The effects of radiation on antitumor efficacy of an oncolytic adenovirus vector in the Syrian hamster model

Abstract

We report that radiation enhances the antitumor efficacy of the oncolytic adenovirus vector VRX-007 in Syrian hamster tumors. We used tumor-specific irradiation of subcutaneous tumors and compared treatment options of radiation alone or combined with VRX-007 and cyclophosphamide (CP). Radiation therapy further augmented the VRX-007-mediated inhibition of tumor growth, in both CP-treated and non-CP-treated hamsters, even though radiation did not lead to increased viral replication in tumors when compared with those treated with VRX-007 alone. Moreover, tumor growth inhibition was similar in tumors irradiated either 1 week before or after injection with VRX-007, which suggests that radiation exerts its antitumor effect independently from vector therapy. Thus, our results demonstrate that these two therapies do not have to be provided simultaneously to enhance their combined effectiveness against subcutaneous hamster tumors.

Figure 1

The effects of combined therapy with radiation and VRX-007 on tumor size. HaK tumors were injected for 6 days with 1×10¹⁰ PFU of VRX-007 and then irradiated with 8 Gy on days 1, 39, and 55 p.i. (arrows). (a) Mean tumor volume. The number of animals per group were: mock (n=9), VRX-007 (n=8), radiation (n=7), VRX-007 + radiation (n=8). There was a significant difference (P< 0.03) between mock and all other groups starting at day 24, and there was a significant difference (P< 0.05) between each single therapy and combined therapy starting at day 41. Error bars represent mean + SE. (b) Serum neutralizing antibody titers. An anti-Ad antibody assay of the serum was performed at time of sacrifice, 61 days p.i. (P=0.6991).

Figure 2

The effect of radiation on infected HaK cells in vivo and in vitro in short-term studies. Animals were irradiated with 8 Gy 24 h before intratumoral injection with VRX-007 for 6 consecutive days. Samples were collected at days 4 and 7 after the last virus injection. There were 3 animals per group. (a) TCID₅₀ assay of virus extracted from HaK tumors. There was no significant difference between both groups on days 4 or 7 (P>0.100). (b) Serum neutralizing antibody titers. An anti-Ad antibody assay of the serum was performed at time of death indicated. There was no significant difference between both groups on days 4 or 7 (P>0.200). (c) Single step growth curve demonstrating the effect of radiation 24 h before or after VRX-007 infection. HaK cells were irradiated with 20 Gy at 24 h before or after infection with VRX-007. The level of infectious virus was tested from the total cells and media from the dish and is represented as TCID₅₀/ml as determined on HEK293 cells.

Figure 3

The combination of VRX-007, cyclophosphamide (CP), and radiation treatments results in the least amount of tumor growth. HaK tumors were irradiated with 8 Gy at 1 day before 6 consecutive days of VRX-007 intratumoral injections. Intraperitoneal injections of CP were given biweekly starting one week before infection and for the duration of the study. The number of animals per group were: mock (n=7), CP (n=7), radiation (n=7), CP + radiation (n=9), VRX-007 (n=9), VRX-007 + CP (n=8), VRX-007 + radiation (n=7), VRX-007 + CP + radiation (n=7). Radiation is abbreviated as “R.” (a) Mean tumor volume measured biweekly throughout the study. (b) Mean tumor volume at time of sacrifice, 44 days p.i. (c) TCID₅₀ of virus extracted from the tumors, collected at time of sacrifice. There was significantly more infectious virus in both CP-treated groups compared to both non-CP treated groups (P=0.0012). There was no difference in the amount of virus in the tumors of the VRX-007 + CP and VRX-007 + CP + R groups.

Figure 4

Radiation and 007-Luc work independently to inhibit HaK tumor growth. HaK tumors were injected once with 1×10¹⁰ PFU of 007-Luc. Irradiated groups were given 8 Gy either immediately before infection (007-Luc + radiation-before) or one week after infection (007-Luc + radiation-after). The number of animals per group were: mock (n=8), radiation-before (n=9), radiation-after (n=6), 007-Luc (n=6), 007-Luc + radiation-before (n=6), 007-Luc + radiation-after (n=9). (a) Mean tumor volume. Error bars represent mean + SE. After 35 days post infection, the single therapy groups (007-Luc only, radiation only) had significantly larger tumors than those in either of the double therapy (007-Luc + radiation) (P<0.02). Importantly, tumor growth suppression was similar (P=0.088) in the 007-Luc + radiation-before and 007-Luc + radiation-after groups. (b) Luciferase expression in tumors, measured by total flux of photons. The gray line suggests the background intensity (approximately 10⁵ photons). The 007-Luc + radiation-after group could not be imaged at the day 1 and day 3 time points because the infection and radiation are done in BSL-2 isolation and the imaging is done in a BSL-3 facility; once the hamsters enter the BSL-3 area for imaging, they cannot be brought into the BSL-2 area for radiation. However, up to the point of receiving radiation treatment, these hamsters were treated identically to those in the vector only (007-Luc) group, so their luciferase expression data is expected to be the same as for animals in the 007-Luc group.

Figure 5

Radiation and 007-Luc work independently to inhibit SHPC6 tumor growth. SHPC6 tumors were injected once with 1×10¹⁰ PFU 007-Luc. Irradiated groups were given 6 Gy either immediately before infection (radiation-before) or one week after infection (radiation-after). The number of animals per group were: mock (n=9), radiation-before (n=8), radiation-after (n=7), 007-Luc (n=9), 007-Luc + radiation-before (n=9), 007-Luc + radiation-after (n=9). (a) Mean tumor volume. Error bars represent mean + SE. Tumors in the mock group were significantly larger (P<0.05) than all other groups on day 28, and tumors in the 007-Luc group were larger than either double combination on day 28 (P<0.05). Tumor suppression was similar in the 007-Luc + radiation-before and 007-Luc + radiation-after groups (P>0.40 at all time points). (b) Luciferase expression in tumors, measured by total flux of photons. The gray line suggests the background intensity (approximately 10⁵ photons).