Volume 42, Issue 5, Pages 728-735 (May 2005) Systemic efficacy of combined suicide/cytokine gene therapy in a murine model of hepatocellular carcinoma  Anna-Lisa Stefani, Luisa Barzon, Ignazio Castagliuolo, Maria Guido, Monia Pacenti, Cristina Parolin, Fabio Farinati, Giorgio Palù  Journal of Hepatology  Volume 42, Issue 5, Pages 728-735 (May 2005) DOI: 10.1016/j.jhep.2004.12.037 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Expression of IL-2 and HSV-TK transcripts in human liver cancer cells (HepG2, Hep3B and PLC/PRF/5) transduced with LIL-2TKSN or MFGIL-2TKSN vectors. Random primed cDNA from total RNA was used for real-time quantitative RT-PCR analysis using SYBR green reagents. Expression of the target genes was normalized to the endogenous control β-actin mRNA, as quantified by real-time RT-PCR analysis using TaqMan β-actin RNA Control Reagent Kit (Applied Biosystem). Data are representative of three separate experiments performed in triplicate. Journal of Hepatology 2005 42, 728-735DOI: (10.1016/j.jhep.2004.12.037) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 In vitro cytotoxicity of GCV in parental cells and cells transduced with LIL-2TKSN or MFGIL-2TKSN vectors. Cells were incubated with various concentrations of GCV for 5 days, followed by cell survival quantitation by XTT assay. IC50 was calculated as the concentration of drug which inhibits cell growth by 50%. Data are representative of at least three separate experiments performed in sestuplicate; each point represents the mean ± SE and is expressed as percentage relative to drug-free cells. Journal of Hepatology 2005 42, 728-735DOI: (10.1016/j.jhep.2004.12.037) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Analysis of apoptosis with Cell Death Detection ELISA Plus kit in GCV treated cells. Results are expressed as fold increments of presence of cytoplasmatic histone-associated DNA fragments with respect to the same cells non GCV treated. Journal of Hepatology 2005 42, 728-735DOI: (10.1016/j.jhep.2004.12.037) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Transduced and non-transduced cells were mixed in different proportions and treated with GCV 1 to 100μM. On day 5, XTT assay was performed to evaluate cell survival. Data are representative of at least three separate experiments performed in triplicate. Survival ratios were expressed as percentages relative to corresponding mixture of untreated control cells. Journal of Hepatology 2005 42, 728-735DOI: (10.1016/j.jhep.2004.12.037) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 In vivo cytotoxic effect of GCV in tumors obtained by sc injection of parental or transduced syngenic cells in balb/c mice. A total of 107 cells was injected in both flanks in balb/c mice. After 10 days, mice were treated by daily i.p. injection of PBS or 100mg/kg GCV for 6 days. Tumor volumes (V) were calculated by the formula of rotational ellipsoid: V=A×B2/2, where A is the longer diameter, and B is the smaller diameter. Each point is the mean (n=6–8) ± SE tumor volume after 6 days-treatment with PBS or GCV and is expressed as a percentage relative to baseline tumor size. Third column of each group (PBS or GCV) represent the mean volume of non transduced tumor inoculated in the contralateral flank of same mice with respect to transduced tumor, after 6 days of PBS or GCV treatment. Journal of Hepatology 2005 42, 728-735DOI: (10.1016/j.jhep.2004.12.037) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Histopathological analysis of murine HCC tumor specimens obtained by s.c. inoculation of cells in syngenic balb/c mice. Tissue specimens were fixed in buffered 4% formalin for 24h and paraffin embedded after dehydration. 5μm tissue slides were cut and stained with haematoxylin and eosin following routine methods. The figures show representative pictures of: (a) non-transduced tumor of mice not receiving GCV; (b) MFGIL-2TKSN-transduced tumor of mice not receiving GCV, with presence of moderate inflammatory cells infiltration (arrows); (c) non transduced tumor of GCV-treated mice; (d) transduced tumor of GCV-treated mice with significant infiltration of inflammatory cells, necrotic areas (e) and presence of several apoptotic bodies (f). Journal of Hepatology 2005 42, 728-735DOI: (10.1016/j.jhep.2004.12.037) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Immunofluorescence of in vivo MFG-ΔLNGFR-transduced tumors. Immunofluorescence was carried out on 5μm frozen section, PFA 4% fixed, stained with biotynilated antiNGFR MoAb and with streptavidn-PE. Sections were examined at confocal Leyca TCS-NT/SP2 microscopy. (a) tumor inoculated with non-virus producing conditioned medium (negative contol); (b) and (c) tumor inoculated with repeated injection of MFGΔLNGFR retroviral vector (magnification, 40× and 63× respectively). The Figure shows representative pictures. Journal of Hepatology 2005 42, 728-735DOI: (10.1016/j.jhep.2004.12.037) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 8 Antitumor effect on estabilished HCC tumors induced by repeated injections of MFGIL-2TKSN retroviruses (second column). First column represent mean volume of tumor inoculated with no-virus containing conditioned medium. Tumor volume was estimated after 6 days of GCV treatment. Each column represent the mean ± SE and is expressed as a percentage relative to baseline tumor size. Journal of Hepatology 2005 42, 728-735DOI: (10.1016/j.jhep.2004.12.037) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions