**5. Understanding Molecular Mechanisms of Responsiveness and Resistance of PDACs to VSV-Based OV Therapy**

VSV is a prototypic nonsegmented negative-strand (NNS) RNA virus (order *Mononegavirales*, family *Rhabdoviridae*). VSV is a promising oncolytic virus against various malignancies, and it has several advantages as an OV [178–180]: (i) its basic biology and interaction with the host have been extensively studied. The oncoselectivity of VSV is mainly based on VSV's high sensitivity to Type I interferon (IFN) mediated antiviral responses (and therefore inability to replicate in healthy cells), while it can specifically infect and kill tumor response cells, most of which lack effective Type I IFN responses; (ii) although WT VSV can cause neurotoxicity in mice, nonhuman primates, several VSV recombinants, including VSV-∆M51, have been generated which are not neurotropic but retain their OV activity; (iii) VSV has a broad tropism for different types of cancer cells (including PDACs), as its primary mode of entry into a host cell utilizes binding of the VSV-G protein to LDLR, which is ubiquitous, and VSV-G is also capable of using other common surface molecules for cell entry [179]; (iv) there is no preexisting immunity against VSV in most humans; (v) replication occurs in the cytoplasm without risk of host cell transformation; (vi) cellular uptake occurs rapidly; (vii) VSV has a small, easily manipulated genome, and novel VSV-based recombinant viruses can be easily engineered via reverse genetics to improve oncoselectivity, safety, oncotoxicity, and to work synergistically with host immunity and/or other therapies in a specific tumor environment (e.g., PDAC); (viii) as other members of the order *Mononegavirales*, and compared to positive-strand RNA viruses, VSV is less likely to mutate, and our recent study demonstrated long-term genetic stability of VSV recombinants carrying large transgenes [62]. All these and other advantages make VSV a promising candidate OV for PDAC treatment, and we have shown that VSV is effective against the majority of PDAC cell lines in vitro and in vivo [50,51]. Importantly, several phase I clinical trials using VSV against different malignancies are in progress (ClinicalTrials.gov for trials NCT03647163, NCT02923466, NCT03120624, NCT03865212, and NCT03017820).

VSV exhibits inherent oncotropism based largely on defective or reduced type I IFN responses, as specific genes associated with type I IFN responses are downregulated or functionally inactive [165,181]. In addition, IFN signaling can be inhibited by MEK/ERK signaling or by epigenetic silencing of IFN-responsive transcription factors IRF7 or IRF5 [182,183]. However, some PDACs do not have these defects and resist VSV infection like normal cells, which are sensitive to IFN-α treatment and capable of secreting type I IFNs following VSV infection [184].

There has been a demonstration of neurotoxicity in mice infected intranasally or intracranially, demonstrating a need for methods of improvement of VSV oncoselectivity and neurotropic safety without compromising oncolytic ability. There are at least eight approaches demonstrated to address these needs [178–180]: (i) mutating the VSV M protein; (ii) VSV-directed IFN-β expression; (iii) attenuation of VSV through disruption of normal gene order; (iv) mutating the VSV G protein; (v) introducing targets for microRNA from normal cells into the VSV genome; (vi) pseudotyping VSV; (vii) experimental adaptation of VSV to cancer cells; and (viii) using semi-replicative VSV. Most of the studies in our laboratory focus on VSV-∆M51 recombinants containing a deletion of the methionine residue at position 51 of the M protein, VSV-∆M51. This mutation results in an inability of VSV-M to inhibit nucleus-to-cytoplasm transport of cellular mRNA, including antiviral transcripts, in normal cells with functional antiviral signaling [185,186].

Our laboratory has characterized numerous human PDAC cells lines and discovered a wide range of susceptibility and permissiveness of different PDAC cell lines to VSV and other tested OVs [50,51,56,58–61]. The range includes "super-permissive" cell lines (such

as MIA PaCa-2 and Capan1), "super-resistant" cell lines (such as HPAF-II, Hs766T), and well as many cell lines in between (such as SUIT2 and AsPC-1). Below we describe different mechanisms associated with the resistance of some PDACs to VSV.
