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Background: Cancer cells of microscopic metastases can be envisaged as ideal constituents for the development of a genetically modified, autologous tumor cell vaccine. However, their extremely low number has thus far blocked this approach. Purpose: The aim of this study was to culture micrometastatic tumor cells present in bone marrow of patients with various forms of epithelial cancer and to thereby establish immortalized cell lines. Methods: Bone marrow aspirates from the upper iliac crest of 152 patients with cancer of the prostate, kidney, lung, breast, or colorectum were cultured at 1 x [10.sup.7] to 6 x [10.sup.7] mononuclear cells (MNC) per flask in fetal calf serum-containing RPMI-1640 medium supplemented with 10 ng/mL epidermal growth factor and 10 ng/mL basic fibroblast growth factor. The proliferation of epithelial cells on extracellular matrix-coated plates was monitored by sampling and staining aliquots with cytokeratin-specific antibodies. After 3-6 weeks in culture, the cells were transferred to Petri dishes, and 200-300 epithelial cells per plate were microinjected with DNA encoding for the simian virus 40 (SV40) large T antigen. Cells were screened at various time points for expression of large T antigen and epithelial markers, such as cytokeratins, prostate-specific antigen, prolactin-inducible protein, or intestinal-specific annexin; their bone marrow-seeking potential was tested in immunodeficient SCID (i.e., severe combined immunodeficiency) mice given subcutaneous transplants of the immortalized cells. Results: Prior to culture, more than 90% of all samples presented with fewer than 10 tumor cells per 8 x [10.sup.5] MNC. In 68 cases (44.7%), the established culture conditions allowed a two to four log transient expansion of these cells with rather small differences among the tumor types studied. Epidermal growth factor and basic fibroblast growth factor were found to be essential for this culture system. After microinjection of the propagated cells with T-antigen DNA, permanent cell lines were obtained; some of these cell lines (prostate and lung cancer cell lines) are now beyond culture passage 80. The cells showed no notable changes in the pattern of expressed epithelial antigens and were able to disseminate into bone marrow in SCID mice. Conclusions: This procedure allows the selective immortalization of micrometastatic carcinoma cells. Integration of SV40 DNA and expression of T antigen did not substantially change the epithelial phenotype of the propagated cells. Implications: The established system will allow an in-depth molecular analysis of human micrometastatic cancer cells and could become a useful source for the generation of autologous tumor cell vaccines.