title: Transfection of primary tumor cells and tumor cell lines with plasmid DNA/lipid complexes.
abstract: Cancer vaccines that utilize genetically modified tumor cells require gene transfer methods capable of producing immunostimulatory doses of transgenes from fresh or short-term cultures of human tumor cells. Our studies optimize in vitro transfection of primary tumor cells using cationic lipids and a plasmid encoding the gene for human interleukin-2 (IL-2). Established tumor cell lines produced 10- to 100-fold more IL-2 than did fresh or short-term tumor cultures as measured by enzyme-linked immunoabsorbent analysis. Importantly, transfection of primary tumor cells produced immunostimulatory levels of IL-2 as determined by increased thymidine incorporation by autologous peripheral blood mononuclear cells and lymphokine-activated killer cell activity. IL-2 secretion by tumor cells persisted for at least 30 days post-transfection and was unaffected by freeze thawing or irradiation to 8000 rads. Multiple solid tumor types were successfully transfected, but normal blood mononuclear cells and leukemic blasts were resistant to transfection. Enzyme-linked immunoabsorbent analysis of the amount of IL-2 secreted into the medium by transfected tumor cells correlated with the percentage of tumor cells expressing intracellular IL-2 as measured by flow cytometry. Plasmids utilizing a cytomegalovirus promoter yielded superior transfection efficiencies compared with plasmids containing a Rous sarcoma virus promoter. These results suggest that a clinical vaccine trial using autologous tumor cells genetically modified to secrete IL-2 is feasible in patients with solid tumors.
authors.affiliation: Arizona Cancer Center, Tucson 85724-5024, USA.
authors.forename: A T
authors.initials: AT
authors.lastname: Stopeck
authors.forename: E M
authors.initials: EM
authors.lastname: Hersh
authors.forename: J L
authors.initials: JL
authors.lastname: Brailey
authors.forename: P R
authors.initials: PR
authors.lastname: Clark
authors.forename: J
authors.initials: J
authors.lastname: Norman
authors.forename: S E
authors.initials: SE
authors.lastname: Parker
mesh_terms: D001358:Avian Sarcoma Viruses / Q000235:genetics; D019496:Cancer Vaccines / Q000235:genetics*; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000008:administration & dosage*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology; D008055:Lipids / Q000008:administration & dosage*; D009369:Neoplasms / Q000276:immunology* / Q000473:pathology / Q000628:therapy; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D019496:Cancer Vaccines; D007376:Interleukin-2; D008055:Lipids; D004247:DNA; D001616:beta-Galactosidase