High efficiency of alphaviral gene transfer in combination with 5-fluorouracil in a mouse mammary tumor model

Abstract

BackgroundThe combination of virotherapy and chemotherapy may enable efficient tumor regression that would be unachievable using either therapy alone. In this study, we investigated the efficiency of transgene delivery and the cytotoxic effects of alphaviral vector in combination with 5-fluorouracil (5-FU) in a mouse mammary tumor model (4 T1).MethodsReplication-deficient Semliki Forest virus (SFV) vectors carrying genes encoding fluorescent proteins were used to infect 4 T1 cell cultures treated with different doses of 5-FU. The efficiency of infection was monitored via fluorescence microscopy and quantified by fluorometry. The cytotoxicity of the combined treatment with 5-FU and alphaviral vector was measured using an MTT-based cell viability assay. In vivo experiments were performed in a subcutaneous 4 T1 mouse mammary tumor model with different 5-FU doses and an SFV vector encoding firefly luciferase.ResultsInfection of 4 T1 cells with SFV prior to 5-FU treatment did not produce a synergistic anti-proliferative effect. An alternative treatment strategy, in which 5-FU was used prior to virus infection, strongly inhibited SFV expression. Nevertheless, in vivo experiments showed a significant enhancement in SFV-driven transgene (luciferase) expression upon intratumoral and intraperitoneal vector administration in 4 T1 tumor-bearing mice pretreated with 5-FU: here, we observed a positive correlation between 5-FU dose and the level of luciferase expression.ConclusionsAlthough 5-FU inhibited SFV-mediated transgene expression in 4 T1 cells in vitro, application of the drug in a mouse model revealed a significant enhancement of intratumoral transgene synthesis compared with 5-FU untreated mice. These results may have implications for efficient transgene delivery and the development of potent cancer treatment strategies using alphaviral vectors and 5-FU.

Comparative protein profiling of B16 mouse melanoma cells susceptible and non-susceptible to alphavirus infection: Effect of the tumor microenvironment

“…Several studies have shown that enhanced alphavirus infectivity is mediated by a decrease in the intracellular level of type I IFNs, which is true both in vitro and in vivo. 27,28 Interesting results, which are consistent with the current research, have been shown for Vesicular Stomatitis Indiana virus (VSV). Ovarian and breast cancer cells were highly susceptible to VSV infection in vitro, but the infection of the developed tumors in vivo was inefficient due to activation of the JAK/STAT pathways and overexpression of interferonstimulated genes induced by tumor-infiltrating macrophages.…”

“…For alphaviruses, it is possible to increase the tissue permeability and promote their intratumoral distribution, simultaneously using synergic chemotherapeutic drugs. 27,61 However, the slow kinetics of viral spread after intratumoral injections is a common disadvantage that has also been reported for other vectors, e.g., adenoviruses 62 and retroviruses. 63 The oncolytic properties of alphaviruses make them a promising tool with the potential to significantly improve the field of cancer treatment.…”

“…The pSFV/EGFP, pSFV/Ds-Red and pSFV/Enh.Luc vectors were generated as described previously. 12,27 The resulting plasmids were used to produce SFV/EGFP, SFV/DS-Red and SFV/Enh.Luc virus particles. Briefly, the pSFV/EGFP, pSFV/DS-Red, pSFV/Enh.Luc and pSFV-Helper 93 plasmids were linearized using the SpeI restriction enzyme.…”

SI‐CLP inhibits the growth of mouse mammary adenocarcinoma by preventing recruitment of tumor‐associated macrophages

“…The cell growth medium containing the infectious recSFV particles was then harvested, filtered through 22 μm filters and rapidly frozen in liquid nitrogen, and subsequently used as a source of recSFV particles for cell infection and SI‐CLP containing supernatant production. Infection of BHK‐21 cells was done as previously described . Briefly, 5–6 × 10 6 BHK‐21 cells were cultured in 75 cm 2 flasks and used for infection with pSFVEnh/Luc and pSFVEnh/mSI‐CLP virus particles.…”

“…Infection of BHK-21 cells was done as previously described. 18 Briefly, 5-6 × 10 6 BHK-21 cells were cultured in 75 cm 2 flasks and used for infection with pSFVEnh/Luc and pSFVEnh/mSI-CLP virus particles. For infection, the cells were washed twice with PBS containing Mg 2+ and Ca 2+ (Invitrogen, UK).…”