Assessing and Overcoming Resistance Phenomena against a Genetically Modified Vaccinia Virus in Selected Cancer Cell Lines

Abstract

Genetically modified vaccinia viruses (VACVs) have been shown to possess profound oncolytic capabilities. However, tumor cell resistance to VACVs may endanger broad clinical success. Using cell mass assays, viral replication studies, and fluorescence microscopy, we investigated primary resistance phenomena of cell lines of the NCI-60 tumor cell panel to GLV-1h94, a derivative of the Lister strain of VACV, which encodes the enzyme super cytosine deaminase (SCD) that converts the prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic compound 5-fluorouracil (5-FU). After treatment with GLV-1h94 alone, only half of the cell lines were defined as highly susceptible to GLV-1h94-induced oncolysis. When adding 5-FC, 85% of the cell lines became highly susceptible to combinatorial treatment; none of the tested tumor cell lines exhibited a "high-grade resistance" pattern. Detailed investigation of the SCD prodrug system suggested that the cytotoxic effect of converted 5-FU is directed either against the cells or against the virus particles, depending on the balance between cell line-specific susceptibility to GLV-1h94-induced oncolysis and 5-FU sensitivity. The data provided by this work underline that cellular resistance against VACV-based virotherapy can be overcome by virus-encoded prodrug systems. Phase I/II clinical trials are recommended to further elucidate the enormous potential of this combination therapy.

Keywords: 5-fluorocytosine; NCI-60 tumor cell panel; chemovirotherapy; oncolytic virotherapy; super cytosine deaminase prodrug system; vaccinia virus; virotherapy resistance.

Figure 1

Tumor cell lines of the NCI-60 panel (n = 54) infected with the virotherapeutic compound GLV-1h94 without (A) and with (B) addition of the prodrug 5-fluorocytosine (5-FC). (a,b) At 96 h post infection, tumor cell masses were analyzed via sulforhodamine B (SRB) assay. Bars in red indicate a remnant tumor cell mass of more than 75% (in comparison to mock-infected cells) and thereby define a “high-grade resistance” to GLV-1h94-mediated oncolysis; bars in orange denote remaining tumor cell masses in the range of 50–75%, defining a “partial permissiveness” to oncolysis by GLV-1h94; bars in green specify a remaining tumor cell mass of less than 50%, categorized as “high-grade permissiveness” to GLV-1h94-mediated oncolysis. Mean ± SD of three independent experiments are shown. Thresholds (100%, 75%, 50%) are indicated by dotted lines. (c) Overview of the 5-FC conversion system (employing enzymes YCD + YUPRT). dUMP: deoxyuridine monophosphate; TS: thymidylate synthase; dTMP: deoxythymidine mono-phosphate; dTTP: deoxythymidine triphosphate; 5-FdUMP: 5-fluorodeoxyuridine mono-phosphate; 5-FUMP: 5-fluorouridine monophosphate; 5-FUTP: 5-fluorouridine triphosphate; 5-FC: 5-fluorocytosine; YCD: yeast cytosine deaminase; 5-FU: 5-fluorouracil; UDK: uridine kinase; UDP: uridine phosphorylase; YUPRT: yeast uracil phosphoribosyltransferase; DPD: dihydro-pyrimidine dehydrogenase; FbAL: 5-fluoro-ß-alanine. Scheme modified from [15].

Figure 2

5-fluorouracil (5-FU) sensitivity of two “high-grade resistant” ((a); NCI-H460, HCT-15) and two “high-grade permissive” ((b); OVCAR-8, DU-145) tumor cell lines. All tumor cell lines were treated with different concentrations of 5-FU (10⁻⁴, 10⁻³, 10⁻², 10⁻¹, 1 mM) and cell masses were analyzed at 24, 48, 72, and 96 h via SRB assay. Mean ± SD of two independent experiments are shown. Red boxes indicate relevant differences in responsiveness to 5-FU between all cell lines at a concentration of 10⁻³ mM 5-FU at which the greatest difference between the tested cell lines with respect to tumor cell masses was observed.

Figure 3

Replication of GLV-1h94 in two “high-grade resistant” ((a); NCI-H460, HCT-15) and in two “high-grade permissive” ((b); OVCAR-8, DU-145) tumor cell lines alone and after the addition of the prodrug 5-fluorocytosine (5-FC). (a,b) All four tumor cell lines were infected with GLV-1h94 at MOI 0.1. At 3 hpi, 5-FC [0.1 mM] was added and replication was analyzed via plaque assay at 3, 24, 48, 72, and 96 hpi. (c) Remaining cell masses of NCI-H460, HCT-15, OVCAR-8, and DU-145 tumor cells measured via SRB assay at 96 hpi with GLV-1h94 (MOI 0.1). Mean ± SD of two independent experiments are shown. Dashed lines indicate a virus concentration of 10⁶ PFU/mL to better illustrate differences between the analyzed cell lines. PFU: plaque-forming units; MOI: multiplicity of infection; hpi: hours post infection with GLV-1h94.

Figure 4

Metabolic conversion of 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) after infection of a “high-grade resistant” ((a); NCI-H460) and a “high-grade permissive” ((b); OVCAR-8) tumor cell line. Both tumor cell lines were infected with GLV-1h94 at MOI 0.01. At 3 hpi, 5-FC [0.1 mM] was added and supernatants were collected at 3, 24, 48, 72, and 96 hpi. Concentrations of 5-FC and 5-FU were determined by mass spectrometry. MOI: multiplicity of infection; hpi: hours post infection with GLV-1h94.

Figure 5

Fluorescence images of the “high-grade resistant” tumor cell line NCI-H460 (a) and the “high-grade permissive” tumor cell line OVCAR-8 (b) infected with GLV-1h94 ± prodrug 5-fluorocytosine (5-FC). Both tumor cell lines were infected with GLV-1h94 at MOI 0.1. At 3 hpi, 5-FC [0.1 mM] was added and brightfield (left panels) as well as fluorescence images (right panels) were taken at 24, 48, 72, and 96 hpi. Images were taken with a Leica DMi8 microscope equipped with a DMC 4500 camera, original magnification 50×.

Figure 6

Fluorescence images of the “high-grade resistant” tumor cell line NCI-H460 (a) and the “high-grade permissive” tumor cell line OVCAR-8 (b) infected with GLV-1h94 ± prodrug 5-fluorocytosine (5-FC). Both tumor cell lines were infected with GLV-1h94 at MOI 0.1. At 3 hpi, 5-FC [0.1 mM] was added and brightfield (left panels) as well as fluorescence images (right panels) were taken at 96 hpi. Images were taken with a Leica DMi8 microscope equipped with a DMC 4500 camera, original magnification 50×.

Figure 7

Fluorescence images of the “high-grade resistant” tumor cell line HCT-15 (a) and the “high-grade permissive” tumor cell line DU-145 (b) infected with GLV-1h94 ± prodrug 5-fluorocytosine (5-FC). Both tumor cell lines were infected with GLV-1h94 at MOI 0.1. At 3 hpi, 5-FC [0.1 mM] was added and brightfield (left panels) as well as fluorescence images (right panels) were taken at 24, 48, 72, and 96 hpi. Images were taken with an Olympus IX50 microscope equipped with an F-view Soft Imaging System camera, original magnification 40×.

Figure 8

Fluorescence images of the “high-grade resistant” tumor cell line HCT-15 (a) and the “high-grade permissive” tumor cell line DU-145 (b) infected with GLV-1h94 ± prodrug 5-fluorocytosine (5-FC). Both tumor cell lines were infected with GLV-1h94 at MOI 0.1. At 3 hpi, 5-FC [0.1 mM] was added and brightfield (left panels) as well as fluorescence images (right panels) were taken at 96 hpi. Images were taken with an Olympus IX50 microscope equipped with an F-view Soft Imaging System camera, original magnification 40×.

Figure 9

Vaccinia virus constructs GLV-1h68 (a) and GLV-1h94 (b). F14.5L encodes a short polypeptide of 49 amino acids, which was found to be highly conserved among different vaccinia strains, as well as other poxviruses; J2R, gene locus for thymidine kinase; A56R, gene locus for hemagglutinin; P_SEL, vaccinia synthetic early/late promoter; P_7.5, vaccinia early late promoter; P₁₁, vaccinia late promoter; ruc-GFP, Renilla luciferase and Aequorea green fluorescent protein; rtfr, genes for human transferrin receptor; lacZ, ß-galactosidase; gusA, ß-glucuronidase; fcu1, yeast-originated transgene-expressing cytosine deaminase and uracil phosphoribosyltransferase.