**2. Major Challenges with Current PDAC Treatments**

Since 1997, gemcitabine-based chemotherapy has been the standard first-line treatment for patients with unresectable locally advanced, or metastatic pancreatic cancer with a median survival rate of 4.4–5.6 months, especially when patients are not healthy enough for combination therapies [14]. Gemcitabine (dFdC) is an analog of deoxycytidine and a pro-drug that, once transported into the cell, must be phosphorylated by cellular deoxycytidine kinase to gemcitabine diphosphate (dFdCTP) and gemcitabine triphosphate (dFdCTP), both of which can inhibit processes required for DNA synthesis. Other commonly used chemotherapies for pancreatic cancer include 5-fluorouracil (5-FU), oxaliplatin, albumin-bound paclitaxel, capecitabine, cisplatin, irinotecan, and docetaxel [15,16]. Although several gemcitabine-based combination treatments exist, most have not considerably improved survival. While some combinatorial chemotherapy treatments, such as gemcitabine with erlotinib, have demonstrated potential for longer patient survival, the majority of patients eventually experience tumor progression due to the development of resistance, and therefore novel therapies are required, especially those that do not rely solely on chemotherapeutic drugs [17,18].

The mechanisms of de novo or inherent resistance of PDACs to chemo- or radiotherapeutics are not well understood. Several factors have been demonstrated to contribute to such resistance, including (i) multiple factors associated with the nature of the PDAC tumor microenvironment (TME) [19,20]; (ii) nucleoside transporters or/and nucleoside enzymes affecting drug uptake and metabolism [21]; (iii) hypoxia-inducible factor-1 alpha (HIF-1α) regulated glucose metabolism [22]; (iv) stromal-derived Insulin-like Growth Factors (IGFs) [23]; (v) abnormal expression of tumor-associated mucin proteins [24]; (vi) IFN-related DNA-damage resistance signature (IRDS) of some tumors [25]. The understanding of chemoresistance of PDACs to chemotherapy is very important, as at least some of these mechanisms could be also contributing to the resistance of PDACs to OV therapy.

The success of any treatment for PDAC is further complicated by the TME of PDAC, which is characterized by dense stroma comprised of abundant fibroblasts, hypoxia, and sparse vasculature. Moreover, the infiltration of tumor-promoting immune cells mediates immune evasion and promotes tumor progression. The stroma surrounding the tumor is primarily composed of pancreatic stellate cells (PSCs) which are activated by secreted factors such as TNFα, TGF-β, and interleukins 1, 2, 10, and themselves secrete mucins, collagen, fibronectin, and laminin in addition to some other factors, forming a thick extracellular matrix (ECM). This composition generates an incredibly dense physical barrier, to both host immune cells and potential therapeutics while also increasing interstitial pressure, which, when combined with sparse vasculature, forms a hypoxic environment, further inhibiting immune cells in terms of recruitment and effectiveness. PI3K/Akt, a key downstream mediator of many receptor tyrosine kinase signaling pathways involved in cell proliferation, migration, and inhibition of apoptosis, is phosphorylated under hypoxic conditions, along with MAPK (Erk), which regulates cell proliferation in response to various growth factors, which have been associated with resistance to gemcitabine [26,27]. The limits on antitumor immune cell recruitment also leads to T-cell exhaustion resulting in loss of cytotoxic effector function and further limits appropriate immune responses. SDF-1α/CXCR4 signaling-induced activation of the intracellular FAK-AKT and ERK1/2 signaling pathways and a subsequent IL-6 autocrine loop in cancer cells can further increase chemoresistance [28].

The low expression of nucleoside transporters (NT) and inactivity of nucleoside enzymes (NE) both affect the activity of gemcitabine. Low expression of a nucleoside transporter hENT1 restricts the uptake of gemcitabine, preventing its incorporation into the DNA of replicating cancer cells, and high expression of hENT1 is related to longer overall survival in pancreatic cancer patients [29,30]. The inactivation of deoxycytidine kinase (dCK), an enzyme responsible for the initial phosphorylation of gemcitabine, also mediates resistance. dCK is often inactivated in gemcitabine-resistant PDAC lines [31], and knockdown of dCK has been shown to lead to the development of resistance [32], while expression of a DCK transgene (along with uridine monophosphate kinase) sensitized pancreatic cancer cells to gemcitabine [33].

Pancreatic cancers metabolize glucose at higher rates and show higher expression of HIF-1α positively correlated with gemcitabine resistance [34,35]. HIF-1α increases glucose uptake and metabolism in the cell and is stabilized by MUC1, a common biomarker for cancers including PDAC [36]. Knockdown of HIF-1α in gemcitabine-resistant cells reduced tumor cell survival following gemcitabine treatment, and treatment with digoxin, and HIF-1α inhibitor, reduced glucose uptake and cell survival in cells treated with gemcitabine [37]. The increased glucose uptake under hypoxic conditions feeds into the glycolysis pathway

and increases biomass; however, the exact mechanisms by which HIF-1α reduces sensitivity to chemotherapeutics have yet to be determined.

In addition, stromal-derived IGFs activate the insulin/IGF1R survival signaling pathway, reducing responsiveness to chemotherapeutics [38]. One proposed mechanism describes crosstalk between activated Insulin/IGF signaling pathways in PDAC. IGF-1 and IGF-1R, which are known to be abundantly expressed in the PDAC tissue, can stimulate β-cell proliferation and increase β-cell mass, increasing basal insulin production which may alter the trophic effects of the endocrine cells on the exocrine cells. Endocrine βcells that express oncogenic K-ras can also be one potential progenitor for PDAC under chronic tissue inflammation [39]. This is further supported by evidence that demonstrates macrophages and myofibroblasts are the two major sources of IGFs within the pancreatic tumor microenvironment, and that chemoresistance is increased when cytotoxic agents increase M2-like macrophage infiltration [23]. For any novel therapies to be effective, they should be able to address most if not all of these challenges.

The structural composition of mucins produced by cells in certain cancers, such as breast and pancreatic cancers, has been suggested to limit immune cell recognition by blocking infiltration [40]. Similarly, the dense mucin mesh prevents cellular uptake of chemotherapeutics like gemcitabine and 5-FU within the tumor. MUC1 and MUV4 are overexpressed and aberrantly glycosylated in the majority of pancreatic tumors [41]. Kalra et al. demonstrated that the inhibition of mucin *O*-glycosylation enhanced the cytotoxic effects of 5-FU against human pancreatic cancer cell lines, but not against the mucin-deficient cell line [40]. They suggest that preventing the formation of the mucin facilitates the diffusion of drugs across the compromised mucus layer, improving intracellular drug uptake and enhancing cytotoxic drug action. Elevated MUC1 and MUC4 expression have also been correlated with greater degrees of resistance to gemcitabine [42]. It was also demonstrated that gemcitabine-resistant cells had accentuated the non-oxidative branch of the pentose phosphate pathway activity and increased pyrimidine biosynthesis, conferring resistance by increased dCTP production. MUC1 and MUC4 overexpression was also shown to upregulate *mdr* genes in pancreatic cancer cells, including *ABCC1, ABCC3, ABCC5,* and ABCB1 genes [41,43]. MUC4 expression was shown to be conversely correlated with the expression of hCNT1 and hCNT3 transporters, preventing uptake of chemotherapeutic drugs like gemcitabine, and hCNT1 is upregulated when MUC4 is inhibited, resulting in increased drug sensitivity [44]. Finally, MUC4-overexpressing CD18/HPAF-Src were not sensitive to gemcitabine, conferring resistance and survival advantages through erbB2-dependent and anti-apoptotic pathways [45]. Altogether, mucins including MUC1 and MUC4 have been demonstrated to be highly overexpressed and aberrantly glycosylated in pancreatic cancer cells, conferring resistance to various chemotherapies and the downregulation of these oncoproteins may represent a promising therapeutic strategy for reversing chemoresistance and reducing tumor progression and mass.

Type I IFN signaling is upregulated in some tumors responding to chemotherapy and can have antitumor as well as pro-tumor effects. The expression of a type I IFN-related DNA-damage resistance signature (IRDS) was reported to correlate with resistance to chemotherapy and radiotherapy in multiple cancer types. In breast cancer, the IRDS has been implicated in the development of chemoresistance, which may be another potential mechanism of resistance in PDACs as well [25]. The STAT1/IFN pathway transmits a cytotoxic signal either in response to DNA damage or to IFNs, such as in the case of viral infection. Cells with an IRDS (+) profile show constitutive activation of the STAT1/IFN pathway. Interestingly, this chronically activated state of the STAT1/IFN pathway may select against transmission of a cytotoxic signal, instead resulting in pro-survival signals mediated by STAT1 and other IRDS genes [25]. In agreement with this mechanism, STAT1 is highly upregulated in many cancers, including PDAC, and protects SCC-61 cells from ionizing radiation-mediated death [46]. STAT1 may also induce resistance with other DNA damage-based treatments, such as gemcitabine, and may transduce survival/growth signals that enhance tumor survival under some conditions [47]. Sensitivity to DNA damage is coupled with sensitivity to IFNs such that selection for resistance to one may lead to resistance to the other [48], which could prove to be a problem with not only chemoand radiotherapies, but OV treatments as well.
