*5.1. Upregulated Type I IFN Signaling and Constitutive Expression of a Subset of IFN-Stimulated Genes (ISGs)*

Our extensive analysis of a large number of human PDAC cell lines demonstrates that PDAC cell lines show surprising diversity with regard to their ability to produce and respond to type I IFNs, and the evaluation of IFN sensitivity and IFN-α and IFNβ production within a cell line may be used to predict its responsiveness to oncolytic treatment [50,51] (Figure 2).

**Figure 2.** Permissiveness of PDAC to VSV: Four Different Phenotypes. This figure demonstrates the variability across PDAC in regard to permissiveness to infection by VSV. Permissiveness refers to the cells allowance for viral attachment, infection, and replication. The figure was created by authors with BioRender software (BioRender.com).

Upregulated or residual expression of antiviral genes display four unique phenotypes (Figure 2): (i) no type I IFN production and not responsive to type I IFN, (ii) no type I IFN production but responsive to type I IFN, (iii) type I IFN production and responsive to type I IFN, (iv) super resistant PDACs: type I IFN production, responsive to type I IFN and constitutive expression of many antiviral IFN-stimulated genes (ISGs) [51,56,58].

We also conducted a transcriptome analysis to identify biomarkers for resistance of PDAC cell lines to VSV-∆M51. Of the genes identified, six demonstrate constitutive co-expression in the VSV-resistant cell lines: MX1, EPSTI1, XAF1, GBP1, SAMD9, and SAMD9L [58]. Most of these genes are known to have an antiviral effect. Moreover, shRNA-mediated knockdown of MX1 showed a positive effect on VSV-∆M51 replication in resistant PDAC cells, suggesting that at least some of the identified ISGs contribute to resistance of PDACs to VSV-∆M51 [58]. Finally, we demonstrated that JAK inhibitors effectively break resistance to VSV-∆M51 while affecting very few non-ISGs, suggesting that the constitutive expression of these genes is likely a causative factor for the phenotype of resistance [50,56]. Further evidence that host antiviral response to VSV-∆M51 infection is the source of resistance has been shown in infection with WT VSV, as even cell lines resistant to VSV-∆M51 are permissive to at least some degree to the WT-VSV, which is better able to evade antiviral responses in the host [50,51].
