The results presented here demonstrate that established medulloblastoma cell lines express the measles receptor CD46. Importantly, we have also shown that medulloblastoma specimens removed from patients have high levels of CD46 expression. Having demonstrated receptor expression, experiments were then performed that revealed significant antitumor activity of the modified measles virus against medulloblastoma cell lines in vitro. Cell killing was accompanied, as expected, by syncytia formation. The cytotoxic effect was complete in 72 h. In addition, replication of the virus in the tumor cells was documented. Killing was seen at MOI as low as 0.1, and essentially complete killing was seen at an MOI of 1.0. Of note, killing of D283med, a suspension cell line, was documented at similar MOIs. This finding may have relevance to potential use of the modified measles virus for the treatment of tumor disseminated in the CSF, a possibility currently being explored in our laboratory.
The data presented here also show that modified measles virus significantly prolongs survival of measles-treated animals in an orthotopic model of medulloblastoma. While none of the animals survived long-term, we believe that the prolongation of survival is promising. Interestingly, when SCID-treated animals were autopsied, no tumor was found in their brains. Indeed, we were unable to determine the cause of death in these animals, despite histological examination of the lungs, heart, liver, kidneys, and GI tract, in addition to the brain. Upon consultation with a radiation oncologist, it was suggested that the whole-body irradiation given to the mice prior to tumor implantation may be the cause of the animal's premature death. Therefore, subsequent studies, including bioluminescent imaging, were performed in nonirradiated athymic nude mice.
Bioluminescent imaging analysis enhanced our ability to observe tumor growth and tumor regression following MV-GFP administration in vivo without having to sacrifice the animal. Pathological review of the animals confirmed the bioluminescent images in that 2 of the animals were free of tumor and the third had a very small amount of residual tumor. The complete abolition of tumor in 2 mice differed from the initial athymic nude mice study. In that study, all animals exhibited residual tumor at the time of pathological examination, although in 8 out of 11 mice the primary tumor was eradicated. It is highly plausible that tumor cells were able to gain access, either through direct injection or through infiltration, to the subarachnoid space via the right lateral ventricle before MV-GFP therapy was administered. It is our experience that when tumor cells gain access to the cerebral spinal fluid and the subarachnoid space, therapeutic efficacy is more challenging. MV therapy could be applied to the tumor bed following surgical resection to target microscopic residual disease. This approach potentially could ameliorate the need for radiation and chemotherapy. In the future, bioluminescent imaging analysis will allow us to better evaluate the effectiveness of our treatment regimen and perhaps tailor a better treatment schedule.
The virus used in these experiments is replication competent. We have demonstrated that virus is produced by infected cells and that infected cells express GFP, encoded by the infecting virus. Replication competent viruses have potential advantages over replication incompetent viruses. For example, in a trial of intrathecal administration of a nonreplicating p53 adenovirus, transgene expression was found only a short distance (5–8 mm) from the injection site, indicating that viral spread is very limited when the virus does not replicate.8
The lack of robust viral spread has profound implications for the treatment of brain tumors, where individual cells may migrate into the brain, away from the main tumor mass. Hopefully, the use of replicating virus will allow further spread of virus through the tumor.
The safety of vaccine strains of measles viruses in humans is well established. The effect of intracerebral injection of the virus in nonhuman primates has also been investigated. In the early 1970s, Albrecht et al20
found no clinical signs of encephalitis in rhesus monkeys injected with low passage Edmonston's strain. In 4 of 12 animals, virus could be isolated from Vero cells cocultured with brain sections for injected animals. No histological evidence of active measles infection, such as intranuclear inclusions or syncytia, could be found in any of the animals. Similarly, injection of the Edmonston strain virus into the thalamus or CSF via cisternal injection of grivet monkeys or cynomologuous monkeys had no clinical toxicity.21
The pathological findings in these monkeys, gliosis and inflammatory infiltrate, were no different from that seen in vehicle-only–injected animals. We have studied the effect of repeated injections of the Edmonston strain in rhesus monkeys previously vaccinated with the virus.22
In these experiments, the animals were extensively evaluated for virus and symptoms systemically and in the central nervous system. With 30 months of follow-up, no animal showed signs or symptoms of viral infection. No biochemical evidence of infection could be detected. Virus could not be recovered from the CSF, blood, or bucchal swabs. MR imaging at 4–5 months after injection showed no evidence of encephalitis. Taken together, these results strongly suggest that intracerebral injection of measles is safe.
A modified Edmonston strain measles virus has been investigated as a potential therapeutic agent for a number of tumor types. In particular, Galanis and co-workers7
have shown that the virus is effective against orthotopic models of GBM. In one study, 6 of 10 mice treated with modified measles virus survived more than 70 days compared with none of 8 surviving past 50 days when treated with UV-inactivated virus (P
These studies have led to the opening of a clinical trial using modified virus for the treatment of adult patients with recurrent GBM. The results presented here differ from those seen in the GBM model in that all of the treated mice in these experiments died. Only one of the treated mice had detectable tumor on histological examination, suggesting that the virus is very effective against the tumor.
Other oncolytic viruses have been explored as possible treatment modalities for medulloblastoma. Lun et al28
reported on the effects of treatment with reovirus in vitro and in an orthotopic model. Reovirus requires activated Ras protein for tumor cell killing. Five of 7 medulloblastoma cells lines were susceptible to killing by reovirus in vitro, and, as expected, susceptibility was related to the level of activated Ras expression in the tumor cells. Similarly, these investigators demonstrated an increased survival in animals injected with Daoy into the cerebellum and cerebrum and subsequently treated with a single or multiple injections of reovirus into the same site. These investigators used Daoy in their in vivo studies because the cell line was the most sensitive to reovirus killing in vitro. We chose to use D283med in the in vivo experiments presented here because this cell line had lower levels of measles virus receptor expression when compared with other medulloblastoma lines tested. In addition, the molecular biology of Daoy is not typical of medulloblastoma, as the cell line is wild-type p53 null and has homozygous deletion of p16.25–27
Because all surgical medulloblastoma specimens expressed the measles virus receptor, measles virus may have some advantages over reovirus, where some tumors may be resistant to cell killing.
In a previous study the use of myxoma virus as a therapeutic agent for medulloblastoma was explored.28
In that study 9 of 10 medulloblastoma cell lines were effectively killed in vitro by myxoma virus. Similarly, they found that treatment of medulloblastoma with myxoma virus in an orthotopic murine xenograft model resulted in a significant prolongation of survival. Because sensitivity to myxoma virus seems related to activated Akt expression and because medulloblastomas frequently contain activated Akt, this virus has promise as a potential therapy. Indeed, concurrent treatment of tumor-bearing animals with rapamycin and myxoma virus resulted in a better survival than seen with drug or virus alone.
One major impediment to the successful use of MV as an anticancer therapy is preexisting antiviral antibodies in immunized (essentially all) patients. However, we do not believe that neutralizing antibodies to MV will have a major effect on the clinical applicability of a single dose of virus for the treatment of medulloblastoma, as the antiviral immune response will take a longer time to become active than the time it takes for the virus to kill the cells. Additionally, in peripheral regions of the tumor, the blood-brain barrier is intact, which is not permeable to antibodies. In a study of the effect of 2 injections of measles virus 1 week apart in immunized macaques, no evidence of brain toxicity was noted.22
In a study evaluating MV antibody status in children with cancer, the findings suggested that cancer and its associated therapy interfered with antibody production in these children.29
However, if multiple episodes of MV treatment are necessary, novel approaches have recently been developed to address this issue. Cell-based carriers, such as mesenchymal stem cells, have been evaluated and are currently being used to deliver MV to tumor cells while protecting them from antibody neutralization.30
If the efficacy of MV therapy is determined to be insufficient due to neutralizing antibodies, then a cell carrier may be an option to circumvent the antibody response.
In summary, we have demonstrated that a modified measles virus has therapeutic potential in the treatment of intracerebral medulloblastoma. These results provide initial data to be pursued with additional studies with the goal of using the virus in a clinical trial for the treatment of medulloblastoma.
Conflict of interest statement. None declared.