نبذة مختصرة : Pancreatic cancer (PC) is the seventh most deadly cancer with a survival rate at five years below 10%. Surgery remains the best treatment for PC but is most often incongruous due to the progressiveness of the disease. Other treatments such as radiotherapy and chemotherapy regimens are also available but are accompanied by numerous side effects and the risk of innate or acquired resistance. In addition, these treatment protocols could be inadequate when faced with the cellular and molecular complexity of pancreatic cancers and their tumour microenvironment (TME). Due to these shortcomings, other treatment options are being studied, including targeted therapies, immunotherapies and gene-based therapies. Oncolytic viruses (OV) are novel cancer gene therapies that are 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 OV replication and efficacy in cancer cells. We selected SG33 as a prototypic, new OV that derives from wild-type Myxoma virus (MYXV) Lausanne Toulouse 1 (T1) that was used as control. We equipped SG33 and T1 genomes with the ANCHOR system and infected a panel of cell lines. The ANCHOR system is composed of a fusion protein (OR-GFP) that specifically binds to a short, non-repetitive DNA target sequence (ANCH) and spreads onto neighbouring sequences by protein oligomerization. Its accumulation on the tagged viral DNA results in the creation of fluorescent foci. 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 clustering into horse-shoe shape replication centres, and (iii) SG33 replicates to higher levels as compared to T1. Lastly, as a translational proof-of-concept, we benchmarked SG33 replication and oncolytic ...
Processing Request
No Comments.