During tumor progression, micrometastases at their earliest stages have been difficult to analyze qualitatively or quantitatively because of a lack of suitably sensitive markers to discriminate small numbers of tumor cells from normal tissue cell populations. To overcome this problem, the Escherichia coli β-galactosidase (lacZ) gene was introduced into human EJ Ha-ras oncogene-transfected BALB/c 3T3 cells with subsequent injection of transfected cells into athymic nude mice. Using a chromogenic substrate (5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside), the lacZ-bearing tumor cells at primary tumor sites as well as at secondary organs stain intensely blue and can be easily distinguished from the host tissue cells hours, days, or weeks postinjection. Staining of lacZ-bearing tumor cells is specific and extremely sensitive in detecting micrometastatic foci in lungs and other organs, including brain and kidney for the first time. Stable integration of the lacZ and ras genes into cultured cells and subsequent tumor cells was verified by Southern blot analyses. The lacZ gene appears to be a stable marker during tumor progression in vivo based both on phenotypic (5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside staining) and on genotypic (Southern blot analysis) evidence. Furthermore, 5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside staining of tumor cells can also be used together with alkaline phosphatase staining relatively specific for endothelial cells to relate the topographies of metastatic cells and host blood vessels in embedded sections. By using the lacZ gene as a sensitive quantitative marker, analyses of micrometastasis development in the lung indicate that the ras oncogene contributes to the metastatic phenotype in this EJ Ha-ras model system, although further genetic and/or phenotypic alterations appear to be necessary for long-term growth and development into overt metastases. These findings demonstrate the effectiveness and sensitivity of the bacterial lacZ gene as a phenotypic marker in tumor progression studies, providing both a qualitative and a quantitative tool in virtually any tumor system for examining micrometastasis formation in target organs and the relationship of tumor cells to host organ microenvironments.
This work is supported by NIH Research Grants R01-CA27755 (L. A. C.) and R01-CA48032 (T. P. P.). Case Western Reserve University is a comprehensive cancer center supported by USPHS Grant P30CA43703, awarded by the National Cancer Institute, Department of Health and Human Services.