Abstract
Cellular repopulation in Lewis carcinoma irradiated with 60Co gamma-rays was examined by performing sequential cell-survival estimations using an in vitro soft-agar-colony assay. Following local irradiation (15--35 Gy) two distinct types of colony were seen: compact colonies with tightly packed cells and diffuse colonies with widely dispersed cells. Maximal diffuse colony formation in vitro was only obtained in the simultaneous presence of adequate numbers of compact colonies. After whole-body irradiation only compact colonies were observed. Only-cell survival data from compact colony counts correlated with cell survival estimated by the lung colony assay and we conclude that compact colonies are produced by clonogenic tumour cells. Cytochemical and immunological evidence showed that diffuse colonies were composed of macrophages. After local irradiation the initial kill of clonogenic tumour cells was dose dependent. At each dose level, repopulation began immediately and proceeded with a doubling time of about 1 day. Macrophage colony-forming cells (macrophage progenitors) per tumour were initially reduced by about 3 decades, but recovered very rapidly to reach pretreatment levels within 2 days. We conclude that at least two populations of clonogenic cells are present in Lewis lung carcinoma, tumour cells that repopulate irradiated tumours by in situ proliferation and host-macrophage progenitors that repopulate locally irradiated tumours by infiltration. The hazards of confusing host and tumour cell colonies in in vitro assay systems are stressed.
Highlights
Summary.-Cellular repopulation in Lewis carcinoma irradiated with 60Co y-rays was examined by performing sequential cell-survival estimations using an in vitro soft-agar-colony assay
Cell-survival data from compact colony counts correlated with cell survival estimated by the lung colony assay and we conclude that compact colonies are produced by clonogenic tumour cells
We have no evidence to suggest that the small-cell density has any influence on the clonal growth of either tumour cells or macrophage progenitors
Summary
Each population was counted separately and the cell yield per g of untreated tumour was 8-5 x 107 (s.d. 2-8 x 107) for the large cells and 1.35 x 107 (s.d. 0.49 x 107) for the small cells. Cell survival.-The survival of cells derived from Lewis lung tumours was assayed in vitro using the soft-agar-colony assay developed by Courtenay (1976). Cultures were incubated at 37°C in an atmosphere of 5% 02, 5% CO2 and 90% N2 for 12 weeks, and colonies of more than 50 cells were counted. Cell identification techniques.-(i) Samples of cell suspensions, or cells from agar colonies, were gently centrifuged with IgG antibodycoated sheep erythrocytes (EA) at room temperature, and after resuspension the rosettes were counted under phase contrast, as a percentage of the total nucleated cells. (iv) Cell preparations were stained for nonspecific esterase and for chloroacetate esterase using the methods of Yam et al (1971)
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