Monoclonal antibody 13A to murine CD44 was used to bind the alpha-particle emitter 213Bi to cell surfaces of cultured EMT-6 or Line 1 tumor cells. Data on kinetics and saturation of binding, cell shape and nuclear size were used to calculate the absorbed dose to the nuclei. Treatment of monolayer cells with [213Bi]MAb 13A produced a classical exponential survival curve with no apparent shoulder. Microdosimetry analyses indicated that 1.4-1.7 Gy produced a 37% surviving fraction (D0). Multicellular spheroids were shown to bind [213Bi]MAb 13A mainly on the outer cell layer. Relatively small amounts of activity added to the spheroids resulted in relatively large absorbed doses. The result was that 3-6-fold less added radioisotope was necessary to kill similar fractions of cells in spheroids than in monolayer cells. These data are consistent with the interpretation that the alpha particles from a single 213Bi atom bound to one cell can penetrate and kill adjacent cells. Flow cytometry was used to sort cells originating from the periphery or from the interior of spheroids. Cells from the outside of the [213Bi]MAb 13A exposed spheroids had a lower surviving fraction per administered activity than cells from the interior. Cells were killed efficiently in spheroids up to 20-30 cells in diameter. The data support the hypothesis that alpha-particle emitters should be very efficient at killing cells in micrometastases of solid tumors.

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The experiments described investigate the potential influence of surrounding normal tracheal epithelial cells on the survival and growth of carcinogen-exposed epithelial cells in tracheal mucosa reconstructed from known cell mixtures. Cell mixtures containing preneoplastic or neoplastic rat tracheal epithelial cells and a small fraction of normal tracheal or esophageal epithelial cells were inoculated into the lumen of previously frozen-thawed tracheas which were then transplanted s.c. into syngeneic hosts. Within 2-3 weeks an intact tracheal mucosa was regenerated from the inoculated cells. At various times after cell inoculation and transplantation, cells were harvested from the repopulated trachea and the number of diploid normal and/or aneuploid carcinogen-exposed tracheal epithelial cells determined by flow cytometry (DNA content) and the frequency of cells with altered in vitro growth capacity determined. The data suggest that normal tracheal epithelial cells have an enhanced 'survival' capacity relative to carcinogen-exposed cell lines in the regenerated tracheal mucosa. When greater than 10(4) normal cells were inoculated with a 2- to 100-fold excess of carcinogen-exposed cell lines the regenerated epithelium was comprised almost entirely of normal-diploid-epithelial cells. Independent of the ratio of normal to altered cells, when less than 10(4) normal epithelial cells were inoculated in the mixture some carcinogen-altered cells and some normal cells were present in the regenerated epithelium. When established repopulated tracheas containing mixtures of normal and neoplastic epithelial cells were left in the animal no tumors developed. In contrast tracheas containing neoplastic cells alone or partially scraped tracheas containing comparable numbers of neoplastic cells covering a contiguous area of the submucosa, tumors developed within 2-5 weeks of cell inoculation and transplantation. These data suggest that normal tracheal epithelial cells have the capacity to modulate expression of the neoplastic phenotype (tumor development) in neoplastic populations. This effect appears to require close contact with the normal cells as inhibition is not observed when the neoplastic cells occupy a single contiguous area of the trachea.

As rat tracheal epithelial cells progress from a normal to a neoplastic phenotype there are systematic changes in their ability to produce and activate latent transforming growth factor type beta (TGF-beta) as well as systematic changes in their response to this growth factor. Using a TGF-beta radioreceptor binding competition assay it was found that normal proliferating rat tracheal cells in early primary culture produced latent TGF-beta. With the emergence of terminally differentiated cell populations active TGF-beta was also detected in the conditioned medium. When normal cells were cultured under conditions allowing for continued proliferation, no active TGF-beta was detected in the conditioned medium. Colonies of proliferating epithelial cells in 4-6 week primary cultures or subculturable tracheal cell lines did not produce detectable levels of active or latent growth factor. With neoplastic progression there was likewise a change in response to active TGF-beta. Normal tracheal cells in primary culture were highly sensitive to growth-factor-induced decreases in thymidine uptake as well as to the induction of terminal differentiation. Proliferating epithelial cells in late (4-6 week) primary cultures and preneoplastic, subculturable cell lines were often as sensitive as normal cells to the growth factor-induced decline in thymidine uptake. None of these altered populations, however, was induced to differentiate (to form cornified, cross-linked envelopes) in the presence of TGF-beta.

A simple magnetic separation technique has been developed using lectins specific for two of the cell types found in the tracheal mucosa. The resulting populations of basal and secretory cells were examined for proliferative capacity in culture and in vivo. The basal cell fraction contains the cells that proliferate in culture and respond to 12-O-tetradecanoylphorbol-13-acetate. In addition, the basal cell fraction exhibited the highest proliferative capacity in vivo during the first few days after transplantation. Repopulation of inverted intestinal segments showed that only with suspensions containing a significant proportion of basal cells could a mucociliary lining be established. Segments receiving the same number of unsorted or predominantly mucous secreting cells did not repopulate in vivo. These data support the hypothesis that the basal cell is most likely the stem cell of the tracheal epithelium.