Oncolytic measles viruses encoding interferon beta and the thyroidal sodium iodide symporter gene for mesothelioma virotherapy

Abstract

Mesothelioma usually leads to death within 8-14 months of diagnosis. To increase the potency of oncolytic measles viruses (MVs) for mesothelioma therapy, we inserted the interferon beta (IFNbeta) gene alone or with the human thyroidal sodium iodide symporter (NIS) gene into attenuated MV of the Edmonston lineage. The corresponding mouse IFNbeta (mIFNbeta) viruses, MV-mIFNbeta and MV-mIFNbeta-NIS, successfully propagated in human mesothelioma cells, leading to intercellular fusion and cell death. High levels of mIFNbeta were detected in the supernatants of the infected cells, and radioiodine uptake was substantial in the cells infected with MV-mIFNbeta-NIS. MV with mIFNbeta expression triggered CD68-positive immune cell infiltration 2-4 times higher than MV-GFP injected into the tumor site. The numbers of CD31-positive vascular endothelial cells within the tumor were decreased at day 7 after intratumoral injection of MV-mIFNbeta or MV-mIFNbeta-NIS, but not after MV-GFP and PBS administration. Immunohistochemical analysis showed that MV-mIFNbeta changed the microenvironment of the mesothelioma by increasing innate immune cell infiltration and inhibiting tumor angiogenesis. Oncolytic MVs coding for IFNbeta effectively retarded growth of human mesotheliomas and prolonged survival time in several mesothelioma tumor models. The results suggest that oncolytic MVs that code for IFNbeta and NIS will be potent and versatile agents for the treatment of human mesothelioma.

Figure 1

Characterization of MVs in vitro. Schematic construction of MV-mIFNβ and MV-mIFNβ-NIS is shown in (a). mIFNβ gene was cloned to take place of the GFP gene in MV genome. p(+)MV-mIFN and p(+)MV-NIS were cut by the same pair of restriction enzymes and religated to contain both the mIFN and NIS gene in the MV genome (a). One-step viral growth curves for MV-GFP, MV-mIFNβ, MV-GFP-NIS, and MV-mIFNβ-NIS in Vero cells (b). Supernatants from the culture were collected to measure mIFNβ secretion (c). Human mesothelioma cells were infected by MVs. Human mesothelioma cells H2596, H2373, H513, M30, and MSTO-211H were infected with MV-GFP or MV-mIFNβ at MOI of 1.0. Pictures were taken 36 h later (d).

Figure 2

Virus progeny and mIFNβ secretion by human mesothelioma cells after infection with MVs. M30 mesothelioma cells were infected by MVs at MOI of 1.0. After 24, 48, or 72 h of incubation, cells were harvested for virus titration (a), and supernatant was collected to measure the mIFNβ secretion (b).The same experiments were performed to the human mesothelioma cells H513 for viral progeny (c) and mIFNβ (d).

Figure 3

¹²⁵I uptake by human mesothelioma cells after infection with MVs. Mesothelioma cells were incubated with MVs at MOI of 1.0 or 0.1 for 48 h. Uptake of ¹²⁵I was assayed in mesothelioma H513 (a) and H2373 (b). Iodide uptake is blocked by perchlorate, a specific inhibitor of NIS. Without NIS, cells infected with MV-mIFNβ do not concentrate iodide.

Figure 4

Administration of MV-mIFNβ increased infiltration of CD68-positive immune cells and inhibition of tumor angiogenesis. mIFNβ induced robust CD68-positive cell infiltration and decreased microvessel density (as indicated by CD31 staining) in the tumor. Mesothelioma H513-bearing nude mice were given PBS, MV-GFP, or MV-mIFNβ intratumorally; (a) 4 or 7 days later, tumor sections were stained for CD68+ and CD31+ cells. Original magnification, × 200. Quantitative analysis was carried out to compare CD68+ infiltration into 0.1 mm² area (b), and the CD31 coverage rate was calculated (c) as described in Material and methods. ^*P<0.05; ^**P<0.01. Scale bar denotes 200 μm (a, right corner).

Figure 5

¹²⁵I uptake images of mesothelioma-bearing mice treated with MVs intratumorally. Mesothelioma H513-bearing SCID mice were treated with MVs intratumorally. On day 7, ¹²⁵I was used to check iodine uptake. Mice were imaged with CT/SPECT 1 h after ¹²⁵I intraperitoneal injection. There was significant uptake of ¹²⁵I in MV-GFP-NIS or MV-mIFNβ-NIS-treated mesothelioma xenografts (white arrow). The pictures were taken at the section with the peak ¹²⁵I uptake in the tumor. In contrast, MV-GFP, MV-mIFNβ, and PBS-treated tumors had much less signals (green arrow). The different physiologic uptake in thyroid gland and stomach and excretion into bladder were observed.

Figure 6

Therapeutic effects of oncolytic MVs for mesothelioma xenografts. Mesothelioma H513 cells were inoculated into the flanks of nude mice. The efficacy of MVs was compared by tumor growth (a) and survival (b). MV with mIFNβ expression arrested tumor growth. The experiment was repeated in SCID mice bearing mesothelioma H513 cells. Both tumor growth (data not shown) and survival (c) showed the superiority of MV-mIFNβ. Mesothelioma H2373 cells were established in the peritoneum of nude mice to mimic the human conditions. MV-mIFNβ significantly extended the survival of mice (d). ^*P<0.05; ^**P<0.01; ^#P<0.001.