Growth characteristics in the initial stage of micrometastasis formation by bacterial LacZ gene-tagged rat prostatic adenocarcinoma cells

A micrometastasis model was established using a rat differentiated prostatic adenocarcinoma, designated PLS301Z, transfected with the lacZ gene encoding a bacterial beta-galactosidase. The morphology, tumorigenicity and metastatic ability of PLS301Z were comparable to those of the parental cells. Micrometastatic foci could be specifically detected at the single cell level after X-Gal staining with a dissecting microscope. After intravenous injection, the number of X-Gal positive foci in the lung decreased progressively to a steady-state level (less than 1% of injected cells) by 4-7 days, while the size of persisting positive foci started to increase from 4 days after inoculation, as demonstrated by image analysis. X-Gal and BrdU double staining revealed that BrdU labeling indices of X-Gal-positive cells decreased transiently at the 2-day time point and increased again from 4 days after inoculation. Type IV collagen immunostaining showed the tumor cells to be surrounded by a basement membrane intravascularly at the time point when they started new growth. Electron microscopy confirmed that, 2 days post injection, most tumor cells were degenerative or dead, but on day 4, persisting tumor cells formed multicellular clumps in contact with the vascular basement membrane inside vessels. These results indicate that PLS301Z cells begin to grow intravascularly depending upon the presence of a basement membrane before extravasation at the initial stage of micrometastasis formation.