Preclinical Evaluation of the Oncolytic Vaccinia Virus TG6002 by Translational Research on Canine Breast Cancer

Abstract

Oncolytic virotherapy is a promising therapeutic approach for the treatment of cancer. TG6002 is a recombinant oncolytic vaccinia virus deleted in the thymidine kinase and ribonucleotide reductase genes and armed with the suicide gene FCU1, which encodes a bifunctional chimeric protein that efficiently catalyzes the direct conversion of the nontoxic 5-fluorocytosine into the toxic metabolite 5-fluorouracil. In translational research, canine tumors and especially mammary cancers are relevant surrogates for human cancers and can be used as preclinical models. Here, we report that TG6002 is able to replicate in canine tumor cell lines and is oncolytic in such cells cultured in 2D or 3D as well as canine mammary tumor explants. Furthermore, intratumoral injections of TG6002 lead to inhibition of the proliferation of canine tumor cells grafted into mice. 5-fluorocytosine treatment of mice significantly improves the anti-tumoral activity of TG6002 infection, a finding that can be correlated with its conversion into 5-fluorouracil within infected fresh canine tumor biopsies. In conclusion, our study suggests that TG6002 associated with 5-fluorocytosine is a promising therapy for human and canine cancers.

Keywords: 5-fluorouracil; FCU1; TG6002; dog; mammary tumor; oncolytic virotherapy; suicide gene therapy; translational research; vaccinia virus.

Graphical abstract

Figure 1

Characterization of In Vitro Infection, Replication, and Oncolytic Activity of the Double-Deleted VACV on Canine Cancer Cells Using 2D Monolayer Cultures (A) Amplification factor of VVTG17990 in two canine tumor cell lines (A72 and REM134) and two human tumor cell lines (HeLa and MDA-MB-231) infected at MOIs of 10⁻⁵ and 10⁻⁴ and collected 4 days post-infection. The results are presented as a mean of triplicate experiments ± SD. (B) Susceptibility of REM134 canine tumor cell line to VACV infection. REM134 tumor cells were infected at the indicated MOI with VVTG17990, and the percentage of GFP-positive cells was determined by cytometry at 24 h post-infection. The results were obtained from three separate experiments ± SD. (C) Viral replication kinetics of TG6002 on canine tumor cells. Replication capacity was analyzed through viral growth curve by recovering infected REM134 monolayers (300 PFUs per well) at different time points. The titers of progeny virus were determined by serial dilution titration on Vero cells. The results are presented as a mean of triplicate experiments ± SD. (D) Oncolytic effect of the double-deleted VACV on REM134 canine tumor cells. Cells were infected at MOIs of 10⁻⁵, 10⁻⁴, 10⁻³, or 10⁻² with TG6002, and cell viability was measured 5 days later by trypan blue exclusion. The results are presented as a mean of triplicate experiments ± SD. (E) Combination of oncolytic and prodrug cytotoxicity. REM134 canine tumor cells were infected at a MOI of 10⁻⁴ with TG6002. After 48 h, 5-FC was added in a range of concentrations, and cell survival was determined 3 days later by trypan blue exclusion. Results were standardized against values for wells lacking virus and drug, which represented 100% viability. Values are represented as means ± SD of three individual determinations.

Figure 2

Characterization of Replication and Oncolytic Activity of TG6002 on a 3D In Vitro REM134 Model (A) Viral replication kinetics of TG6002 on REM134 spheroids. Replication capacity was analyzed through viral growth curve by recovering infected REM134 spheroids (100 PFUs per well) at different time points. The titers of progeny virus were determined by serial dilution titration on Vero cells. The results are presented as a mean of triplicate experiments ± SD. (B) Combination of oncolytic and prodrug cytotoxicity on REM134 spheroids. Spheroids were infected with the indicated doses of TG6002. After 48 h, 5-FC was added at 1 mM, and cell survival was determined 8 days later using the CellTiter-Blue Cell Viability Assay. Results were standardized against values for wells lacking virus and drug, which represented 100% viability. The asterisks indicate a significant difference (p < 0.05) between groups (Student’s t test). Values are represented as means ± SD of three individual determinations.

Figure 3

In Vivo Anti-tumor Activity of VACV on a REM134 Canine Tumor Xenograft Model (A) In vivo detection of VACV and apoptosis in the REM134 xenograft tumors. Untreated mice (1, 2, and 3) and mice treated intratumorally (10⁶ PFUs; 4, 5, and 6) or intravenously (10⁷ PFUs; 7, 8, and 9) with VVTG17990 were sacrificed at day 14 post-infection, and macroscopic tumors were removed, formalin fixed, and analyzed by immunohistochemistry. Cellular DNA was stained in blue with DAPI (1–9), GFP was stained in green (1, 4, and 7), CD31 was stained in red (1, 4, and 7), VACV was stained in purple (2, 5, and 8), and activated caspase-3 was stained in orange (3, 6, and 9). Scale bars, 100 μm. (B) Mean tumor volume after intratumoral or systemic administration of TG6002 with 5-FC. Mice bearing REM134 subcutaneous xenografts were treated with three intratumoral (10⁶ PFUs) or intravenous (10⁷ PFUs) injections of TG6002 (indicated by vertical arrows). Three days after the last virus injection, the animals were treated twice daily with per os administration of water or 5-FC (200 mg/kg/day) for 3 weeks. The data represent mean ± SD of 11 animals.

Figure 4

In Vitro Evaluation of VACV Transduction in Canine Mammary Tumor Explants (A) Transduction of VACV in canine mammary tumor explants by fluorescence microscope detecting GFP. Three biopsies were infected with 10⁶ PFUs of VVTG17990 per well, and transduction was monitored 2, 3, and 4 days after infection. From left to right, each column corresponds to days 0, 2, 3, and 4. From top to bottom, each line fits to a canine mammary tumor explant (first tumor, 1–4; second tumor, 5–8; and third tumor, 9–12). GFP expression was observed and increased during 4 days after infection with VVTG17990, confirming the susceptibility of three canine grade I tubular complex mammary adenocarcinomas to VACV (magnification, 200×). (B) Microphotographs: immunohistochemistry for VACV on uninfected canine mammary tumor explant (1) and two canine mammary tumor explants infected with 10⁶ PFUs of VVTG17990 (2 and 3); viral antigen is detected in the cytoplasm of tumoral cells (brown staining). Scale bars, 20 μm.

Figure 5

In Vitro Evaluation of the Oncolytic Potency of TG6002 with 5-FC in Canine Mammary Tumor Explants (A) Histological microphotographs of uninfected canine mammary tumor explants and infected canine mammary tumor explants with TG6002 and 5-FC. (1) Microphotograph: biopsy of canine mammary tumor collected after surgical excision diagnosis of tubular complex mammary adenocarcinoma, grade I, without necrosis. (2) Microphotograph: explant of canine mammary tumor cultured in culture medium for 6 days after surgical collection. (3 and 4) Microphotographs: explants of canine mammary tumor cultured with TG6002 and 5-FC. Six days after infection by TG6002—10⁶ PFUs (3) or 10⁷ PFUs (4)—with 5-FC, nuclear pyknosis (arrowheads), cell-to-cell detachment (arrows), and cell debris (asterisks) in the lumen of tubules were noticed. Tumor necrosis ranged from 75% to 100%, regardless of TG6002 dosage. Hematoxylin-eosin-saffron (HES) staining was used. Scale bars, 20 μm. (B) 5-FU generated by canine mammary tumor explant infected by TG6002 upon conversion of 5-FC. A canine mammary tumor biopsy was infected with 10⁶ or 10⁷ PFUs of TG6002 and then incubated with 1 mM 5-FC from day 2 to day 6 post-infection. The relative concentration of 5-FC and 5-FU in the culture supernatant was measured by high-performance liquid chromatography. The results are expressed as the percentage of 5-FU in the media relative to the total amount of 5-FC plus 5-FU.