Transient infection of freshly isolated human colorectal tumor cells by reovirus T3D intermediate subviral particles
Reovirus T3D preferentially kills tumor cells expressing Ras oncogenes and has shown great promise as an anticancer agent in various preclinical tumor models. Here, we investigated whether reovirus can infect and kill tumor cell cultures and tissue fragments isolated from resected human colorectal tumors, and whether this was affected by the presence of endogenous oncogenic KRAS. Tissue fragments and single-cell populations isolated from human colorectal tumor biopsies were infected with reovirus virions or with intermediate subviral particles (ISVPs). Reovirus virions were capable of infecting neither single-cell tumor cell populations nor small fragments of intact viable tumor tissue. However, infection of tumor cells with ISVPs resulted in transient viral protein synthesis, irrespective of the presence of oncogenic KRAS, but this did not lead to the production of infectious virus particles, and tumor cell viability was largely unaffected. ISVPs failed to infect intact tissue fragments. Thermolysin treatment of tumor tissue liberated single cells from the tissue and allowed infection with ISVPs, but this did not result in the production of infectious virus particles. Immunohistochemistry on tissue microarrays showed that junction adhesion molecule 1, the major cellular reovirus receptor, was improperly localized in the cytoplasm of colorectal tumor cells and was expressed at very low levels in liver metastases. This may contribute to the observed resistance of tumor cells to reovirus T3D virions. We conclude that infection of human colorectal tumor cells by reovirus T3D requires processing of virions to ISVPs, but that oncolysis is prevented by a tumor cell response that aborts viral protein synthesis and the generation of infectious viral particles, irrespective of KRAS mutation status.
[Show abstract] [Hide abstract] ABSTRACT: Oncolytic virotherapy is a new strategy for cancer treatment for humans and dogs. Reovirus has been proven to be a potent oncolytic virus in human medicine. Our laboratory has previously reported that canine mast cell tumor and canine lymphoma were susceptible to reovirus. In this study, canine solid tumor cell lines (mammary gland tumor, osteosarcoma and malignant melanoma) were tested to determine their susceptibility towards reovirus. We demonstrated that reovirus induces more than 50% cell death in three canine mammary gland tumors and one canine malignant melanoma cell line. The reovirus-induced cell death occurred via the activation of caspase 3. Ras activation has been shown to be one of the important mechanisms of reovirus-susceptibility in human cancers. However, Ras activation was not related to the reovirus-susceptibility in canine solid tumor cell lines, which was similar to reports in canine mast cell tumor and canine lymphoma. The results of this study highly suggest that canine mammary gland tumor and canine malignant melanoma are also potential candidates for reovirus therapy in veterinary oncology.0Comments 3Citations
- "On the other hand, CIP-p, CIP-m, CTB-p, CNM-m, MacKinley, Moresco, KMeC and CMGD2, that have low or no expression of Ras activity, are highly susceptible to reovirus. These discrepancies were also observed in some of the human cancers [21, 27, 28, 31], canine mast cell tumor  and canine lymphoma  . Indeed, we cannot completely deny that there exist other mechanisms of reovirus susceptibility besides the Ras signaling pathway. "
[Show abstract] [Hide abstract] ABSTRACT: The usage of reovirus has reached phase II and III clinical trials in human cancers. However, this is the first study to report the oncolytic effects of reovirus in veterinary oncology, focusing on canine mast cell tumor (MCT), the most common cutaneous tumor in dogs. As human and canine cancers share many similarities, we hypothesized that the oncolytic effects of reovirus can be exploited in canine cancers. The objective of this study was to determine the oncolytic effects of reovirus in canine MCT in vitro, in vivo and ex vivo. We demonstrated that MCT cell lines were highly susceptible to reovirus as indicated by marked cell death, high production of progeny virus and virus replication. Reovirus induced apoptosis in the canine MCT cell lines with no correlation to their Ras activation status. In vivo studies were conducted using unilateral and bilateral subcutaneous MCT xenograft models with a single intratumoral reovirus treatment and apparent reduction of tumor mass was exhibited. Furthermore, cell death was induced by reovirus in primary canine MCT samples in vitro. However, canine and murine bone marrow-derived mast cells (BMCMC) were also susceptible to reovirus. The combination of these results supports the potential value of reovirus as a therapy in canine MCT but warrants further investigation on the determinants of reovirus susceptibility.0Comments 11Citations
- "This negative correlation was also suggested by Twigger et al., where the oncolytic effects of reovirus was shown to be independent of the epidermal growth factor receptor (EGFR)/ Ras signaling pathway in head and neck cancer cell lines . Furthermore, the lack of association between reovirus susceptibility and the Ras signaling pathway has also been reported in human hematopoietic cancers , colon cancer  and non-small cell lung cancer . Moreover, it is known that the ras mutation in canine tumors is rare , . "
[Show abstract] [Hide abstract] ABSTRACT: Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D) is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A) on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin) mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA) inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.0Comments 14Citations
- "To test whether the jin-1 virus is still dependent on cellular proteases the protease inhibitor E64d was used. If cells are exposed to E64d prior to infection, intact virions are trapped in the endosome while ISVPs can complete the replication cycle [30,31]. To confirm that wt ISVPs are JAM-A independent we exposed the U118MG cells to wt T3D ISVPs and to intact T3D virions (fig. "