Engineered measles virus Edmonston strain used as a novel oncolytic viral system against human hepatoblastoma

Abstract

Background: Hepatoblastoma (HB) is the most common primary, malignant pediatric liver tumor in children. The treatment results for affected children have markedly improved in recent decades. However, the prognosis for high-risk patients who have extrahepatic extensions, invasion of the large hepatic veins, distant metastases and very high alpha-fetoprotein (AFP) serum levels remains poor. There is an urgent need for the development of novel therapeutic approaches.

Methods: An attenuated strain of measles virus, derived from the Edmonston vaccine lineage, was genetically engineered to produce carcinoembryonic antigen (CEA). We investigated the antitumor potential of this novel viral agent against human HB both in vitro and in vivo.

Results: Infection of the Hep2G and HUH6 HB cell lines, at multiplicities of infection (MOIs) ranging from 0.01 to 1, resulted in a significant cytopathic effect consisting of extensive syncytia formation and massive cell death at 72-96 h after infection. Both of the HB lines overexpressed the measles virus receptor CD46 and supported robust viral replication, which correlated with CEA production. The efficacy of this approach in vivo was examined in murine Hep2G xenograft models. Flow cytometry assays indicated an apoptotic mechanism of cell death. Intratumoral administration of MV-CEA resulted in statistically significant delay of tumor growth and prolongation of survival.

Conclusions: The engineered measles virus Edmonston strain MV-CEA has potent therapeutic efficacy against HB cell lines and xenografts. Trackable measles virus derivatives merit further exploration in HB treatment.

Figure 1

Expression of the CD46 receptor in human HB cells and a normal liver cell line. High levels of the CD46 receptor were observed in the human HB cell lines with the rate of 90.82% in Hep2G cells and 80.03% in HUH6 cells. A relatively low level of the CD46 receptor was detected in L-02 cells. Analyses were performed by flow cytometry using a monoclonal CD46 antibody.

Figure 2

Infectivity, induction of syncytia, and CPE of MV-Edm in human HB cells. (a) Serial analysis to determine the CPE of recombinant MV-Edm was performed every 24 hours on the human HB cell lines Hep2G and HUH6 and normal liver cell line L-02. Seventy-two hours after infection at MOIs of 0.1 and 1, the cells were stained with crystal violet representing viable, attached cells. (b) The time course of cell viability of the human HB cell lines Hep2G and HUH6 and normal liver cell line L-02 (n = 8) after infection with recombinant MV-Edm at MOIs of 0.1 and 1 was analyzed using a Cell Titer 96 Aqueous nonradioactive cell proliferation assay kit. ***Group_MOI=1.0versus Group _MOI=0, P < 0.05; ** Group_MOI=0.1versus Group _MOI=0, P < 0.05. * Group_MOI=0.01versus Group _MOI=0, P < 0.05.

Figure 3

Replication of MV-CEA in human HB cells. CEA level of the (a) intracellular and (b) culture supernatant in the Hep2G cell line was detected by ELISA from 24 to 96 hours after infection at an MOI of 0.1. The intracellular CEA levels increased with time and peaked at 72 hours post infection, and the supernatant CEA level peaked at 84 hours post infection in Hep2G cells but not in L-02 cells. This phenomenon provides strong evidence for MV-CEA replication and cell lysis.

Figure 4

Apoptosis induced by MV-CEA in the human Hep2G and HUH6 cells. The cells were infected with MV-CEA at an MOI of 0.1. Adherent and detached cells were harvested at 24, 48 and 72 hours post infection. (a) The percentage of apoptotic cells was measured by FACS and is shown in this figure. In the L-02 cells, there was no dramatic apoptosis, but the Hep2G cells demonstrated dramatic apoptosis at 72 hours postinfection. (b)The significant difference is significant between the HB and L-02 groups but not between the two HB cell lines. * Group _Hep2GVersus Group _L-02, P < 0.01; **Group _HUH-6Versus Group _L-02, P < 0.01. (c) We further examined the poly(ADP-ribose) polymerase expression by Western blot and found that the 85-kd cleaved poly(ADP-ribose) polymerase fragment was expressed in the Hep2G and HUH-6 cells at 72 h after infection with MV-CEA. This finding was in agreement with the FACS results.

Figure 5

The serum CEA concentrations and tumor suppression after intratumoral MV-CEA therapy of human HB xenografts. Mice bearing HB xenografts (Hep2G) were injected intratumorally with 2 × 10⁶ TCID50 of MV-CEA every other day a total of five times (1.0 × 10⁷ total TCID50/mouse). Treatment groups (n = 8 per group) received active MV-CEA; the untreated group received UV-inactivated MV-CEA. (a) The time course of serum CEA concentration in HB xenograft-bearing mice after MV-CEA therapy. *Group _untreatedversus Group _treated, P < 0.05; (b) The increase in tumor volume after initiation of the MV-CEA therapy. The data points are given as the median with positive standard error. *Group _untreatedversus Group _treated, P < 0.05; (c) The Kaplan-Meier survival curves of the treated and untreated mice. The results show significant suppression of tumor growth in the MV-CEA treated animals (p < 0.05) and statistically significant prolongation of survival (p < 0.05).