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. HSVtk-expressing 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 HSVtk− and their respective βgal-expressing cells displayed a cell-type specific bystander effect that was higher in monolayers than in MCS. However, HSVtk−expressing cells in two- or three-dimensional cultures were always significantly more sensitive to GCV than the βgal-expressing counterparts, supporting the feasibility of this suicide approach in vivo. We present evidence showing that HSVtk-expressing 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.
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We thank Gabriela Sobrido and Ana Bihary for technical assistance, Fernanda Roca for histological preparations, and Dr S Klein and Dr M Diament for mice adenocarcinomas. This work was partially supported by a grant from FONCYT: BID802/OC-AR — PICT 05-00037-00709, CABBIO and a grant from BioSidus SA. LMEF, ALK and GCG are members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina). VFB and CCC are fellows of CONICET and GLF is a fellow of BioSidus SA.
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Finocchiaro, L., Bumaschny, V., Karara, A. et al. Herpes simplex virus thymidine kinase/ganciclovir system in multicellular tumor spheroids. Cancer Gene Ther 11, 333–345 (2004). https://doi.org/10.1038/sj.cgt.7700682
- suicide gene therapy
- murine adenocarcinoma
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