ONYX-411, a conditionally replicative oncolytic adenovirus, induces cell death in anaplastic thyroid carcinoma cell lines and suppresses the growth of xenograft tumors in nude mice

Abstract

Anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer variant, accounting for 1-2% of all cases, but 33% of deaths, and exhibiting an average life expectancy of 5 months. ATC is largely unresponsive to radioactive iodine, chemotherapy, external beam radiation or surgery, underscoring the need for new and effective therapies. We evaluated the therapeutic potential of an oncolytic adenovirus, ONYX-411, that replicates selectively in and kills cells with dysfunction of the retinoblastoma (RB) pathway. In the present study, we report that ONYX-411 is able to induce cell death in eight human anaplastic carcinoma cell lines in vitro. The cytopathic effect of the virus is specific to cells with RB dysfunction, which appears to be frequent in ATC. We confirmed the expression of the coxsackie adenovirus receptor, CAR, in all ATC cell lines, demonstrating the potentially universal application of this oncolytic viral therapy to ATC. In addition, the growth of xenograft tumors induced in athymic mice with the ARO and DRO cell lines was significantly reduced by ONYX-411 treatment. These results indicate that ONYX-411 can be a potential therapeutic agent for the treatment of ATC, rendering this class of conditionally replicating adenoviruses an attractive candidate for clinical trials.

Figure 1

Determination of RB and p53 dysfunction and coxsackie adenovirus receptor (CAR) expression in ATC cell lines. Western blots of 50 μg of whole-cell lysate from each ATC cell line and normal primary thyrocytes as a control were evaluated for RB (top panel), p53 (second panel), CAR (third panel) and β-actin expression as a loading control (bottom panel). The blot is representative of results obtained from three independent experiments. RB, retinoblastoma.

Figure 2

Efficacy of ONYX-411 is specific to RB dysfunction. (a) Dose effect of ONYX-411 adenovirus treatment of ARO, DRO and BHT101 cells. Cells were infected with ONYX-411 virus at 25, 50, 100 and 200 MOIs. Cell viability was assessed 3 days post-infection by MTT assay. Values are plotted as a percentage of mock-infected controls. The data shown here are the means ± s.d. from two independent experiments with six replicates for each variable. (b) Cell viability in various ATC cell lines after ONYX-411 infection at an MOI of 50 was assessed 3 and 6 days postinfection by MTT assay. Values are plotted as a percentage of mock-infected controls. The data shown here are the mean values ± s.d. from two independent experiments with six replicates of each variable. MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RB, retinoblastoma.

Figure 3

Tumoricidal effect of ONYX-411 on ATC cell line-induced xenografts. Cells (10⁶) from each of the cell lines, ARO (a and b) and DRO (c and d) were injected s.c. into nude mice to generate xenograft tumors. Three weeks post-injection, mice were divided into two groups per cell line, and 5×10⁷ pfu per 50 μl of either UV-inactivated control or intact ONYX-411 virus was injected intratumorally per day per tumor for five consecutive days. Tumor volumes were monitored every week for 6–7 weeks and plotted as a function of time (a and c). Animals were killed in cases where tumor volumes exceeded 1000 mm³. The values represent means ± s.e. from seven (ARO) and 10 (DRO) independent tumors. (b and d) Representative images of animals with tumors demonstrating the suppressive effect of ONYX-411-treated tumors (animals on the right) in comparison to their UV-inactivated virus-treated controls (animals on the left). Analysis of variance for repeated measures indicated that the treatment differences between UV-inactivated and ONYX-411 virus with both ARO and DRO cells were statistically significant at the P=0.0442 and 0.0042 levels, respectively. S.c., subcutaneously.

Figure 4

Hematoxylin and eosin-stained sections of ARO (a–d) and DRO (e–h) xenograft tumors from nude mice treated with UV-inactivated (a, b, e and f) or intact ONYX-411 virus (c, d, g and h). In each case, the panels (b, d, f and h) are higher magnification images of the cells. Arrows indicate regions of blood vessel formation. The circled structures represent empty follicles with poorly organized follicular thyroid cells, which were frequently observed in ARO cell tumors (b). Similar structures were not observed in the case of DRO cell tumors (f). UV, ultraviolet.