Herpes simplex virus thymidine kinase/ganciclovir system in multicellular tumor spheroids

Abstract

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...

Suicide gene therapy on spontaneous canine melanoma: correlations between in vivo tumors and their derived multicell spheroids in vitro

“…5,10,19 The cytotoxicity of increasing concentrations (0.01-1000 mg ml À1 ) of the pro-drug GCV was tested on monolayers and Sphs of transiently bgal-or HSVtk-expressing melanoma cells. As shown in Figure 3, sparse growing monolayers derived from different tumors showed very different sensitivities to the HSVtk/GCV system.…”

“…10,15 Then, it could be inferred that the inverse correlation between Sphs MCR and monolayers survival would be due to a faster repopulation, through recruitment of the surviving quiescent cells to the proliferative fraction after suicide gene treatment. 10,15,20 If this were the case, the MCRi should correlate with the rate of Sph radial growth. This was, in fact, observed (as shown in Figure 7a), because the daily radial increase of Sphs during the linear phase of growth was proportional to that of the MCRi, following a linear tendency (R 2 ¼ 0.98).…”

“…These three-dimensional assemblies have been used as experimental models to better reflect the cellular environment that is found in vivo. [5][6][7][8][9][10] Being highly complex systems, their cellular properties are dependent on the origin of the tumor cells, their transformation state, medium and growth conditions. They offer a versatile in vitro system of intermediate complexity between monolayer cultures in vitro and tumors in vivo.…”

“…[5][6][7][8][9][10] Sphs combine the relevance of organized tissues with the controlled environment of in vitro methodology. [5][6][7][8][9][10] These cell aggregates organized in vitro have a great potential for a number of clinical and biomedical applications. In brief, they represent a more realistic experimental model for optimizing and predicting the efficacy of cancer therapies in the respective in vivo tumors.…”

“…In brief, they represent a more realistic experimental model for optimizing and predicting the efficacy of cancer therapies in the respective in vivo tumors. [5][6][7][8][9][10] Most of the cancer gene therapy studies carried out on animal models use established tumor cell lines that were kept in culture for many generations, making them very different from the original tumors. We here isolated and characterized 15 canine melanoma cell lines derived from surgically excised melanoma tumors, in order to evaluate potential in vivo responses of individual spontaneous canine melanomas to suicide gene therapy.…”