We have developed multicellular spheroids (MCS) established from LM05e and LM3 spontaneous Balb/c-murine mammary adenocarcinoma and B16 C57-murine melanoma derived cell lines as an in vitro model to study the efficacy of the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide system. We demonstrated for the first time that HSVtk-expressing cells assembled as MCS manifested a GCV resistance phenotype compared to the same cells grown as sparse monolayers. HSVtkexpressing LM05e, LM3 and B16 spheroids were 16-, three-and nine-fold less sensitive to GCV than their respective monolayers, even though they could express transgenes 10-, eight-and five-fold more efficiently. Mixed populations of HSVtkÀ and their respective bgal-expressing cells displayed a cell-type specific bystander effect that was higher in monolayers than in MCS. However, HSVtkÀexpressing cells in two-or three-dimensional cultures were always significantly more sensitive to GCV than the bgal-expressing counterparts, supporting the feasibility of this suicide approach in vivo. We present evidence showing that HSVtkexpressing tumor cells, when transferred from monolayers to MCS, displayed: (i) lower GCV cytotoxic activity and bystander effect; (ii) higher and efficient expression of genes transferred as lipoplexes; (iii) lower cell proliferation rates; and (iv) changes in intracellular Bax/Bcl-xL rheostat of mitochondria-mediated apoptosis. Cancer Gene Therapy ( A ntitumor suicide gene therapy is one of the emerging strategies against cancer. 1 It consists of the introduction into cancer cells of a gene, whose product is capable of converting a nontoxic prodrug into a cytotoxic drug. 2 One of such suicide genes, the thymidine kinase gene from the herpes simplex virus (HSVtk), in combination with the prodrug ganciclovir (GCV), has been extensively and successfully used for the treatment of a variety of cancers in some animal models. HSVtk can efficiently phosphorylate the guanosine analogue GCV and allows its further transformation, after subsequent phosphorylation by cellular kinases, into cytotoxic ganciclovir-triphosphate, which inhibits cellular DNA polymerases. 3 GCV-induced apoptosis is due to incorporation of the drug into DNA resulting in replication-dependent formation of DNA double-strand breaks and, at later stages, S and G 2 /M arrest. 4 As this therapeutic gene cannot be easily introduced into the whole cell population of a tumor, the successful eradication of tumors depends on a phenomenon called the bystander effect, by which the unmodified adjacent tumor cells are also sensitive to the GCV cytotoxic effect. 5,6 This bystander effect permits that the transfection of only a minority of tumor cells may lead to effective tumor regression. 6 Despite extensive preclinical evaluation both in vitro and in vivo in several experimental models, no studies have been undertaken examining a nonviral HSVtk/GCV suicide system in spheroids, a model that mimics the microregions of solid tumors. 7 Multicellular spheroids (MCS) have been us...