Sendai virus as a novel oncolytic virus for urothelial carcinoma
Andrew J. Charap, BS1, John Heard, MS2, Matthew Lin, BS1, Jorge Daza, MD1, John Sfakianos, MD1, Amir Horowitz, PhD1.
1Icahn School of Medicine at Mount Sinai, New York, NY, 2SUNY Downstate College of Medicine, New York, NY.
Introduction: The use of oncolytic viruses is an active area of investigation in the field of urologic oncology. Several clinical trials have been completed investigating recombinant adenovirus as a second-line treatment for non-muscle invasive bladder cancer after BCG-failure. Sendai virus is a murine paramyxovirus with no innate virulence in humans but with documented oncolytic activity and tumor cell specificity. Sendai virus treatment has been investigated in several murine solid tumor models, but to date has not been studied in bladder cancer.
Methods: We modified the genome of the Sendai virus to express green-fluorescent protein (GFP) as an indicator of successful viral infection. We then infected the urothelial carcinoma cell line 639V with Sendai virus at various titrations. Three days post-infection we performed a flow cytometric analysis to quantify oncolytic activity and changes in cell-surface protein expression. Finally, we infected primary urothelial carcinoma cells derived from pathologic T1, high grade urothelial tumor in vitro.
Results: At three-days post-infection, we observed GFP fluorescence in 639V cells exposed to the Sendai virus at titers greater than 1000 infectious units (IU). Successful viral infection, defined as a positive GFP-signal during cell acquisition via flow cytometry, directly correlated with increasing viral titer (Figure 1). Higher viral titers associated with increased 639V cell death. We observed upregulation of the immune checkpoint PD-L1, in addition to increased expression of the MHC class I proteins HLA-A, B, and C (Figure 2). We observed gross fluorescence of primary tumor cells 72-hours post-infection (Figure 3) using fluorescence microscopy.
Conclusion: Functionally wild-type Sendai virus was capable of both infecting and killing 639V urothelial carcinoma cells in vitro. Flow cytometric analysis revealed dynamic changes in expression of important surface proteins after infection, including PD-L1. These proof-of-concept experiments demonstrate the feasibility of Sendai virus as an alternative to adenovirus for the treatment of non-muscle invasive bladder cancer.
Figure 1: 639V cells exposed to GFP-expressing Sendai virus were analyzed 72 hours post-infection to determine successful infection and cell death. A: Values indicate the median fluorescence intensity (MFI) of 10000 639V cells at a given quantity of infectious units (IU). B: Cell death was determined using an amine viability dye. C: Histograms displaying MFI of HLA-A/B/C and PD-L1 of 639V cells 72-hours post exposure to Sendai virus, gated on live cells.
Figure 2: Gross fluorescence of Sendai-infected primary tumor cells derived from a pathologic T1, high-grade tumor after cystectomy, visualized using fluorescence microscopy.
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