ESTABLISHMENT OF MICROMETASTATIC CARCINOMA CELL-LINES - A NOVEL SOURCE OF TUMOR-CELL VACCINES

Background: Cancer cells of microscopic metastases can be envisaged as ideal constituents for the development of a genetically modified, aut ologous tumor cell vaccine. However, their extremely low number has th us far blocked this approach. Purpose: The aim of this study was to cu lture micrometastatic tumor cells present in bone marrow of patients w ith various forms of epithelial cancer and to thereby establish immort alized cell lines. Methods: Bone marrow aspirates from the upper iliac crest of 152 patients with cancer of the prostate, kidney, lung, brea st, or colorectum were cultured at 1 x 10(7) to 6 x 10(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 ext racellular matrix-coated plates was monitored by sampling and staining aliquots with cytokeratin-specific antibodies. After 3-6 weeks in cul ture, the cells were transferred to Petri dishes, and 200-300 epitheli al 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 protei n, or intestinal-specific annexin; their bone marrow-seeking potential was tested in immunodeficient SCID (i.e., severe combined immunodefic iency) 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(5) MNC. In 68 cases (44.7%), the established culture conditions allowed a two to four log transient ex pansion of these cells with rather small differences among the tumor t ypes studied. Epidermal growth factor and basic fibroblast growth fact or were found to be essential for this culture system. After microinje ction of the propagated cells with T-antigen DNA, permanent cell lines were obtained; some of these cell lines (prostate and lung cancer cel l lines) are now beyond culture passage 80. The cells showed no notabl e changes in the pattern of expressed epithelial antigens and were abl e to disseminate into bone marrow in SCID mice. Conclusions: This proc edure allows the selective immortalization of micrometastatic carcinom a cells. Integration of SV40 DNA and expression of T antigen did not s ubstantially 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 usef ul source for the generation of autologous tumor cell vaccines.