Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus

Abstract

Oncolytic adenoviruses as a treatment for cancer have demonstrated limited clinical activity. Contributing to this may be the relevance of preclinical animal models used to study these agents. Syngeneic mouse tumor models are generally non-permissive for adenoviral replication, whereas human tumor xenograft models exhibit attenuated immune responses to the vector. The cotton rat (Sigmodon hispidus) is susceptible to human adenovirus infection, permissive for viral replication and exhibits similar inflammatory pathology to humans with adenovirus replicating in the lungs, respiratory passages and cornea. We evaluated three transplantable tumorigenic cotton rat cell lines, CCRT, LCRT and VCRT as models for the study of oncolytic adenoviruses. All three cells lines were readily infected with adenovirus type-5-based vectors and exhibited high levels of transgene expression. The cell lines supported viral replication demonstrated by the induction of cytopathogenic effect (CPE) in tissue culture, increase in virus particle numbers and assembly of virions seen on transmission electron microscopy. In vivo, LCRT and VCRT tumors demonstrated delayed growth after injection with replicating adenovirus. No in vivo antitumor activity was seen in CCRT tumors despite in vitro oncolysis. Adenovirus was also rapidly cleared from the CCRT tumors compared to LCRT and VCRT tumors. The effect observed with the different cotton rat tumor cell lines mimics the variable results of human clinical trials highlighting the potential relevance of this model for assessing the activity and toxicity of oncolytic adenoviruses.

FIG 1

Transgene expression in cotton rat tumor cells infected with a non-replicating adenovirus expressing GFP. CCRT, LCRT and VCRT cotton rat tumor cells, the A549 and Saos-2 human cancer cell lines and TS/A and 15-12RM murine cancer cells were infected with Ad.GFP at MOI 1, 10 and 100 and the percentage of cells expressing GFP was determined at 48 h by flow cytometry.

FIG 2

Lytic activity of Ad.OW94 (E1⁺) or Ad.GFP (E1⁻) on CCRT, LCRT, VCRT, and HEK-293 cells. Each of the cell lines were seeded at 2 × 10⁵ cells per well onto 6 well plates and infected with Ad.GFP or Ad.OW94 at MOI of 10 or 100 PFU per cell, or treated with a PBS control. After 7 days, cells were: (A) stained with 1% crystal violet, (B) examined microscopically for CPE, and (C) examined by fluorescence microscopy for GFP expression.

FIG 3

Viral burst assays to determine the quantity of adenovirus released by (A) LCRT, (B) VCRT and (C) CCRT cells infected with Ad5.wt. The titer of infectious adenovirus was assessed at 4 h and 72 h following infection with Ad5.wt at MOI 0, 1, 10, or 100. Error bars represent SD of the mean.

FIG 4

Transmission electron micrographs of CCRT, LCRT and VCRT cells performed 4 h and 72 h after infection with adenovirus. (A & B), CCRT (C & D) LCRT and (E & F) VCRT cotton rat cell lines were infected with Ad5.wt at MOI 1 and examined by electron microscopy 4 h (A, C & E) and 72 h (B, D & F) after infection. Arrows indicate adenoviral particles.

FIG 5

Time-dependent increase in functional viral titer after infection with replicating adenovirus. LCRT, VCRT and CCRT cells were infected with Ad.OW94. At 5 h and 72 h cell-free media supernatants were transferred to HeLa cell monolayers. HeLa cells were analyzed 24 h later for GFP expression.

FIG 6

Quantification of adenoviral genomes in LCRT, VCRT and CCRT tumors in vivo. CCRT, LCRT and VCRT tumors were established in cotton rats and infected with Ad5.wt or Ad.null. Five and eight days after the initial injection, tumors were removed from the animals from both groups, total DNA was extracted and the number of adenovirus genomes measured using real-time PCR. The amount of virus was determined against a standard curve of known adenovirus. Error bars represent the SD of the mean for 8 tumor samples.

FIG 7

Growth curves for (A) LCRT, (B) VCRT and (C) CCRT tumors in vivo following infection with Ad.null (open circle), Ad5.wt (closed circle) or PBS (triangle). Cotton rats were implanted subcutaneously with 1 × 10⁶ infected tumor cells. Tumor size was measured daily with calipers and the tumor volume expressed the area of a rotational ellipse. Error bars represent the SEM.