Introduction: Bladder cancer is associated with high risk of tumour recurrence and therapeutic resistance. Cancer stem cells (CSC) within a particular tumour are postulated to drive tumorigenesis and influence tumour behaviour. Recent studies have shown that Newcastle disease virus (NDV) is able to selectively kill and exert a strong oncolytic effect against various cancer types. However little is known about the oncolytic effect of NDV against CSC. In this study, the oncolytic effect of NDV against putative bladder CSC was examined. Methods: Putative bladder CSC was selectively grown in the form of 3D-spheroids from six different bladder cancer cell lines. The spheroid cells were characterised for their stemness properties to ensure that these cells truly represent CSC. This was conducted via the analysis of CSC associated genes and cell surface markers expression. Subsequently, the oncolytic effect of the wild-type NDV-AF2240 strain against the bladder cancer spheroids was investigated. Results: All the spheroids expressed significantly high levels of CSC-associated genes. Flow-cytometry analysis revealed that the expression pattern of the CSC-associated surface markers was different in the spheroid cells; suggesting heterogeneity in the expression signatures of these cells. The infection of spheroids with NDV showed that the NDV was able to target bladder cancer spheroids but there was a spectrum of response across the different spheroids. Intriguingly, NDV was able to persistently infect bladder cancer spheroids that were not sensitive towards NDV infection as the presence of NDV viral genes were detected in the spheroid cells. The NDV persistently infected bladder cancer spheroids were resistant to superinfection and developed an antiviral state by expressing low levels of interferon-beta (IFN-b). NDV persistency of infection affects the process of epithelial to mesenchymal transition (EMT) of cancer cells as the spheroid forming ability of an established NDV persistently infected bladder cancer cell line, EJ28-PI was shown to be impaired. The EJ28-PI cells expressed significantly high levels of the EN2 gene. Knockdown of the EN2 expression reduced the viability of EJ28-PI cells; suggesting a role for EN2 in mediating NDV persistency of infection in cancer cells. Conclusion: Bladder CSC gene expression signatures influence the efficacy of NDV-mediated oncolysis. Our current work is focused on identifying genes and signalling pathways that influence NDV-mediated oncolysis us-ing whole-transcriptomic sequencing. The findings of this study can potentially be used to enhance the efficacy of NDV-mediated oncolysis and accelerate the translation of NDV as an oncotherapeutic agent in the clinic.
Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme which catalyzes the formation of cyclodextrin from starch. The production of CGTase using lactic acid bacterium is an attractive alternative and safer strategy to produce CGTase. In this study, we report the construction of genetically modified Lactococcus lactis strains harboring plasmids that secrete the Bacillus sp. G1 β-CGTase, with the aid of the signal peptides (SPs) SPK1, USP45 and native SP (NSP). Three constructed vectors, pNZ:NSP:CGT, pNZ:USP:CGT and pNZ:SPK1:CGT, were developed in this study. Each vector harbored a different SP fused to the CGTase. The formation of halo zones on starch plates indicated the production and secretion of β-CGTase by the recombinants. The expression of this enzyme is shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. A band size of ∼75 kDa corresponding to β-CGTase is identified in the intracellular and the extracellular environments of the host after medium modification. The replacement of glucose by starch in the medium was shown to induce β-CGTase production in L. lactis. Although β-CGTase production is comparatively low in NZ:SPK1:CGT, the SP SPK1 was shown to have higher secretion efficiency compared to the other SPs used in this study.