3.1.1. PDAC Cell Lines

Numerous human PDAC cell lines have been established and can be characterized by their distinctive genotypic and phenotypic variations, including their relative permissiveness or resistance to OV infection [50–52]. Utilizing cell lines as a model system offers several advantages for studying PDAC, including easy propagation and indefinite growth. These features represent a cost-effective and consistent model that can easily be used to study molecular mechanisms and biomarkers of resistance or permissiveness of PDAC cells to OVs [50,52]. While cell line-based approaches represent quick, straightforward, and consistent models, several features reduce their clinical translatability. First, the homogeneous nature of cell line models fails to accurately represent the heterogeneous nature of typical in vivo tumors, including PDAC [53]. Indeed, cell lines are under selection

for mutations and phenotypes allowing growth advantage in a monolayer, however, the selection mechanisms in vivo are different [54]. In fact, established PDAC cell lines not only lose the heterogeneity present in the primary tumor, but the evolution of these cell lines to grow in culture may obscure genetic aberrations present in the primary tumor [52]. Additionally, many PDAC cell lines are originated from metastasized disease, so the ability to study PDAC progression is severely limited. Secondly, cell lines cultured in a monolayer lack the important three-dimensional structure and function as seen in vivo [54]. Thirdly, the PDAC cell line model fails to represent the TME, which is understood to be a dynamic player in PDAC tumor progression [54]. Lastly, cultured cell lines lack selection pressure from the host adaptive immune system, thus leaving mutations necessary for evading host immunity underrepresented. The outcome of the OV therapy depends on the complex interaction between tumor cells, virus, and innate and adaptive immune systems of the host. One of the desirable outcomes of this interaction is OV-mediated stimulation of immune response against tumor cells. However, normal PDAC stromal cells can induce innate antiviral responses against OV replicating in tumor cells, and adaptive immune response can prematurely clear virus infection instead of targeting tumor cells. Unfortunately, cell culture-based models cannot address these important issues.

Even given the disadvantages of the cell line model, it is a good starting proofof-principle platform that has allowed our group to investigate mechanisms regarding responsiveness or resistance to OV therapy [50,51,55–62]. For example, our group is interested in understanding why/how certain PDAC cell lines are more resistant to VSV infection than other PDAC cell lines [50]. The cell line model in this aspect allows for reliable comparative measurements of virus replication, spread, and cell lysis. Additionally, the cell line model allows for relatively straightforward screening of both cellular and viral genes and proteins of interest. Cell line models allow for efficient virus tractability through reporter genes such as GFP [63]. Additionally, cell culture-based systems allow innovative imaging approaches for single-cell real-time analysis of OV replication and efficacy in pancreatic cancer cells [64].

Depending on the nature of the investigation, either human or murine PDAC cell lines can be used. Human PDAC cells, derived from primary pancreatic tumors or "cell linederived xenograft (CDX)" models, have been used since as early as 1963 to characterize and test anti-cancer drugs [65]. The use of human PDAC cells provides the obvious benefit of having the same genetic makeup of the human disease, including key PDAC mutations in KRAS, CDKN2A, p53, and SMAD4 [3]. Although using human PDAC cell lines as a model has numerous informative applications, this model has a limited ability for consequent in vivo studies. If using human PDAC cell lines, researchers are limited to T cell-deficient nude athymic (nude), or B and T cell-deficient severe combined immunodeficient (SCID) mice [66,67]. As will be later discussed in this review, while such in vivo models have many applications, they lack the ability to assess the role of the adaptive immune system against PDAC as well as OV, both important when determining the efficacy of potential OV therapeutics.

To circumvent this caveat, murine PDAC cell lines can be used. Using murine PDAC cells derived from murine PDAC tumors allows researchers to establish PDAC in immunocompetent mice, allowing for the study of OV therapy in the presence of the functional adaptive immune system. One notable drawback to this model is the potential genetic dissimilarity (and thus clinical translatability) between mouse and human PDAC cells.

Generally, murine PDAC cell lines are originated from mice that have PDAC due to either chemical induction or genetic modifications in genetically engineered mouse model (GEMM). Once commonly used PDAC cell line that was cultured from a chemically induced PDAC tumor is Panc02, which has been extensively used for PDAC research [68]. The PDAC tumor from which it was derived was established by implanting 3-methylcholanthrene (3-MCA)-saturated threads of cotton in the pancreas of C57BL/6 mice. Despite its long-term use in evaluating various therapeutic strategies, Panc02 cells lack clinical significance for PDAC due to the absence of some common mutations found in human

PDAC. More relevant murine PDAC cell lines are originated from the KPC mouse model of PDAC (LSL- KrasG12D; LSL-Trp53R173H; *Pdx*1-*Cre*) [68]. KPC mice develop spontaneous PDAC which closely resemble the genetics, physiology, tumor progression, and metastatic hallmarks of human PDAC [69], and will be described in more detail later in this review.
