Gene Therapy and Molecular Biology Vol 10, page 207 Gene Ther Mol Biol Vol 10, 207-222, 2006
Effects of spatial configuration on tumor cells transgene expression Research Article
Cecilia C. Casais, Armando L. Karara, Gerardo C. Glikin, and Liliana M. E. Finocchiaro* Unidad de Transferencia Genética, Instituto de Oncología "Ángel H. Roffo", Universidad de Buenos Aires, Argentina
__________________________________________________________________________________ *Correspondence: Liliana M. E. Finocchiaro, Ph.D, Unidad de Transferencia Genética, Instituto de Oncología "A. H. Roffo" UBA, Av. San Martín 5481, 1417 Buenos Aires, Argentina; Telephone/FAX: 54 (11) 4580-2813; Email: gglikin@bg.fcen.uba.ar Key words: multicellular tumor spheroids, persistent gene expression, non-viral vectors Abbreviations: !-galactosidase, (!gal); analysis of variance, (ANOVA); cytomegalovirus immediate early promoter, (CMVie); LM05e spheroids, (LM05e/S); monolayers, (/M); simian virus 40 early promoter/enhancer, (SV40e); spheroids, (/S); three-dimensional, (3D)
Received: 10 April 2006; Revised: 26 June 2006 Accepted: 10 July 2006 electronically published: August 2006
Summary We investigated the impact of the multicellular architecture on transgene expression of LM05e and LM3 spontaneous Balb/c-mammary adenocarcinoma and HEp-2 human laryngeal squamous carcinoma cell lines. When transferred from monolayers to spheroids, tumor cells strongly enhanced transient transgene expression, which surprisingly was still detectable 75 days after lipofection. The cytomegalovirus immediate early promoter (CMVie) yielded a very high !-galactosidase (!gal) transgene expression, which resulted 8-, 6- and 3-fold greater in LM05e, LM3 and HEp-2 spheroids than the corresponding monolayers. The SV40 early promoter displayed both, a lower spheroids/monolayers ratio and about 10% of !gal expression driven by CMVie. Cis-addition of Epstein Barr virus EBNA-1/oriP cassette enhanced the CMVie-driven transgene expression only in human HEp-2. Deletion of a 325 bp 5’ fragment of the CMVie promoter dropped spheroids !gal expression to 5%. This effect was restored to 10-25% or 25-60% by the insertion of one KCS (18 bp) or four myc-max consensus sequences (67 bp) respectively. When spheroids disassembled and grew as monolayers, !gal activity dropped accordingly. Our results demonstrated that the spatial configuration determined the expression enhancement and persistence in spheroids, being an event: fully reversible, proportional to spheroid compactness and independent of plasmid integration into the host genome.
intermediate complexity between monolayer cultures in vitro and tumors in vivo. In brief, spheroids combine the relevance of organized tissues with the controlled environment of in vitro methodology (Mueller-Klieser, 1997; Bates et al, 2000). Furthermore, they mirror the radius and chemosensitivity of differentiating tumors in vivo more closely than conventional cell cultures (Olive and Durand, 1994; Kolchinsky and Roninson 1997; Fehlauer et al, 2004). Being highly complex systems, their cellular properties are dependent on the origin of the tumor cells, their transformation state, and medium and growth conditions. Non-viral vectors such as cationic lipids have important safety advantages over viral approaches, including their reduced immunogenicity, low cytotoxicity and minimal capacity for insertional mutagenesis (Glover et al, 2005). Although the efficacy of new cationic lipids
I. Introduction Multicellular spheroids are tissue-like structures of cells, with no artificial substrate for cell attachment (Mueller-Klieser, 1997). These cell aggregates organized in vitro have a great potential for a number of clinical and biomedical applications (Sutherland, 1998; Santini and Rainaldi, 1999). This three-dimensional (3D) cell system has been widely used as a model for microenvironmental effects on basic biological mechanisms, such as the regulation of proliferation, metabolism, differentiation, cell death, invasion, angiogenesis or immune response (Bates et al, 2000; Fehlauer et al, 2004). Compared to conventional monolayer cultures, 3D-cell aggregates resemble more closely the in vivo situation with regard to cell shape and cell environment, which in turn can affect gene expression and biological behavior of the cells. These 3D-structures offer a versatile in vitro system of 207