Sequential observation of micrometastasis formation by bacterial lacZ gene-tagged Lewis lung carcinoma cells.

Sequential events in micrometastasis formation including entry into the blood circulation and arrest, extravasation and initial growth in the lung was investigated using bacterial lacZ gene-tagged Lewis lung carcinoma cells (4A1-1). Micrometastases in the lung could thereby be specifically detected at the single cell level by X-Gal staining. After intravenous injection, X-Gal positive tumor cells appeared to extravasate within hours, but most cells then degenerated or died in the alveolar space by 2-3 days postinjection. A decreased BrdU labeling index to a negligible level at 2 days postinjection and reduction of X-Gal positive foci to a basal level (less than 0.1% of injected cells) by 4 days are in line with rapid clearance of tumor cells from the lung. The size and BrdU labeling indices of the persisting X-Gal positive foci, however, started to increase from 4 days postinjection. Type IV collagen immunostaining demonstrated loss of pre-existing basement membranes with growth of micrometastases: When 4A1-1 cells were inoculated subcutaneously, lung micrometastases from resulting tumors were detected as single or small numbers of X-Gal positive cells at 2 weeks postinjection. Progressive development of micrometastasis to macroscopic metastasis was noted by 4-5 weeks postinjection. The results indicate that micrometastasis formation by Lewis lung carcinoma cells involves a sequence of events starting with rapid extravasation after arrest in the lung within 1 day, followed by death of most cells at 2-3 days and subsequent new growth and expansion of persisting tumor cells from 4 days postinjection.