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First-in-class imaging approach for single-cell, real-time analysis of oncolytic virus replication and efficacy in cancer cells
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Edité par CCSD -
International audience. Purpose: Oncolytic viruses (OV) are novel cancer gene therapies that are quickly moving toward the forefront of modern medicines. However, their full therapeutic potential is hindered by the lack of convenient and reliable strategies to visualize and quantify OV growth kinetics and therapeutic efficacy in live cells. Here, we present a first-in-class imaging approach for single-cell, real-time analysis of oncolytic virus replication and efficacy in cancer cells.Experimental Design: OV are produced in a BSL-2 facility. The Anchor system is composed of a fusion protein (OR-GFP) that specifically binds to a short, nonrepetitive DNA target sequence (ANCH) and spreads onto neighboring sequences by protein oligomerization. Its accumulation results in fluorescent foci2,3. Imaging for viral genomes is performed using Incucyte Zoom, Thermo CX7, and Ivis spectrum.Results: We selected SG33 as a prototypic, not previously published, OV that derives from wild-type Myxoma virus (MYXV). Lausanne Toulouse 1 (T1) MYXV strain1 was used as control. We equipped SG33 and T1 genomes with the ANCHOR system and infected a panel of cell lines. We found that (i) SG33 and T1-Anchor DNA can be readily detected and quantified by live imaging, (ii) both OVs generate perinuclear replication foci after infection, and (iii) SG33 replicates to higher levels compared to T1. We next performed a pilot, high content study using the Prestwick library and identified compounds regulating SG33-Anchor and T1-Anchor infection. Last, as a translational proof-of-concept, we infected a panel of primary cells derived from patients with pancreatic cancer (PDAC), a disease with no cure, with SG33-Anchor. We robustly and rapidly classified primary cancer cells as resistant or permissive, both at the population and the single-cell levels. We engrafted human PDAC primary cells in athymic mice and found that SG33-Anchor infection and replication is readily quantifiable in tumors, in vivo.Conclusion: Collectively, we provide herein for the first time a novel strategy to quantify each step of OV infection in live cells in real-time. First successful examples are side-by-side comparison of OV efficacy, robust and rapid identification of positive regulators of viral replication, but also viral antidotes, and first steps toward theranostic platforms for patient with incurable neoplasms. Thus, this approach has the potential to rationalize the use of OV for the benefit of patients with diseases with no cure.
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