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.[1-14] Further, several of these studies suggest that lytic strains of NDV are also oncolytic, and one study has demonstrated that expression of the RAC1 gene is necessary for NDV replication.
Lytic strain 73-T 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]. It killed normal human lung fibroblasts in vitro at the same rate that it killed cancer cells. However, this strain did not kill human B-cell lymphoma (i.e., Burkitt lymphoma) cells in vitro  and did not kill normal, proliferating human white blood cells or normal human skin fibroblasts in vitro.[3,6,8]
Lytic strain Roakin has been reported to kill human lymphoma B cells and T cells transformed in vitro from a Hodgkin lymphoma patient four to five times faster than it killed normal, resting human white blood cells.[4,5] This strain killed normal, proliferating human white blood cells in vitro, although at a lower rate than in cancer cells.
Overall, these results suggest that the lytic strains of NDV replicate well in some types of normal cells and replicate poorly in some types of cancer cells. These data and the absence of serious illness in individuals infected with NDV [1-3,10,13,17-22]) are consistent with the view that NDV may replicate 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”).
This strain does not replicate efficiently in normal human white blood cells in vitro. Other experiments have shown that NDV Ulster can kill infected cells [14,26] and that it can replicate in human cancer cells regardless of cell cycle.[7,21]
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. Injection produced 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 inhibited 68% to 96% 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 was not affected, suggesting that the virus was unable to disseminate widely throughout the body.[11,14,21]
In the above-mentioned neuroblastoma xenograft study, strain 73-T replicated over time in tumor tissue but replicated poorly when injected into the thigh muscle of athymic, nude mice. This finding is consistent with the proposal that NDV replicates more efficiently in cancer cells than in most normal cells.
In another nude mouse study, strain V4UPM inhibited the growth of some cell lines of subcutaneously injected human glioblastoma multiforme 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 yet another nude mouse study, a single intraperitoneal injection of strain 73-T in mice bearing human neuroblastoma xenografts resulted in complete, durable tumor regressions in 9 of 12 (75%) of the treated mice.
Athymic, nude mice make small numbers of T cells, and they produce interferons, natural killer cells, and macrophages.[11,27,28] It is possible 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.
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.[6,8,11,21,26,29-48] A few of these studies used human cells,[6,8,21,30,31,39,42,43,45,48] but most used animal cells and animal tumor models.[6,8,11,21,26,29,31-38,40,44-47]
Two of these in vitro studies demonstrated that infection of human immune cells with NDV causes the cells to produce and release cytokines interferon-alpha and tumor necrosis factor (TNF)-alpha.[6,8] In one of these studies, infection of human cancer cells with NDV made the cells more sensitive to the cytotoxic effects of TNF-alpha.
Some 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.[8,30,31,49] 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.[21,31]
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.[21,30,39,45,48,50,51] It has been reported that this improved interaction leads to better T cell activation.[21,30,39,45,48] 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.[21,30,39] 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.[21,30,39,45,48]
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;[11,21,26,29,32,36-38,40,41,44-48] however, additional experiments have utilized one or more of the following tumor models: mouse B16 melanoma, mouse Lewis lung carcinoma,[33,36] 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, 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. 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.
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.[11,21,26,29,32,37,38,40,44,46,47] At least four studies [26,29,38,40,46,47] 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 [11,32] 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.[11,32,37,44,46,47] 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.[32,37] 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.
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