns were observed between individual lines arising separately from the same stock of pre-immortal cells, suggesting a common progenitor. Retroviral integration analysis of pre-immortal cells at different stages of pre-crisis growth showed changes indicative of a progressive transition from polyclonality to clonality as the cells approached crisis. Each of the immortal lines contained a sub-set of the integration sites of their pre-immortal progenitors, with individual combinations and copy numbers of sites. Since all the cell lines appeared to originate from single foci in separate flasks, it is likely that each set arose from a common clone of pre-immortal cells as the result of separate genetic events. There was no evidence from this analysis to suggest that specific integration sites played any part either in the selection of pre-crisis clones or in the subsequent establishment of immortal lines.

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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.

The immunohistochemical localization of basement membrane type IV collagen was investigated with a mouse monoclonal antibody in major surgical resections from 25 patients with invasive squamous carcinomas of the head and neck. Irrespective of site, size or stage of the disease, the 16 primary invasive tumours were almost completely surrounded by a layer of type IV collagen. Focal abnormalities were regularly present, consisting of thickening and aggregation of type IV collagen together with attenuation and segmental loss. Similar changes were seen in metastatic squamous carcinomas in 36 cervical lymph nodes. It is suggested that the probable formation of a normal basement membrane protein by these squamous carcinomas indicates the preservation of a normal function of differentiating squamous epithelia. The results indicate that a major basement membrane component, type IV collagen, continues to co-exist with invasive and metastatic squamous carcinomas.