To validate the use of multicellular spheroids to predict the efficacy of herpes simplex thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene therapy in the respective in vivo tumors, we established and characterized 15 melanomaderived cell lines from surgically excised melanoma tumors. Three HSVtk-lipofected cell lines were not sensitive to GCV in any culture configuration, other five displayed similar sensitivity as monolayers or spheroids, and only one resulted more sensitive when grown as spheroids. Other six cell lines manifested a relative multicellular resistance (MCR) phenotype growing as spheroids, compared with the same cells growing as monolayers. The reverse correlation between the MCR and the monolayers survival to HSVtk/GCV suggests that one of the main causes of MCR would be the rapid cell repopulation after suicide gene treatment. The high correlation of MCR with the spheroids radial growth and with the mitotic index of the respective originary tumors supported this re-growth involvement. A remarkable finding was the high correlation in HSVtk/GCV sensitivity between in vivo tumor and the corresponding derived cell lines growing as spheroids (R 2 ¼ 0.85). This strongly encourages the implementation of spheroids as highly realistic experimental model for optimizing and predicting the in vivo response of the respective tumors to therapeutic strategies.

We present here a nonviral immunogene therapy trial for canine malignant melanoma, an aggressive disease displaying significant clinical and histopathological overlapping with human melanoma. As a surgery adjuvant approach, it comprised the co-injection of lipoplexes bearing herpes simplex virus thymidine kinase and canine interferon-b genes at the time of surgery, combined with the periodic administration of a subcutaneous genetic vaccine composed of tumor extracts and lipoplexes carrying the genes of human interleukin-2 and human granulocyte-macrophage colony-stimulating factor. Following complete surgery (CS), the combined treatment (CT) significantly raised the portion of local disease-free canine patients from 11% to 83% and distant metastases-free (M0) from 44% to 89%, as compared with surgery-only-treated controls (ST). Even after partial surgery (PS), CT better controlled the systemic disease (M0: 82%) than ST (M0: 48%). Moreover, compared with ST, CT caused a significant 7-fold (CS) and 4-fold (PS) rise of overall survival, and > 17-fold (CS) and > 13-fold (PS) rise of metastasis-free survival. The dramatic increase of PS metastasis-free survival ( > 1321 days) and CS recurrence-and metastasis-free survival (both > 2251 days) demonstrated that CT was shifting a rapidly lethal disease into a chronic one. In conclusion, this surgery adjuvant CT was able of significantly delaying or preventing postsurgical recurrence and distant metastasis, increasing disease-free and overall survival, and maintaining the quality of life. The high number of canine patients involved in CT (301) and the extensive follow-up ( > 6 years) with minimal or absent toxicity warrant the long-term safety and efficacy of this treatment. This successful clinical outcome justifies attempting a similar scheme for human melanoma.

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Interferon-b lipofection II. Mechanisms involved in cell death and bystander effect induced by cationic lipid-mediated interferon-b gene transfer to human tumor cells MS Villaverde, ML Gil-Cardeza, GC Glikin and LME FinocchiaroWe evaluated the cytotoxic effects (apoptosis, necrosis and early senescence) of human interferon-b (hIFNb) gene lipofection. The cytotoxicity of hIFNb gene lipofection resulted equivalent to that of the corresponding addition of the recombinant protein (rhIFNb) on human tumor cell lines derived from Ewing's sarcoma (EW7 and COH) and colon (HT-29) carcinomas. However, it was stronger than rhIFNb on melanoma (M8) and breast adenocarcinoma (MCF7). To reveal the mechanisms involved in these differences, we compared the effects of hIFNb gene and rhIFNb protein on EW7 and M8 (sensitive and resistant to rhIFNb protein, respectively). Lipofection with hIFNb gene caused a mitochondrial potential decrease simultaneous with an increase of oxidative stress in both cell lines. However, rhIFNb protein displayed the same pattern of response only in EW7-sensitive cell line. The great bystander effect of the hIFNb gene lipofection, involving the production of reactive oxygen species, would be among the main causes of its success. In EW7, this effect killed 460% of EW7 cell population, even though only 1% of cells were expressing the transgene. As hIFNb gene was effective even in the rhIFNb protein-resistant M8 cell line and in a way not limited by low lipofection efficiency, these results strongly support the clinical potential of this approach.

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