SCATTERED MICROMETASTASES VISUALIZED AT THE SINGLE-CELL LEVEL - DETECTION AND RE-ISOLATION OF LACZ-LABELED METASTASIZED LYMPHOMA-CELLS

To study metastasis at the single-cell level we transduced highly meta static ESb lymphoma cells with a retroviral expression vector containi ng the lacZ (bacterial beta-galactosidase) gene. This allowed single E Sb-lacZ tumor cells to be detected in infiltrated target organs by mea ns of X-Gal staining. Despite expression of the lacZ gene, the tumor c ells were still tumorigenic, highly metastatic, unchanged in phenotype and therefore comparable to parental ESb cells. After spontaneous met astasis, 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-o rgan staining, histological examination revealed scattered distributio n of tumor cells throughout the organ, which was not evident with pare ntal ESb cells. In addition, clusters with diffuse or dense (focal) ap pearance were found, in correlation with the whole organ staining. Exp ression of the foreign lacZ gene allowed the metastatic spread of tumo r cells to liver and spleen to be quantified approximately by FACS ana lysis. Furthermore, it was shown that the newly expressed beta-gal was expressed not only intercellularly but also at the cell surface. Ther e it could be recognized by MAbs and cytotoxic T-cells (CTL). beta-gal did not affect CTL recognition of the ESb tumor-associated antigen. I n conclusion, lacZ could be used as a genetic marker for a highly meta static lymphoma, to define scattered metastatic spread in the liver at the single-cell level and to quantify the tumor load by FACS analysis . (C) 1994 Wiley-Liss, Inc.