SEQUENTIAL OBSERVATION OF MICROMETASTASIS FORMATION BY BACTERIAL LACZGENE-TAGGED LEWIS LUNG-CARCINOMA CELLS

Sequential events in micrometastasis formation including entry into th e blood circulation and arrest, extravasation and initial growth in th e lung was investigated using bacterial lacZ gene-tagged Lewis lung ca rcinoma cells (4A1-1). Micrometastases in the lung could thereby be sp ecifically detected at the single cell level by X-Gal staining, After intravenous injection, X-Gal positive tumor cells appeared to extravas ate within hours, but most cells then degenerated or died in the alveo lar space by 2-3 days postinjection. A decreased BrdU labeling index t o a negligible level at 2 days postinjection and reduction of X-Gal po sitive foci to a basal level (less than 0.1% of injected cells) by 4 d ays 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 colla gen immunostaining demonstrated loss of pre-existing basement membrane s with growth of micrometastases. When 4A1-1 cells were inoculated sub cutaneously, lung micrometastases from resulting tumors were detected as single or small numbers of X-Gal positive cells at 2 weeks postinje ction. Progressive development of micrometastasis to macroscopic metas tasis was noted by 4-5 weeks postinjection. The results indicate that micrometastasis formation by Lewis lung carcinoma cells involves a seq uence 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 sub sequent new growth and expansion of persisting tumor cells from 4 days postinjection.