**7. Future Directions and Conclusions**

As discussed previously, several factors pose a series of challenges that will determine whether OV is suitable for cancer treatment, especially for PDAC, where any treatment is further complicated by the TME, which is characterized by dense stroma comprised of abundant fibroblasts, hypoxia, sparse vasculature, as well as infiltration of tumor-promoting immune cells mediating immune evasion and tumor progression. The ideal OV treatment should allow for sufficient delivery and penetration of PDACs with the virus, induction of adaptive antitumor responses, and prevention of premature OV clearance by host antiviral response. It is unlikely that any monotherapy could address all these challenges, and future effective OV-based treatments will likely be combinatorial (chemo-virotherapy, radiovirotherapy, chemo-radio-virotherapy, chemo-radio-immuno-virotherapy, etc.). Many of VSV-based combinatorial approaches have been described in our previously published reviews [178,180], and some additional approaches will be discussed below [195].

First of all, to study and address all these challenges, the ideal model systems should employ immunocompetent animals (to examine antiviral as well as antitumor immune responses) and be able to monitor not only tumor growth and spread, but also OV spread.

Figure 3 illustrates the system that we currently use in our laboratory to investigate various VSV-based OV treatments against PDAC.

**Figure 3.** Syngeneic Model of PDAC in Mice. This figure shows the method of development for the murine model of PDAC. In this model, C57BL6 mice are used both for the development of the tumors and to evaluate the treatment in vivo. The figure was created by authors with BioRender software (BioRender.com).

PDAC is a highly heterogenic disease, and our studies have demonstrated dramatic differences between different human PDAC cell lines in their permissiveness to VSV and other OVs [50,51]. Future studies should define distinct subtypes of PDACs to develop personalized treatment strategies for different types of PDACs [3,196,197]. Although there is still no consensus classification for clinical application, some treatments work better against a particular PDAC subtype. For example, patients who had a germline BRCA mutation had significantly longer progression-free survival with maintenance a PARP inhibitor olaparib than with placebo [198]. It could be interesting to test combinations of OVs with olaparib against PDACs that have the BRCAness phenotype [10]. Interestingly, at least two studies showed the increased efficacy of OV therapy for thyroid carcinoma [199] and glioblastoma [200] when OV was combined with olaparib.

One of the major challenges for any PDAC treatment is insufficient drug delivery into the tumors because PDACs are hypovascular, densely packed with ECM components, have a high intratumoral tissue pressure, and very low tumor perfusion. Several previously

developed approaches could be used to improve OV delivery into PDAC. For example, administration of a combination of cilengitide (angiogenesis inhibitor) and verapamil (Ca2+ channel blocker) promoted tumor angiogenesis, while improving gemcitabine delivery and therapeutic efficacy in mice [201]. Additionally, the angiotensin inhibitor losartan was shown to increase perfusion, drug and oxygen delivery [202]. A more recent study highlighted the potential importance of ROCK inhibition using the oral inhibitor fasudil for dual targeting of tumor tension and vasculature [203]. The administration of fasudil, a Rho-kinase inhibitor, and vasodilator, reduced intratumoral fibrillar collagen, improved sensitivity towards gemcitabine/nab-paclitaxel, and reduced metastasis formation on gemcitabine/abraxane treatment [203]. At least some of these drugs could potentially improve OV therapy when used in combination with VSV or other OVs.

The role of the stromal cells during OV therapy is still unclear, and it is likely dependent on the subtype of the particular PDAC. At least under certain conditions, the stromal cells could play a positive role during OV therapy by dampening antiviral responses within tumor and thus stimulating OV replication and OV-mediated oncolysis [204].

Other areas for development include approaches with a focus on antitumor immune stimulation. The TME of many cancers, including PDAC, is known to be immunosuppressive, due to various factors including a dense, fibrotic composition and a hypoxic environment that prevent access and activation of immune cells within the tumor [205,206]. Adoptive T-cell therapy augments the potency of T-cells by chaperoning virus into the tumor [120], overcoming the stromal barrier. Antigen-specific T-cells that were loaded with VSV-∆M51 can also be used to produce viral infection, replication, and subsequent oncolysis, as well as producing a proinflammatory environment that helped suppress the immunosuppressive nature of the TME. Immune tolerance mechanisms have been implicated as the main barrier to effective antitumor immunotherapy [207], and the natural flora of the gut has been indicated to possess the ability to exert influence over the immune response of the TME, resulting in immune tolerance that promotes tumor growth and development. Future experiments should examine the role of the natural flora in the efficacy of OV therapy for PDAC.

**Author Contributions:** M.C.H., D.W.G., and V.Z.G. contributed to the design, writing and editing of this manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by grant 1R15CA238864-01 to V.Z.G. from the National Cancer Institute, National Institutes of Health (Bethesda, MD).

**Acknowledgments:** All figures were created by authors with BioRender software (BioRender.com).

**Conflicts of Interest:** The authors declare no conflict of interest.
