Development of micrometastases: earliest events detected with bacterial lacZ gene-tagged tumor cells.

For the study of micrometastases at their earliest stages, we transfected the lacZ gene, which codes for beta-D-galactosidase in Escherichia coli, into BALB/c 3T3 cells transformed by the Ha-ras oncogene (also known as HRAS1) of a human EJ bladder carcinoma. These cells were subsequently injected into 6-week-old, female athymic NCR-NU nude mice by several routes. With chromogenic detection of the product of the lacZ gene (a heterologous gene not observed in animal cells) by use of 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside, we easily identified tumor cells implanted in the lungs minutes after intravenous injection by the intensely blue staining of the cells harboring the lacZ gene. The number of lung-associated tumor cells remained constant for several hours after intravenous injection but then decreased to a stable level by 24 hours. At most sites of lung invasion, multiple tumor cells, rather than single cells, were identified; this finding suggests that cooperation among multiple cells may be important in the early stages of micrometastasis development. Within several days, a few foci of micrometastases were expanding by proliferation and/or migration of individual tumor cells among host lung cells. These results confirm that the lacZ gene is an ultrasensitive histochemical marker for analyzing both qualitatively and quantitatively the earliest stages of micrometastasis development in the lung and in other organs where micrometastases may ensue.