A novel immunocompetent murine model for replicating oncolytic adenoviral therapy

Abstract

Oncolytic adenoviruses are under investigation as a promising novel strategy for cancer immunotherapeutics. Unfortunately, there is no immunocompetent mouse cancer model to test oncolytic adenovirus because murine cancer cells are generally unable to produce infectious viral progeny from human adenoviruses. We find that the murine K-ras-induced lung adenocarcinoma cell line ADS-12 supports adenoviral infection and generates infectious viral progeny. ADS-12 cells express the coxsackie and adenovirus receptor and infected ADS-12 cells express the viral protein E1A. We find that our previously described oncolytic virus, adenovirus TAV-255 (AdTAV-255), kills ADS-12 cells in a dose- and time-dependent manner. We investigated ADS-12 cells as an in-vivo model system for replicating oncolytic adenoviruses. Subcutaneous injection of ADS-12 cells into immunocompetent 129 mice led to tumor formation in all injected mice. Intratumoral injection of AdTAV-255 in established tumors causes a significant reduction in tumor growth. This model system represents the first fully immunocompetent mouse model for cancer treatment with replicating oncolytic adenoviruses, and therefore will be useful to study the therapeutic effect of oncolytic adenoviruses in general and particularly immunostimulatory viruses designed to evoke an antitumor immune response.

Figure 1

Adenovirus TAV-255 (AdTAV-255) infects and replicates in ADS-12 cells. Western blot analysis of adenoviral E1A and CAR expression in different murine cell lines. (a) CT26 cells and ADS-12 cells were infected with AdTAV-255 at multiplicity of infection (MOI) of 10 for indicated times and assessed for E1A and CAR expression, with β-tubulin as a loading control. (b) Murine cell lines were infected with AdTAV-255 at MOI of 3 for 4 days and assessed for E1A expression. (c) ADS-12 cells were infected with AdTAV-255 at MOI of 10 for 72 and 96 h. The cells were collected and lysed, and total viral yields were determined with plaque assays. Each value is the average from three independent measurements. (d) ADS-12 cells were infected with AdTAV-255 at MOI of 1. Media was collected at 1, 4 and 7 days after infection without disturbing the cell monolayer, and the viral titer in the media was determined with plaque assays.

Figure 2

TAV-255 induced cell death of ADS-12 cells but not other murine cells. (a) Growth of ADS-12 cells in cell culture. (b) ADS-12 cells and CT26 cells were infected with AdTAV-255 at multiplicity of infection (MOI) of 10 for 6 days and cell viability was assessed with MTT assays. (c) Crystal violet staining assays of ADS-12 cells and other murine cell lines infected with adenovirus TAV-255 (AdTAV-255) for 6 days.

Figure 3

Adenovirus TAV-255 (AdTAV-255) kills murine ADS-12 cells in a dose- and time-dependent manner. (a) ADS-12 cells were treated with different doses of AdTAV-255 for 3 and 6 days. Cell viability (expressed as a percentage related to the controls) was determined with MTT assays. (b) Clonogenic assays of ADS-12 cells infected with AdTAV-255. The graph shows relative numbers of colonies formed by ADS-12 cells after infection.

Figure 4

Adenovirus TAV-255 (AdTAV-255) downregulates c-Myc expression in both ADS-12 and A549 cells. ADS-12 and A549 cells were infected with AdTAV-255 at an multiplicity of infection of 10. Lysates were collected at 24, 48 and 72 h post infection and probed for E1A and c-Myc.

Figure 5

Adenovirus TAV-255 (AdTAV-255) inhibits subcutaneous tumor growth in immunocompetent mice. 129S4/SvJaeJ mice were injected with 5 × 10⁵ ADS-12 cells subcutaneously and allowed to form tumors. When the average tumor size reached ~150 mm³, ADS-12 tumors were treated with three intratumoral (i.t.) doses of AdTAV-255 (5 × 10⁷ PFU per dose) or phosphate-buffered saline (PBS) every 3 days. (a) Mean tumor volume of each group is shown through day 12 after starting treatment, when four out of six mice treated with PBS were sacrificed due to tumor size. (b) Representative tumor images from PBS-treated and AdTAV-255-treated group. (c) A survival curve of mice. Mice were euthanized if their tumor diameter exceeded 1.5 cm or tumor volume exceeded 600 mm³. The quantitative data shown are the mean±s.e.m. *P<0.05, **P<0.01.