Effects of Newcastle Disease Virus on Human Cancer Cells
The ability of Newcastle disease virus (NDV) to replicate efficiently in human cancer cells has been demonstrated in both laboratory studies and animal studies.[1-12] Reviewed in [13,14] Several of these studies have provided much of the evidence that lytic strains of NDV are also oncolytic.[3-6,8-10,12] Reviewed in 
Strain 73-T, which is lytic, has been shown to replicate efficiently in human tumor cells  and kill the following types of human cancer cells in vitro : fibrosarcoma, osteosarcoma, neuroblastoma, bladder carcinoma, cervical carcinoma, melanoma, Wilms tumor, and myeloid leukemia;[3,6,8,9] however, this strain did not kill human B-cell lymphoma (i.e., Burkitt lymphoma) cells in vitro. In addition, strain 73-T did not kill normal, proliferating human white blood cells or normal human skin fibroblasts in vitro,[3,6,8] but it killed normal human lung fibroblasts in vitro at the same rate that it killed cancer cells.
Lytic strain Roakin has been reported to kill human B-cell lymphoma cells and T cells transformed in vitro from a Hodgkin lymphoma patient four to five times faster than it kills normal, resting human white blood cells.[4,5] This strain, however, has also been reported to kill normal, proliferating human white blood cells in vitro, though at a lower rate than it kills cancer cells.
Lytic strain Italien (or Italian) has been shown to kill human squamous cell lung carcinoma, melanoma, breast carcinoma, and larynx carcinoma, but not cervical carcinoma, cells in vitro. The replication efficiency of this strain in normal human cells was not tested.
Overall, these results show that there are some types of human cancer cells in which individual lytic strains of NDV do not replicate very well and that there are some types of normal human cells in which they replicate very efficiently. Nonetheless, these data and the absence of serious illness in individuals infected with NDV Reviewed in [1-3,10,13,16-21] are consistent with the view that NDV replicates much more efficiently in human cancer cells than it does in most types of normal human cells (i.e., “DBTRG.05MG human glioblastoma,” “U-87MG human astrocytoma,” “rat F98 glioblastoma cells,” and “mouse Ehrlich ascites carcinoma”).
NDV strain Ulster, which is nonlytic, has also been shown to replicate efficiently in human cancer cells in vitro, including cells of the following types of human tumors: colorectal carcinoma, gastric carcinoma, pancreatic carcinoma, bladder carcinoma, breast carcinoma, ovarian carcinoma, renal cell carcinoma, lung carcinoma, larynx carcinoma, cervical carcinoma, glioblastoma, melanoma, B-cell lymphoma, and T-cell lymphoma. This strain does not replicate very efficiently in resting or proliferating normal human white blood cells in vitro. Other experiments have shown that NDV Ulster can kill infected cells, Reviewed in  and it can replicate in human cancer cells whether they are actively proliferating or not. Reviewed in 
The ability of lytic strains of NDV to kill human cancer cells in vivo has also been examined. In xenograft studies, human cancer cells were injected either subcutaneously or intradermally into athymic, nude mice (i.e., mice that do not reject tumor cells from other animals because they have a defective immune system), and tumors were allowed to form. NDV was injected directly into the tumors, and tumor growth and animal survival were monitored.
Intratumoral injection of strain 73-T was associated with complete tumor regression in 75% to 100% of mice bearing human fibrosarcoma, neuroblastoma, or cervical carcinoma tumors.[1-3,10] Intratumoral injection of 73-T was also associated with more than 80% tumor regression in 66% of mice bearing human synovial sarcoma tumors. In addition, intratumoral injection of 73-T was associated with 68% to 96% inhibition of tumor growth in mice bearing human epidermoid, colon, lung, breast, or prostate carcinoma tumors.
Intratumoral injection of strain Italien was associated with complete tumor regression in 100% of mice bearing human melanoma tumors. The growth of metastatic tumors in these animals, however, was not affected, suggesting that the virus was unable to disseminate widely throughout the body. Reviewed in [14,20]
Replication of strain 73-T in the above-mentioned neuroblastoma xenografts was demonstrated by showing an increase in the amount of virus that could be recovered from tumor samples over time. When this strain was injected into the thigh muscle of athymic, nude mice, the amount of virus that could be recovered decreased with time, a finding consistent with the proposal that NDV replicates much more efficiently in cancer cells than in most normal cells.
Another study of intratumoral injection used the V4UPM strain of NDV in a nude mouse model of tumors produced by subcutaneous injection of human glioblastoma multiform cells. All four mice with tumors from the U-87MG cell line experienced sustained complete responses after one injection. However, no complete responses were observed in mice with tumors from the DBTRG.05MG cell line despite a similar in vitro cytotoxicity compared with U-87MG.
In one study, mice bearing human neuroblastoma xenografts were given single intraperitoneal injections of strain 73-T, and 9 (75%) of 12 treated mice exhibited complete, durable tumor regressions.
It is important to note that athymic, nude mice still make small numbers of T cells, and they produce interferons, natural killer cells, and macrophages. Reviewed in [11,26,27] The possibility that these residual components of the immune system, which may be activated by the presence of NDV, contributed to the antitumor effects observed in the xenograft studies cannot be ruled out.NDV and Cancer Immunotherapy
Other laboratory and animal studies have shown that NDV and NDV-infected cancer cells can stimulate a variety of immune system responses that are essential to the successful immunotherapy of cancer.[6,8,25,28-42] Reviewed in [11,20,43-47] A few of these studies used human cells,[6,8,29,30,38,41,42] Reviewed in [20,44,47] but most used animal cells and animal tumor models.[6,8,25,28,30-37,39] Reviewed in [11,20,43-46]
Two in vitro studies have shown that infection of human immune system cells with NDV causes the cells to produce and release the cytokines interferon-alpha and tumor necrosis factor (TNF)-alpha.[6,8] In one of these studies, it was shown further that infection of human cancer cells with NDV makes the cells more sensitive to the cytotoxic effects of TNF-alpha.
Additional in vitro studies have shown that NDV-infected human cancer cells are better at activating human cytotoxic T cells, helper T cells, and natural killer cells than uninfected cancer cells.[8,29,30,48] The NDV protein hemagglutinin-neuraminidase, which is present in the plasma membrane of virus-infected cells, appears to play a role in the enhancement of T cell activation. There is evidence that this protein makes infected cells more adhesive, thereby promoting the interaction between virus-infected cells and immune system cells. Reviewed in 
Other laboratory studies have shown that the interaction between NDV-infected cancer cells and T cells can be improved if monoclonal antibodies that bind the hemagglutinin-neuraminidase protein on the cancer cells and either the CD3 protein or the CD28 protein on T cells (i.e., bispecific monoclonal antibodies) are also used.[29,38,49,50] Reviewed in [20,44,47] It has been reported that this improved interaction leads to better T cell activation.[29,38] Reviewed in [20,44,47] T cells exposed to NDV-infected human colon cancer cells and bispecific monoclonal antibodies showed not only an increased ability to kill the virus-infected cells but also an ability to inhibit the proliferation of uninfected colon cancer cells.[29,38] Reviewed in  On the basis of these and other in vitro findings, it has been proposed that vaccines consisting of NDV-infected cancer cells and bispecific monoclonal antibodies be tested in humans.[20,29,38,44,47]
As noted above, animal cells and animal tumor models have also been used to explore the immunotherapy potential of NDV. ESb, a mouse model of metastatic T-cell lymphoma has been employed in most of this work;[25,28,31,35-37,39,40] Reviewed in [11,20,43-47] however, additional experiments have utilized one or more of the following tumor models: mouse B16 melanoma, mouse Lewis lung carcinoma,[32,35] mouse P815 mastocytoma, mouse Ca 761-P93 mammary carcinoma, and guinea pig L10 hepatocellular carcinoma.
In one study, it was shown that anticancer activity could be induced in mouse macrophages both in vitro and in vivo by infection with NDV strain Ulster. Similar activation of mouse macrophages in vitro was observed after infection with the NDV lytic strain Lasota. In this study, the activated macrophages showed cytotoxic activity toward ESb, P815 mastocytoma, and Ca 761-P93 mammary carcinoma cells in vitro. Other experiments demonstrated that much of the observed anticancer activity could be attributed to the production and release of TNF-alpha by the infected macrophages. In addition, the infected, activated macrophages showed anticancer activity in vivo when they were injected into mice bearing Ca 761-P93 mammary carcinoma or Lewis lung carcinoma tumors. Human macrophages stimulated with NDV Ulster have also been shown to kill various types of human tumor cells.
In another study, Reviewed in  intratumoral injection of NDV strain Ulster into growing ESb tumors in immunocompetent mice led to a cessation of tumor growth and an absence of metastases in 42% of treated animals. In the remaining mice, tumor growth and metastatic spread continued at the same rate as in control animals. Reviewed in  Additional results from this study indicated that the anticancer effect in the responding animals was due primarily to the activation of T cells directed against a tumor-specific antigen on ESb cells rather than a virus antigen. Reviewed in 
Other studies with NDV Ulster and the ESb tumor model support the idea that virus proteins inserted in the plasma membrane of NDV-infected cancer cells may help the immune system recognize tumor-specific antigens better, potentially leading to an increased ability to kill uninfected cancer cells and virus-infected cells.[25,28,31,36,37,39] Reviewed in [11,20,43,45,46] At least four studies [25,28,37,39] Reviewed in [45,46] have shown that T cells isolated from mice that have growing ESb tumors can be activated in vitro by co-culture with NDV-infected ESb cells and that the resulting activated T cells possess an enhanced ability to kill uninfected ESb cells in vitro. In addition, two in vivo studies  Reviewed in  have shown that mice injected with NDV-infected, irradiated ESb cells are 30 to 250 times more resistant to later injection with proliferating ESb cells than mice that are initially injected with uninfected, irradiated ESb cells. Furthermore, at least two in vivo studies have demonstrated that vaccination of mice with NDV-infected, irradiated ESb cells after surgery to remove a growing ESb primary tumor can prevent the growth of metastatic tumors in approximately 50% of treated animals.[31,36] Reviewed in [11,43,45,46] When the surviving mice were subsequently injected with proliferating ESb cells, they all remained free of cancer, indicating that the NDV/tumor cell vaccine had conferred anticancer immunity.[31,36] Reviewed in [11,45,46] Similar results were obtained from in vivo studies that employed the mouse B16 melanoma model, the mouse Lewis lung carcinoma model, or the guinea pig L10 hepatocellular carcinoma model.
One factor that may influence the effectiveness of NDV/tumor cell vaccines is overall tumor burden. Results obtained with the B16 mouse melanoma model suggest that these vaccines are less effective in individuals with advanced metastatic disease.References
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