Abstract
To study metastasis at the single-cell level we transduced highly metastatic ESb lymphoma cells with a retroviral expression vector containing the lacZ (bacterial beta-galactosidase) gene. This allowed single ESb-lacZ tumor cells to be detected in infiltrated target organs by means of X-Gal staining. Despite expression of the lacZ gene, the tumor cells were still tumorigenic, highly metastatic, unchanged in phenotype and therefore comparable to parental ESb cells. After spontaneous metastasis, whole-organ staining revealed metastatic foci at the surface of the liver. In histological liver sections, metastatic clusters and single dispersed tumor cells could be detected. In contrast to whole-organ staining, histological examination revealed scattered distribution of tumor cells throughout the organ, which was not evident with parental ESb cells. In addition, clusters with diffuse or dense (focal) appearance were found, in correlation with the whole-organ staining. Expression of the foreign lacZ gene allowed the metastatic spread of tumor cells to liver and spleen to be quantified approximately by FACS analysis. Furthermore, it was shown that the newly expressed beta-gal was expressed not only intercellularly but also at the cell surface. There it could be recognized by MAbs and cytotoxic T-cells (CTL). beta-gal did not affect CTL recognition of the ESb tumor-associated antigen. In conclusion, lacZ could be used as a genetic marker for a highly metastatic lymphoma, to define scattered metastatic spread in the liver at the single-cell level and to quantify the tumor load by FACS analysis.
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