Recent advances in molecular diagnostics and therapeutic targets for pancreatic cancer

CHAPTER 16

Recent advances in molecular diagnostics and therapeutic targets for pancreatic cancer Ryan Clay, Shadab A. Siddiqi Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, United States

Abstract Pancreatic adenocarcinoma is one of the most aggressive cancers and has a rising incidence and high mortality. The high mortality rate is attributed to a lack of adequate diagnostics, which limits the efficacy of the few currently available treatment options. The poor prognosis of pancreatic cancer emphasizes an increasing demand to develop efficient diagnostic markers and therapeutic approaches. The current review focuses on advances made in the development of robust molecular biomarkers that can be used for diagnosis and targeted therapies. Here, we discuss three major types of molecular markers for the detection of pancreatic adenocarcinoma: serum, tumor tissue, and cancer stem cell markers. Substantial advances have been made to improve our understanding of pancreatic adenocarcinoma at both the cellular and molecular levels, strengthening the possibilities of early detection and targeted therapies; they are discussed in this chapter.

Keywords: Intraductal papillary mucinous neoplasms (IPMNs); Mucinous cystic neoplasms (MCNs); Pancreatic cancer (PC); Pancreatic ductal adenocarcinoma (PDAC); Pancreatic intraepithelial neoplasms (PanINs).

Background Despite an incidence of only 3.2%, pancreatic cancer (PC) is third in overall cancer deaths with an average 5-year survival of only 8.5% [1]. However, mortality can vary widely depending on the size and stage of the tumor at the time of detection [2,3], with 5-year survival of over 80% for cancers smaller than 10 mm [3]. Despite the advantage of early detection for prognosis and survival, the lack of molecular markers for detecting early or premetastatic PC combined with the invasiveness and cost of existing screening methods prohibit routine screening [2e7]. As a result, over 80% of cancers have already metastasized upon initial discovery [1,3]. G.P. Nagaraju, S. Ahmad (eds.) Theranostic Approach for Pancreatic Cancer ISBN 978-0-12-819457-7 https://doi.org/10.1016/B978-0-12-819457-7.00016-5

Copyright © 2019 Elsevier Inc. All rights reserved.

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More advanced molecular diagnostics and treatments are necessary to reduce the burden of this disease. Exocrine tumors of the pancreas are by far the most common (over 95%) [8], with pancreatic ductal adenocarcinoma (PDAC) making up over 90% of those cases [9]. Other, rarer types of PC include acinar cell carcinomas, pancreatoblastomas, and neuroendocrine tumors [10,11]. Despite improvements in diagnostics and therapeutics in other cancers, the process of detecting PC remains difficult and relies largely on visual screening methods [4,5,7]. Although the average exocrine tumor will take more than a decade to metastasize [12], both early-stage cancers and the precancerous lesions from which they arise are largely asymptomatic and, in the case of pancreatic intraepithelial neoplasms (PanINs), they can be too small to detect reliably with magnetic resonance imaging (MRI) or ultrasound [3,5,13]. Most cases are discovered after the manifestation of nonspecific symptoms associated with late-stage metastatic tumors, such as jaundice, abdominal pain, and weight loss [3e5]. Less than 10% of PCs are discovered before they have spread beyond the pancreas, and are often discovered inadvertently or because the tumor has formed in close proximity to and has compressed the common bile duct, pancreatic duct, or mesenteric and celiac nerves, producing symptoms at an earlier stage [14e16].

Pathogenesis of pancreatic ductal adenocarcinoma PDAC is known to arise from one of three precursor lesions: PanINs, intraductal papillary mucinous neoplasms (IPMNs), and mucinous cystic neoplasms (MCNs) [3,13,17]. PanINs are the most common of the three and their progression into PDAC has been well-characterized [3,13,18,19]. They are a promising target for early detection for multiple reasons: they are relatively common and usually benign [3,13,18]; of the estimated 1% of PanINs that will metastasize [18], they will take well over a decade on average to do so [12]; and those that do progress tend to accumulate characteristic mutations and undergo morphological changes in a predictable manner [3,11e13,17,19e21]. There are four hallmark genetic alterations in the progression of PanIN to PDAC: a gain of function mutation in KRAS (84%e96% of PDACs), and the downregulation of tumor suppression genes p16/CDKN2A (90%e95%), TP53 (40%e90%), and SMAD4/DPC4 (50%e55%) [3,7,9,11e13,16,17,22e25]. Interestingly, the pathogenesis of both familial PC, which accounts for less than 10% of cases, and sporadic PC is driven by

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these same four genes [3,19,22]. KRAS mutates early in PanIN pathogenesis and is associated with grade 1 PanINs [3,11,13,17,19]. The most common mutation occurs in codon 12 of exon 2 (with other common mutations in codon 13 and 61), inhibiting KRAS’s GTPase activity and causing it to become constitutively active [11,13,17]. KRAS constitutive activation drives carcinogenesis through the activation of multiple wellknown downstream pathways related to cell growth and proliferation, including the RAFemitogen-activated protein kinase (MAPK) and PI3KAKT signaling cascades [11,17]. It also causes the shortening of telomeres, leading to chromosomal instability [11,13,17]. Next, inactivation of the CDK-inhibiting p16/CDKN2A is seen in PanIN-2 and allows for unchecked progression through the G1-S phase checkpoint [11,13,17]. Later, the loss of TP53 and SMAD4/DPC4 in PanIN-3 results in the loss of DNA damage-induced G2-M cell cycle arrest and the interrupted transduction of proapoptotic signals by transforming growth factor b (TGF-b) and activin, respectively [11,13,17]. Many other less common driver mutations have been reported, including in known protooncogenes Her2 and Akt2 and tumor suppressor genes BRCA1/2, TGFBR1/2, FHIT, serine/threonineprotein kinase 11 (STK11), and ALK4 [16,17,22]. There is variability among the quantity of driver mutations accumulated between tumor cells, with the number of accumulated mutations negatively affecting prognosis [7,26]. IPMNs and MCNs are thought to progress molecularly in a manner similar to that of PanINs, but with some minor differences that will be relevant in the development of future screening and treatment techniques. IPMNs will exhibit loss of function of STK11/liver kinase B1 more commonly than SMAD4, and may express mucin-2 (MUC2) rather than MUC1, which is overexpressed on most PanINs [13,17]. IPMNs also will tend to possess mutations in GNAS either instead of or in addition to KRAS mutations in early-stage lesions [13]. On the other hand, MCNs tend to accumulate mutations in RNF43 but not GNAS [13,15]. Moreover, unlike PanINs and IPMNs, most MCNs (85%) do not grow within or interact with the main pancreatic duct [13,15,17], likely decreasing the feasibility for developing stool-based screenings. One in three IPMNs and MCNs will become invasive [13], compared with roughly 1% of PanINs [18]. Both IPMNs and MCNs are cystic in nature and larger than PanINs, which makes them easier to detect using abdominal or endoscopic ultrasound, whereas PanINs are solid tumors and are too small to be detected using these methods [13].

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Desmoplastic reaction Pancreatic tumors rely heavily on generating a niche microenvironment to maintain disease progression. Desmoplasia is the mechanism by which PDAC achieves this, and consists of a complex paracrine signaling scheme that takes place between the tumor cells, tumor-associated macrophages (TAM), neutrophils, regulatory T cells (Treg), and pancreatic stellate cells that promotes the carcinogenesis, mutagenesis, and immune evasion of the tumor cells and directly contributes to the infiltrative and metastatic potential of the tumor [27e29]. Both primary and metastatic PDACs exhibit significant desmoplasia and immune cell infiltration from an early stage, and a higher level of extracellular matrix (ECM) deposition in these tumors is associated with a negative prognosis [27,30]. The process starts when pancreatic stellate cells, which are normally quiescent, are induced to differentiate into an activated myofibroblast-like state and proliferate substantially in part as a result of aberrant KRAS and Sonic Hedgehog (SHH) signaling from early cancer cells [7,31] and later by the secretion of growth factors such as platelet-derived growth factor and reactive oxygen species (ROS) from TAM and neutrophils, respectively [27e29]. These activated stellate cells are then stimulated by growth factors secreted from TAMs and neutrophils, including TGFb, fibroblast growth factor, and connective tissue growth factor, to deposit a variety of ECM components such as collagen, hyaluronic acid, osteonectin/ secreted protein acidic and rich in cysteine (SPARC), and other glycosaminoglycans [28,29]. This produces a dense, fibrous, and hypovascularized stroma that provides the perfect environment for carcinogenesis while resisting autoimmunity and chemotherapeutics [27e29]. In addition to activating stellate cells, the secretion of ROS from TAMs and neutrophils has been implicated in multiple aspects of PC, including directly promoting the carcinogenesis and mutagenesis of the cancer cells through inducing DNA strand breaks and base modifications, thereby contributing to the genetic heterogeneity of the cancer cells [28]. Although Tregs do not directly promote carcinogenesis or mutagenesis, they secrete TGFb and suppress autoimmunity, allowing cancer and stellate cells to proliferate [28]. In addition to expressing cell surface and secreted TGFb, some additional markers for tumoral Tregs have been identified, such as CD25 and FoxP3, which are not expressed on other CD4 T cells [28,32]. Taken together, these cancer and immune cells work synergistically to hijack the stellate cells to promote carcinogenesis

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and eventual metastasis of the tumor by generating this niche microenvironment, which represents a major hurdle in the development of novel theranostics [27e29,33].

Molecular markers for pancreatic cancer and their significance in diagnosis and treatment Substantial progress has been made in identifying potential molecular markers to diagnose and treat PC. These markers can be classified into three major classes based on the biological source: serum markers, tumor tissue markers, and cancer stem cell (CSC) markers (Fig. 16.1). We discuss these three types of molecular markers and their importance in theranostics for PC in this chapter. Table 16.1 briefly summarizes the current status of molecular markers and therapeutic targets of PC.

Serum markers Cancer antigen 19.9 Cancer antigen 19.9 (CA 19.9), or sialyl Lewis A, is the best-known and only Food and Drug Administration (FDA)-approved biomarker for monitoring the disease progression and treatment response of PC [34]. Sialyl Lewis A is an aberrant form of disialyl Lewis A, which is normally expressed in pancreatic epithelial cells and participates in immunosuppression [35]. Sialyl Lewis A originates as a result of the epigenetic silencing of a-2,6-sialyltransferase and concurrent hypoxia-induced activation of sialyl Lewis A synthesis enzymes; it is highly overexpressed, accumulated, and secreted in pancreatic and other gastrointestinal tumors [35]. Because CA19.9 is not specific to PC, it cannot be used alone to diagnose PC [34]. However, it has shown some possible diagnostic ability when used in conjunction with other serum markers such as carcinoembryonic antigen (CEA) [36e38], MUC5AC [39], and others. Some antibodies have been developed to target CA19.9, including human monoclonal antibodies (mAbs) 5B1 and 7E3, which demonstrated complement-dependent and antibody-dependent cell-mediated cytotoxicity [34,40,41]. Other antibodies and antibody fragments have been developed for diagnostic, computed tomography (CT), positron emission tomography (PET), and fluorescent visualization applications, including anti-CA19-9 scFv-Fc H310A [42]; an anti-CA19.9 diabody [43,44]; and three 5B1 derivatives, DFO-5B1, FL-5B1, and dual-5B1 [45].

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Molecular Markers and Therapeuc Targets for PC

Serum Markers

Tumor Markers

Cancer Stem Cell Markers

CA19.9

Wilms Tumor 1

CD44

CEA

SPARC

CD24

Mesothelin

VISTA

CD133

IL-6

Hyaluronic Acid

MIC-1/GDF15

Mucins 1, 2, 4, 5AC, 16

Tissue Factor

CD40

Survivin

Her2

TAG-72

Syndecan-1

Osteoponn

Claudin 18.2

Figure 16.1 Molecular markers and therapeutic targets for pancreatic cancer (PC). Many markers are being explored for potential diagnostic and therapeutic applications against PC. Some are expressed on the surface of PC tumor cells and then shed into the serum and could be useful for both detection and tissue-specific targeting, such as mesothelin, tumor-associated glycoprotein 72 (TAG-72), carcinoembryonic antigen (CEA), or cancer antigen (CA)19.9. Others are being targeted with monoclonal antibodyebased immunotherapy, such as the immune checkpoint V-domain immunoglobulin-containing suppressor of T-cell activation (VISTA), which was implicated in PC; or with cancer vaccines and engineered T cells, such as survivin and Wilms tumor 1. Still others are useful targets for stromal depletion therapies, such as CD40 and hyaluronic acid, or for targeted delivery of drugs or visualization agents, such as secreted protein acidic and rich in cysteine (SPARC) and CD44. Her2, human epidermal growth factor receptor 2; IL, interleukin; MIC-1, macrophage inhibitory cytokine 1.

Carcinoembryonic antigen Carcinoembryonic antigen (CEA) is a glycoprotein expressed on the luminal surface of pancreatic epithelial cells and participates in cell adhesion and signaling [34]. CEA has been identified as a potential prognostic

Table 16.1 Molecular markers for diagnosis and therapeutic targets. Target Antibody/agent

CA19.9

Carcinoembryonic antigen

5B1 and 7E3 Anti-CA 19-9 scFv-Fc H310A Anti-CA19.9 diabody DFO-5B1 FL-5B1 Dual-5B1 Labetuzumab

KAb201

Mesothelin

M5A T84.66 Amatuximab Anetumab ravtansine DMOT4039A SS1P (Fv fragment) LMB-100 (Fab fragment) CRS-207 (Listeria live attenuated vaccine) JNJ-64041757 (Listeria live attenuated vaccine) CAR T treatments (various)

Utilization

Source

Therapeutic Diagnostic Diagnostic Diagnostic Diagnostic Diagnostic Therapeutic, diagnostic Drug conjugation Radioimmunotherapy conjugation Radioimmunotherapy conjugation Immunofluorescence Immunofluorescence Therapeutic Therapeutic Drug conjugation Drug conjugation Drug conjugation Drug conjugation Vaccine Vaccine

[34,40,41] [42] [43,44] [45] [45] [45] [46,47] [47e49] [50]

[52,53] [54] [55e58] [60,61] [62] [59,64] [59,65] [59,66] [59,67] [59,68]

Therapeutic

[59,69e73]

[51]

Continued

Table 16.1 Molecular markers for diagnosis and therapeutic targets.dcont'd Target

Antibody/agent

Utilization

Source

MUC1

1B2 12D10 Anti-hMUC1 PankoMab Clivatuzumab Antibodies (various) Vaccines (various) Vaccines (various) B72.3 Indium-111 satumomab pendetide 3E8.scFv.Cys-IR800 3E8 (124)I-mCC49 CAR T treatments (various) Zolbetuximab Claudiximab CAR T treatments (various) LY2181308 (antisense oligonucleotide) YM155 (inhibitor) Survivin-2B80-88 (peptide vaccine) Dendritic cell vaccine Engineered cytotoxic T cells Trastuzumab

Diagnostic Diagnostic Diagnostic Therapeutic Therapeutic, diagnostic Diagnostic Therapeutic Therapeutic Therapeutic, diagnostic Imaging Imaging Therapeutic, diagnostic Imaging Therapeutic Therapeutic Therapeutic Therapeutic Therapeutic Therapeutic Therapeutic Therapeutic Therapeutic Therapeutic

[179] [179] [180,181] [182e184] [185e187] [188e191] [170,188,192,193] [194e197] [101] [101] [103] [104] [105] [106] [212e217] [212,213] [218e221] [92e94] [95] [96] [97] [98] [205]

Siltuximab Clazakizumab sgp130Fc (soluble IL-6R analog) Various additional mAbs

Therapeutic Therapeutic Therapeutic

[123] [126] [127] [128]

MUC5AC MUC4 MUC2 TAG72

Claudin 18.2

Survivin

Human epidermal growth factor receptor 2 IL-6

IL-6R Tissue factor

Osteopontin Secreted protein acidic and rich in cysteine CD44

V-domain immunoglobulincontaining suppressor of T-cell activation Hyaluronic acid CD40

Tocilizumab Sarilumab (89)Zr-Df-ALT-836 64Cu-FVII 1849-ICG (111)In antietissue factor 1849 CAR T treatment HuMax-TF-ADC hu1A12 Osteopontin-VEGF bispecific mAb Abraxane (paclitaxel-conjugated albumin) Paclitaxel/curcumin double-tagged albumin

Therapeutic Therapeutic Imaging Imaging Immunofluorescence Diagnostic Therapeutic Drug conjugation Therapeutic Therapeutic Drug conjugation Drug conjugation

[124] [125] [85] [86] [87] [88] [89] [90] [111] [112] [139] [141e145]

Multiple HA-BODIPY HA-40 -(aminomethyl)fluorescein HA-2,3,5-triiodobenzoic acid HA- tris(hydroxypyridinone) amine JNJ-61610588

Drug conjugation Imaging Imaging Imaging Imaging Therapeutic

[237e245] [238] [245] [244] [245] [153]

PEGPH20 CP-870,893 Chi Lob 7/4

Stromal depletion Therapeutic Therapeutic

[158e168] [201,202] [202,203]

CA, cancer antigen; CAR, chimeric antigen receptor; HA, hyaluronic acid; IL, interleukin; mAb, monoclonal antibody; MUC, mucin.

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indicator during chemotherapeutic treatments because the serum level of CEA is directly associated with the progression of tumor invasion and malignancy [37] and increases the specificity of the diagnosis of PC when used in combination with CA19.9 [36e38]. Some clinically relevant antibodies have been generated against CEA. First, labetuzumab is a humanized mAb that shows diagnostic and therapeutic potential and is currently undergoing clinical trials [46,47]. It has been studied in the context of antibodyedrug conjugates [47e49] and was used in radioimmunotherapy conjugation [50]. KAb201 is another humanized anti-CEA antibody that has similarly high pancreatic tumor specificity and was investigated for the delivery of radioisotopes [51] and conjugation to fluorophores for development in immunofluorescenceguided surgery [52,53]. A third antibody, M5A, is being developed for fluorophore conjugation [54]; a different version of that antibody, T84.66, has been humanized [55] and is undergoing clinical trials [56e58]. Mesothelin Mesothelin is a cell surface molecule that is highly overexpressed in PC [59,60]. Its purpose is unclear but is thought to participate in cell adhesion and binding to CA125 and MUC16 in mesothelial cells and aids metastasis in many cancers [59]. Many mesothelin-targeting treatments are under development [59]. Amatuximab is one such chimeric mAb that is in a phase II clinical trial and interferes with mesothelin’s binding to CA125 and MUC16 [60,61]. There are also many antibody-drug conjugates in development: anetumab ravtansine is in phase I and II clinical trials [62] and consists of a human mAb that delivers the linked microtubule-targeting toxin DM4 to the cancer cell, which internalizes it and secretes a metabolite that in turn kills surrounding cells [63]. DMOT4039A is another antibody-drug conjugate that delivers the antimitotic monomethyl auristatin E to target cells; it has been tested in PC patients [59,64]. SS1P is an Fv antibody fragment that delivers Pseudomonas exotoxin A to mesothelin, which is endocytosed and targets eIF-2a [59,65]. Similarly, LMB-100 is an Fab-PE24 immunotoxin that is entering clinical trials [59,66]. Other therapies include Listeria live attenuated vaccines CRS-207 [59,67] and JNJ-64041757 [59,68]; and various chimeric antigen receptor (CAR) T therapies [59,69e73]. Macrophage inhibitory cytokine 1 Macrophage inhibitory cytokine 1 (MIC-1), also known as growth differentiation factor 15, is a secretory growth factor that is implicated in many

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aspects of tumor invasion, including migration [74] and immunosuppression [75], and is overexpressed within tumors and serum of PDAC patients [76e79]. MIC-1 is of particular interest because of its ability to detect earlystage PDAC before CA19.9 is detectable [76,78], and in the roughly 10% of the patients who do not express CA19.9 at all [78,79]. It can differentiate between early-stage PDAC and IPMN [80] and was able to distinguish between PDAC and pancreatitis when used in conjunction with UL16 binding protein 2 [76] or CA125 [81], but not when MIC-1 was used alone [77]. Therefore, MIC-1 is an important marker for early detection and monitoring of PC and warrants further investigation. Tissue factor Tissue factor (TF) is a cell surface receptor normally expressed in subendothelial cells that binds soluble Factor VII (FVII) upon rupture of the blood vessel to initiate clotting rapidly [82]. It is also aberrantly expressed in PDAC tumor cells, which use its secondary effect of activating proteinaseactivated receptor-2-integrin signaling to initiate angiogenesis, but with the side effect of triggering clotting and thrombosis within the tumor and subsequently causing thromboembolisms at distant sites within the body [83]. Because the expression within PDAC tumors is correlated with disease progression [84] and the increased occurrence of thromboembolic comorbidities in PC patients [83], TF is a promising target for both therapeutics and diagnostics with many theranostic applications being developed. For example, TF has been explored as a target in PET imaging using both radiolabeled anti-TF antibodies [85] and FVII ligand [86]. Other applications targeting TF include immunofluorescence [87], an anti-TF application of immune-SPECT/CT [88], a TF-CAR T cell [89], and an antibody-drug conjugate that showed regression of patient-derived xenografts with minimal off-target effects [90], among others. Survivin Survivin is an antiapoptotic protein normally expressed in embryonic and fetal tissues but not in adult tissues [91]. It was found to be expressed in 81% of PDAC tumors; elevated serum concentrations were associated with metastasis and invasiveness, but not the size or location of the tumor [91,92]. Because survivin is expressed in tumors as well as in the serum, it is unique in that it could serve both as a therapeutic target and diagnostic marker for PDAC. Many attempts at targeting survivin have not panned out, such as with antisense oligonucleotide LY2181308 [92e94] and

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Theranostic Approach for Pancreatic Cancer

inhibitor YM155 [95], but trials involving survivin, peptide vaccines have seen greater success, with prolonged survival and disease stabilization in one phase I trial of the survivin-2B80-88 peptide vaccine [96]. Another cancer vaccine trial is also being attempted using peptide-pulsed dendritic cells [97]. In addition, a study involving engineered cytotoxic T cells targeting survivin is currently under way [98]. Tumor-associated glycoprotein 72 (TAG72) TAG72 is a cell surface glycoprotein that functions similar to an MUC and is often overexpressed in and shed from PC [99]. It is known to be a better prognostic indicator than CA19.9 or CEA [100]. Despite this and the fact that Indium-111 satumomab pendetide, a derivative of the anti-TAG72 antibody B72.3, was the first monoclonal antibody to be approved for tumor imaging [101], other attempted applications of anti-TAG72 mAbs fizzled out. Luckily, new attempts are being made to target TAG72 for prognostic evaluation [102], immunofluorescence-guided surgery [103], the development of new antibodies such as 3E8 [104], improved targeting in PET and CT [105], and CAR T treatments [106]. Osteopontin Osteopontin is an ECM glycoprotein secreted in PDAC; it is implicated in PDAC disease progression. It is expressed as one of three splice variants in PDAC, with osteopontin-a expressed in nearly all PDAC, osteopontin-b expression correlating with survival, and osteopontin-c correlating with metastatic disease [107]. Because PDAC secretes alternatively spliced forms of osteopontin, it shows potential for tumor- and disease stage-specific targeting. Although the exact mechanisms of osteopontin signaling in PDAC are unknown, it binds to CD44 and integrins to trigger processes such as tumor progression and complement inhibition [108]. Osteopontin also drives metastasis by triggering the release of vascular endothelial growth factor (VEGF) and matrix metalloprotease (MMP), which is inhibited by knocking down osteopontin [109]. This process is also stimulated by nicotine, which is the proposed mechanism by which smokers experience elevated PC risk [109]. Osteopontin is being explored as a marker for PC. It was found to perform better than CA19.9 in discerning IPMN [80] and resectable PDAC from pancreatitis [110]. Antiosteopontin antibodies are being developed, including hu1A12, which inhibited metastasis in an in vivo study [111] and also when hybridized with the anti-VEGF antibody bevacizumab [112]. At least one clinical trial is exploring the use of

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osteopontin as a marker of intratumoral hypoxia [113]. However, this marker remains relatively unexplored. Interleukins 6, 8, and 10 Feng, et al. and others showed a correlation between high serum levels of interleukins (ILs) 6, 8, and 10 and poor prognosis of PC [114e116]. IL-6 and IL-1b have similarly been shown to indicate prognosis of PC patients undergoing gemcitabine therapy [117]. IL-6 and IL-8 are proinflammatory cytokines expressed by many PDAC tumoral cell types; they are required for PDAC progression, metastasis, and angiogenesis [118,119]. IL-10 is expressed by Tregs within the tumor and is important for cell proliferation and immune suppression [120,121]. These cytokines work synergistically to promote PDAC disease progression and avoid autoimmune responses [114,122]. They are an attractive target for both therapeutics and diagnostics because they are both expressed within the tumor and also secreted into the serum. Three IL-6 or IL-6R antibodies have been approved: siltuximab, tocilizumab, and sarilumab. A clinical trial using siltuximab, an anti-IL-6 chimeric mAb, against solid tumors including PC proved inefficacious [123]. Tocilizumab, which is a humanized anti-IL-6R mAb, is undergoing a clinical trial for PDAC [124]. Sarilumab, an anti-IL-6R human mAb, has shown efficacy against solid tumors in preclinical trials [125] but has not yet been tested against PC. Other anti-IL-6 antibodies are in development, such as clazakizumab, an anti-IL-6 human mAb [126], and sgp130Fc, a soluble IL-6R analog that inhibits IL-6 trans-signaling [127]. Many other IL-6 antibodies are in various stages of development and are reviewed here [128].

Tumor tissue markers Wilms tumor 1 antigen Wilms tumor 1 antigen (WT1) is expressed in around 75% of PDAC tumors but is absent from normal pancreatic ductal epithelium [129]. Knockdown of WT1 impairs tumor growth in vitro, which indicates that it has an unknown role in PDAC pathogenesis [129]. However, no association has been made between WT1 expression and stage or location of the tumor, which makes it a better target than prognostic indicator [129]. WT1 has been identified as the number one target for developing cancer vaccines against PC by the National Cancer Institute [130,131], and is the subject of multiple cancer vaccine [132,133] and CAR T clinical trials [134,135].

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Secreted protein acidic and rich in cysteine (SPARC) Pancreatic stellate cells maintain the dense stroma in PDAC partially through constant remodeling of the ECM, which requires deposition of new collagen and organization of fibronectin fibrils [136]. SPARC expression from these stellate cells drives aberrant ECM remodeling in PDAC in part through stimulating expression of MMP [137]; it is also involved in tumor invasiveness [136]. SPARC is being investigated as a means of targeting drugs to PDAC tumors because of its high affinity for binding albumin [138]. One such drug conjugate, Abraxane or nabpaclitaxel, was approved by the FDA in 2005 [139]. Applications of this technology targeting PC cancer include coadministration with gemcitabine [140], a paclitaxelecurcumin double-tagged albumin [141], along with other methods of complexing and delivering these agents [142,143]. Studies demonstrated the efficacy of this approach, showing significant improvements in response and survival [144,145]. Another group approached this method from an even more innovative direction by incorporating a maleimide group into the drugs, creating prodrugs that readily conjugated with albumin in vivo [146]. At least one other clinical trial using albuminconjugated paclitaxel is ongoing [147]. Overall, these successes demonstrating the targeting of SPARC within PDAC tumors represent an avenue of therapeutic design that should be explored further. V-domain immunoglobulin-containing suppressor of T-cell activation (VISTA) Immune checkpoint inhibitors have yielded tremendous success in other cancers but have seen little success in PC [148]. The coinhibitory receptor PD-1 is expressed on immune cells and normally binds to programmed death (PD) ligand 1 (PD-L1) expressed on host cells to prevent autoimmunity [149], but although it is commonly upregulated in other tumor cells as a method of immune evasion, it is nearly absent from PCs owing to the lack of T-cell infiltration, rendering current PD1/PD-L1 treatments ineffective [148,149]. On the other hand, pancreatic tumors express CTLA-4 on Treg cells [150], which rely on its coinhibitory function to evade the antitumor immune response. Despite the expression of this immune checkpoint, however, anti-CTLA-4 antibodies have proven ineffective against PC tumors [150]. Blando et al. discovered that this is because a second immune checkpoint, V-domain immunoglobulin-containing suppressor of T-cell activation (VISTA), is expressed on the surface of CD68þ TAMs within PC tumors, which enables them to bypass single-agent

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anti-CTLA-4 therapies [151,152]. Janssen Pharmaceuticals is developing an anti-VISTA antibody, JNJ-61610588, which is under clinical trial [153], and more treatments targeting VISTA are sure to come. This discovery may soon revolutionize the way immune checkpoint therapy is pursued in PC. Hyaluronic acid Hyaluronic acid (HA) is an important ECM component that is crucial to PDAC pathogenesis. HA is secreted from both tumor and stromal cells [154] and is a long glycosaminoglycan consisting of numerous disaccharide repeats [155]. It is one of the ECM components responsible for the high interstitial pressure within the PDAC tumor, because it draws huge amounts of water (roughly 15 molecules per disaccharide) into the tumor, resulting in subsequent blood vessel collapse [155,156]. The level of HA content within PDAC tumors is also associated with epithelial to mesenchymal transition (EMT) and poor prognosis and survival [27,157]. HA has been evaluated for its ability to target and deplete the PDAC stroma [158]. After an initial half-life of 3 min, PEGPH20 was engineered for greater therapeutic efficiency via PEGylation of the human hyaluronidase PH20, which extended its half-life to over 10 h [158]. Many clinical trials have been conducted using PEGPH20 in combination with existing chemotherapeutics, such as dexamethasone or oxaliplatin, irinotecan, fluorouracil, and leucovorin, and many more are under way [159e165]. In general, these trials have shown substantial benefits including decreases in tumor HA level and increased drug perfusion [166]. However, there are significant incidences of thromboembolism in multiple study participants [167,168], which it is hoped may be overcome in subsequent trials. Mucins 1, 2, 4, 5AC, and 16 MUCs are glycoproteins that form a protective layer on normal epithelial cells and perform a variety of tasks that are exploited in PC [169,170]. MUCs contain a tandem repeat polypeptide backbone and up to 90% by weight of N-acetyl-galactosamine oligosaccharide chains branching from the main peptide [170]. Of importance to PDAC, MUC1 and MUC4 are primarily membrane-bound, whereas MUC2, MUC5AC, and MUC16 are secretory [170]. Mucins aid in immunosuppression and metastasis by PDAC tumors in a variety of ways [169e174]. For example, MUC1 and MUC16 can block antigen presentation through binding to siglecs and Toll-like receptors [169]. Overexpression of MUCs can also block cell surface antigens, or they can induce anergy on T cells through binding

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ICAM-1 [169]. MUC4 also appears to produce a splice variant that aberrantly activates the integrin-b1/FAK/ERK pathway to participate directly in the metastasis of PDAC [175]. MUCs are differentially expressed in PanINs, IPMNs, and invasive PDACs, and thus offer the potential to differentiate these lesions at the molecular level [13]. Of these MUCs, only MUC1 is expressed in normal pancreatic ductal epithelium, but is highly overexpressed in PanIN-2/3 and PDAC, which makes it a popular target among emerging therapies [170]. MUC2 is not expressed on PanINs or PDAC but are in IPMN, which could be exploited for the molecular differentiation of these lesions [13]. MUC4, MUC5AC, and MUC16 (as well as MUC3) are expressed increasingly as PanIN progresses from early to infiltrative lesions and are highly expressed in PDAC; they could be useful in staging these tumors [13]. It was shown that combining MUC5AC with CA19.9 screening improved the accuracy of diagnosing resectable PC and allowed for differentiation among PC, pancreatitis, and benign lesions [39]. Further exploiting these expression differences in the precursors of PDAC will be crucial to the development of diagnostics and therapeutics in the near future. Given their importance to the pathogenesis of multiple cancers and the frequent occurrence of tumor-specific neoantigens in these glycoproteins [169e174], MUCs are the target of many therapeutics in development. Early attempts in both antibody and vaccine development failed because most of them recognized the polypeptide structure of the glycoproteins rather than the glycosylations, or because the antigens used to produce them had patterns of glycosylation different from those of the cancers they intended to target [170,176]. The importance of tumor-specific glycosylations has since become more clear [170,177], and the increasing capability to generate tumor-specific anti-MUC mAbs and the possibility of better mapping the epitopes of new antibodies using glycopeptide libraries [178,179] has allowed for greater characterization and exploitation of MUC tumor-specific antibodies. Two such antibodies, 1B2 and 12D10, were engineered using glycopeptide libraries to possess specificities for both glycan and peptide sequences [179]. Another antibody, anti-hMUC1, was found to bind to pancreatic tumor-specific MUC1 while avoiding MUC1 expressed on normal tissues in vivo. It was found to be internalized by the PC cells [180], opening up the potential for further developing a toxin- or radioisotopeconjugated version of this antibody for therapeutic or diagnostic use, an

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area in which previous attempts have failed [181]. PankoMab is another MUC1-reactive antibody designed to bind preferentially to tumorassociated MUC1 [182]; it is being evaluated in phase I and II trials [183,184]. Clivatuzumab is an anti-MUC5AC antibody in clinical trials that is reactive against PanINs and PDAC. It shows promise for use as a serum biomarker [185e187]. MUC5AC is a secretory MUC that is not expressed in normal pancreatic epithelia, but it is expressed in early-grade PanINs, which makes this antibody one of the most promising in allowing early biochemical detection of precancerous lesions of the pancreas [185e187]. MUC4 remains relatively unexplored in PC therapies, but some preliminary investigations into antibodies [188e191] and vaccines [170,188,192,193] are under way. MUC2 also has not been widely explored other than a series of vaccine clinical trials for prostate cancer [194e197], but it could be useful in the early detection of IPMNs [13]. CD40 Normally, helper T cells should activate macrophages to attack cancer cells by stimulating CD40 on the macrophage surface [198]. However, this process does not normally occur in PDAC owing to the prevalence of immune suppressive cells such as Tregs, tumor-associated macrophages, and myeloid-derived suppressor cells, which results in an immunosuppressive tumor environment and subsequent lack of effector T-cell infiltration or activation [30,198]. Beatty et al. were able to demonstrate that stimulating CD40 activation using an agonist antibody resulted in stromal depletion and tumor regression by means of noneT cellemediated macrophage infiltration [198]. Since then, others have explored the use of CD40 agonists and found that they are capable of producing stromal depletion through both T celledependent andeindependent responses against PDAC tumors [199]. Multiple CD40 agonist antibodies are under development [200], including human mAb CP-870,893 [201,202] and chimeric mAb Chi Lob 7/4 [203], but so far they have shown limited efficacy in clinical trials [202,203], and remain an area of active investigation. Human epidermal growth factor receptor 2 (Her2) Human epidermal growth factor receptor 2 (Her2) has garnered particular interest because anti-Her2 therapies are already on the market with more in development. This epidermal growth factor receptor is not expressed on normal pancreatic ductal epithelium but is found in some PanINs and

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PDAC, although the prevalence and extent are debated. Studies have reported Her2 overexpression in as little as 2% [204] to 11% [205], 45% [206], and nearly 70% [16,207] of patient samples. Results from clinical trials involving anti-Her2 inhibitors and mAb have also been hit or miss [208]. One drug, trastuzumab, targets only highly overexpressed Her2, and although Her2 was expressed in 60% of PDAC tumors in one trial, only 11% were deemed to be adequately overexpressed to the extent required for the treatment to be successful [205]. Many other anti-Her2 therapeutics are still under development; whether this receptor can be successfully targeted in PC remains to be determined. Syndecan-1 PDAC cells rely heavily on the mass import of extracellular nutrients through macropinocytosis to sustain their growth [209]. It was discovered that syndecan-1, an important mediator of PDAC macropinocytosis, is expressed on the surface of PDAC tumor cells as a downstream response to mutant KRAS [210]. Syndecan-1 is a heparan sulfate proteoglycan that was previously shown to mediate macropinocytosis, but it is now known that this surface marker is upregulated starting early in PC disease progression and is found in pancreatitis and premalignant PanIN lesions [210]. The same study also showed that inhibition of syndecan-1 resulted in suppressed tumor growth, which indicates its potential utility as a drug target [210]. Although the clinical utility of syndecan-1 and macropinocytosis in treating PDAC remains unknown, it will undoubtedly be an area of intense interest in the future. Claudin 18.2 Claudin 18.2 is an emerging target for PDAC. It was shown to be aberrantly expressed in 59% of tumor specimens but not in normal pancreatic epithelia [211]. Claudin 18.2 is a gastric epithelial tight junction protein normally expressed on luminal regions of polarized cells, which should offer minimal exposure to antibody-based therapies [211,212]. Zolbetuximab, also known as IMAB362 or claudiximab, is an anti-Claudin 18.2 antibody entering phase III clinical trials [212]. It has been shown to target PDAC cancer cells and induce cytotoxicity through antibody-dependent and complement-dependent pathways [213]. Trials using zolbetuximab combined with existing chemotherapeutics are ongoing [214e217]. Many CAR T Claudin 18.2 trials are also enrolling [218e221].

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Cancer stem cell markers Although they make up only around 1% of cells within the tumor and their origin is still not completely understood, CSCs are increasingly recognized as a key driver of metastasis, drug resistance, and disease recurrence in PDAC [222e224]. Since their discovery in 2007 [225,226], PC stem cells (PSCS) rely on multiple common developmental pathways such as SHH, Wnt, Notch, PTEN, and Bmi-1 [222,223]. The prevalence of these developmental pathways in CSCs of many origins has prompted their targeting with various drugs, but the cells frequently compensate for the loss of one of these pathways by upregulating another and quickly develop resistance to these inhibitors [224]. It has also been reported that gemcitabine promotes its own resistance by inducing stemness in PC cells [227e229]. Many groups have shown efficacy in vitro by targeting some of these developmental pathways [222,230], but it remains to be seen whether these repurposed drugs or other efforts to reengineer some chemotherapeutics to stave off resistance will pan out [222,223,231e234]. CD44, CD24, and CD133 As with other stem cells, PCSCs are self-renewing and can produce multiple lineages of differentiated progeny [222e224]. Early characterization in vitro fell along similar lines as with induced pluripotent stem cells in that CSCs can form spherical clusters in low-adhesion conditions and can seed new tumors when implanted [222,223]. Several surface markers have since been identified, such as CD24, CD44, CD133, epithelial-specific antigen, c-Met, aldehyde dehydrogenase (ALDH), and C-X-C chemokine receptor type 4, which are associated with stemness and gemcitabine resistance in PCSCs [222e224]. Despite the usefulness in these markers for new applications such as microfluidic chip liquid biopsy [235], these markers are specific to many cell types [236] and it remains to be seen whether they will be useful in targeted drug delivery. CD44 is a membrane glycoprotein that is associated with PC cells, specifically PCSCs [237]. CD44 expression is associated with poor prognosis and radiation therapy resistance and is expressed in two isoforms: CD44s and CD44v [237]. CD44v is associated with metastasis, and isoform switching between s- and v-forms has also been observed with EMT [237]. Aside from being a marker for PCSCs as discussed earlier, CD44 has also been explored extensively for targeted drug delivery [237e245] and tumor visualization, including HA-BODIPY [238] and HA-40 -(aminomethyl)

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fluorescein [245] for confocal imaging, an HA-2,3,5-triiodobenzoic acid conjugate used for fluorescence and CT imaging [244], and HAtris(hydroxypyridinone)amine for SPECT/CT [245]. HA has also been conjugated to gemcitabine-containing liposomes for targeting CSCs [246]. As noted earlier, a plethora of HAedrug conjugates are under development.

Current diagnostics Once PC is suspected, a variety of noninvasive or minimally invasive diagnostic procedures may be used to locate and stage the tumor [3e5,7,247]. CT and MRI are recommended as the first modes of visualization because both offer fine resolution of the surrounding tissues with similar accuracy [5,7,248,249]. However, CT is considered inadequate for determining tumor resectability because of its low sensitivity in detecting small metastases, which must instead be characterized laparoscopically [250]. The use of MRI has increased over time, owing to the inherent advantage of not exposing the patient to ionizing radiation as in CT [2,7]. MRI is superior in its resolution of soft tissue and the ability to visualize simultaneously the ducts, vasculature, liver, and peritoneum, in which metastases are common [5,250,251]. Abdominal ultrasound is also commonly used as a precursor to CT because it is noninvasive, available, and affordable and can differentially diagnose conditions with similar symptoms such as gallstones and ulcers while avoiding the use of ionizing radiation [2,5,21]. However, because the pancreas lies deep within the abdominal cavity, abdominal ultrasound cannot easily visualize small lesions or discern between benign and metastatic tumors [2,21]. Endoscopic ultrasound is widely used upon initial diagnosis of PDAC because of its high sensitivity and accuracy compared with MRI and CT, especially in diagnosing small, premetastatic, early-stage tumors, and because of its useful applications such as stenting, endoscopic fine needle aspiration, and endoscopic retrograde cholangiopancreatography [7,249]. PET can also be useful in staging and diagnosis because of the ability to discern between benign and metastatic lesions by measuring the rate of glucose uptake through the use of 18F-fluorodeoxyglucose [252] and many PC-specific markers are under development [42e45, 85,86,88,105,247,250,253].

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Emerging diagnostics Despite the usefulness of current diagnostic techniques in identifying laterstage tumors, their high cost and invasiveness or risk for comorbidities prevent their use in preemptive screening for PC in those without a genetic predisposition [2]. Currently no approved serum molecular markers can be used to diagnosis PC [3e5,7]. Although the serum marker CA19-9 is commonly used to determine the prognosis of PCs during therapeutic regimens, it is not specific to PC and cannot be used for initial diagnosis [5,7,37,254]. Identifying minimally invasive, reliable, and affordable screening methods is crucial to reducing the burden of PC. The focus on developing liquid biopsies for various cancers has yielded some promising results, and these advances are moving the reliable early detection of PC closer to reality.

Blood-based DNA screening The development of a routine blood-based PC screen would revolutionize this field, but little progress had been made until recently. An expansive study in 2014 by Bettegowda et al. found that circulating tumor DNA was detectable in most patients with advanced PC and 48% of patients with localized tumors [255]. They also found that 87% of patients that had KRAS mutant tumors could be identified using circulating tumor DNA [255]. This cell-free circulating DNA is released into the bloodstream by dying cancer cells, and because most cancers have at least one trademark somatic mutation [256], the detection of cancer-specific alleles diluted by wild-type DNA in the blood can be accomplished using polymerase chain reaction (PCR)-based methods [255,257,258]. Two subsequent studies by Cohen et al. focused on detecting early-stage (stages IeIII) cancers using circulating tumor DNA, culminating in the development of the CancerSEEK PCR panel [257,258]. CancerSEEK uses 61 sites within 16 common cancer genes to identify a wide range of cancers. It is innovative in its additional combination of protein markers and machine learning to predict the tissue of origin of the cancer accurately [257,258]. It is estimated that this panel could detect up to 95% of PC [258]. This ability to detect premetastatic PC accurately is unprecedented, and the possibility of combining other markers such as cancer-specific promoter methylation offers the possibility to expand and refine this technology [256,259].

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Marker panels To date, numerous markers have been identified that can detect early lesions, track disease progression, and differentiate among PDAC, PanIN, IPMN, and benign lesions, pancreatitis, and nonpancreatic diseases [79]. Several serum panels have been announced that will target some of these markers. The first is an antibody microarray that targets 29 markers including Cdk2, IL-6, VEGF, and sialyl Lewis X, and had an accuracy of 96% in detecting early-stage (stage I and II) PDAC [260]. The second is a sandwich enzyme-linked immunosorbent assayebased assay of three markers, CA19.9, MUC5AC, and MUC16, to detect stage I and II cancers with 75% accuracy and only a 5% false-positive rate [261]. Finally, the third is under development by the GRAIL company, which hopes to develop a liquid biopsy panel capable of detecting multiple cancers [262]. PC marker panel development remains an active area of research; more such panels are sure to come.

Cell-based screening The use of microfluidic devices has revolutionized the isolation of circulating tumor cells (CTCs). Previous attempts at detecting CTCs proved difficult because these cells are heavily diluted by circulating blood cells and are also prone to shearing, anoikis, and attack by immune cells [263]. Because of their high surface area and the ability to coat them with an antibody of choice, rather than pretagging the CTCs within the sample, microfluidic chips have become increasingly useful in isolating CTCs [264,265]. This technology has been used to isolate and then successfully culture CTCs [264] and to isolate CTCs and circulating PCSCs from patient samples [235], which shows that it is gentle enough to isolate cells without destroying them, allowing for further use of biochemical assays downstream. It can also be specific enough to distinguish between similar cell types, which would be important for applying this technology to staging diseases. Many variations are also being studied that integrate various physical methods to maximize contact with the chip surface and methods of mechanical sorting to increase the efficiency and purity of the isolated cells [266e268]. Although this technology is still early, the development of cellbased screening using microfluidic chips show great potential for the early diagnosis of PCs as well as improved monitoring of disease progression and response to treatments.

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Stool-based DNA screening Developing a noninvasive and cost-effective procedure for detecting PDAC and invasive pancreatic lesions is the holy grail of PC diagnostics. Success in detecting other gastrointestinal cancers through stool-based DNA screening has encouraged a similar search for genetic targets in the screening of PC [269]. Early attempts at detecting KRAS and P53, the two most common genetic alterations in PC, in stool samples of PDAC patients were not sensitive or specific enough to detect PDAC reliably [270]. In a subsequent study, Kisiel et al. succeeded in identifying nine aberrantly methylated genes in PDAC stool samples that could be used to identify PC accurately [271]. Of these, bone morphogenetic protein 3 was the most promising because it was more discriminant than the next best three markers combined and had a sensitivity of 67% when combined with KRAS [271]. A clinical trial investigating both genetic mutations and hypermethylation in stool samples of patients with PC and IPMN is also under way [272]. Ultimately, the proximity of PDAC, PanIN, and IPMN lesions to the pancreatic duct combined with limited options for early detection and tracking disease response to treatment make stool-based detection an important area of research.

MicroRNA screening Compared with to DNA screens, relatively little work has been done to identify a microRNA (miRNA) profile for PC. miRNAs are short, singlestranded RNA fragments that regulate gene expression posttranslationally [273]. Since the discovery that cancers often use miRNAs to regulate protein expression [274], many groups have investigated the usefulness of targeting cancer-specific miRNAs for therapeutic purposes [275]. Ultimately, miRNA-based therapies have been hit or miss, hindered by a lack of tumor-specific delivery, low uptake of the sequence, and the tumor’s ability to adapt quickly to the drug [275]. However, profiling tumorspecific miRNA expression patterns may still be useful for detecting PC. Of many studies identifying miRNA signatures in cancers of the pancreas [273,275e280], a few good candidates have emerged for detection and differentiation of pancreatic lesions and cancers, including miR-103, miR-107, and miR-155 to tell tumors apart from normal pancreatic tissue, miR-204 to detect insulinomas, and miR-21 liver metastasis [276]; miR-1290 to detect early cancers and IPMN lesions [278]; and miR-133a

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to distinguish primary from metastatic tumors [279]. Because of the lack of ability to identify and differentiate easily between early and benign lesions of the pancreas, miRNA profiling may prove to be an important tool in the staging and monitoring of the progression of PC in the near future.

Drug resistance PDAC tumors are heterogeneous in cell type and genetic composition and use a variety of mechanisms for developing and maintaining disease progression. One such mechanism is the PC’s innate and adaptive drug resistance capabilities. The first line of defense is the dense, hypovascularized stromal environment that maintains a high interstitial pressure to resist the perfusion of therapeutics [7,27e29,33]. Next, each tumoral cell type can employ various mechanisms for overcoming the drug, including reducing the mechanism of uptake, relying on hypoxia-induced autophagy to avoid uptake, increasing the efflux of the drug via ABC transporters and nucleoside transporters, inducing senescence in response to cues from the tumor environment, or circumventing the drug’s effects by downregulating the targeted pathway, increasing catabolism of the drug, and increasing expression of antiapoptotic proteins [28,281]. A study by Halbrook et al. showed that TAMs also actively participate in resistance to gemcitabine, a long-standing frontline therapy for PC, by releasing species of pyrimidine metabolites that mimic and outcompete gemcitabine for uptake into the cancer cells [282]. Pancreatic tumors especially rely on the adaptive chemoresistance abilities of CSCs, which either are innately resistant to most current therapeutic regimens or can quickly develop resistance through the same mechanisms as listed earlier [222e224]. They also have an important role in engaging cross-talk between tumoral cell types to initiate EMT, which allows the tumor cells to alter their proteome to resist treatment and promotes posttherapy immune escape, ultimately reseeding tumor colonies once the treatment subsides [222e226,283].

New insights into mechanisms of radioresistance CSCs are also a focus of intense interest in PC because of their ability to resist chemotherapy. Ionizing radiation relies in part on the generation of ROS to induce DNA damage in target cells, but CSCs can resist this generation of ROS through multiple mechanisms. For example, CSCs normally maintain characteristically low levels of ROS; this is known to be mediated in part through the upregulation of free radical scavengers such as

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glutathione [284] and the increased production of reduced nicotinamide adenine dinucleotide phosphate that occurs through an aberrant glutamine pathway driven by KRAS and Nrf2 [285]. However, other mechanisms for radiotherapy resistance are suspected, including increased DNA damage repair. Although the full host of mechanisms is unknown, there have been some advances in our understanding of PCSC radioresistance; these discoveries could inform attempts to develop pretreatments or radiotherapy sensitization therapies for PCSCs in the near future. A report by one group shed light on the subject: ALDH is known to be overexpressed in many CSCs and is associated with both chemotherapeutic and radiotherapeutic resistance [286]. However, although the chemoresistant function of ALDH is understood to rely on its ability to catabolize many different chemotherapeutic drugs, its association with radioresistance is less understood [286,287]. Harati et al. discovered that Nanog regulates ALDH expression in CSCs, and that Nanog also induces postradiation double-stranded break DNA repair by regulating Notch1 and Akt [287]. In other words, an increase in ALDH expression observed by many in radioresistant CSCs actually appears to be evidence of upstream Nanog-Notch1-Akt DNA repair in those cells [287]. This discovery may reveal the mechanism by which coadministration of ALDH inhibitors has been observed to sensitize cells to chemotherapy and radiotherapy [288,289], and may result in more effective strategies for targeting PSCSs in vivo. In another discovery, Tsubouchi et al. demonstrated that x-ray radiation directly upregulates the expression of CD44, which in turn maintains ERK phosphorylation (which is involved in cell proliferation) and drives EMT to promote the adaptation of tumor cell protein expression profiles to become more resistant to the therapy [290]. PCSCs that express CD44 are known to be particularly resistant to radiation therapy. Its expression in PDAC tumors has long been associated with poor prognosis [291]. However, although it was assumed that the increase in CD44-expressing PCSCs after x-ray radiation was caused by the relative depletion of non-CD44 cells, this observation may indicate that radiotherapy directly promotes therapeutic resistance and metastasis [290,292].

Penetration enhancers Many groups are currently developing technologies to enhance the penetration of drugs and imaging agents into tumors, including the use of amphiphilic molecules that increase the efficiency of drug delivery.

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One such technology is the penetration enhancer INT230-6, which is a combinatorial therapy in phase I/II clinical trial targeting PDAC [293]. INT230-6 is composed of the chemotherapeutics cisplatin and vinblastine sulfate combined with a proprietary compound that facilitates penetration into the tumor tissue when injected directly into the tumor [294]. An earlier clinical trial demonstrated successful retention of the drugs within the tumor and morphological changes in the lesions with few side effects [295]. Previous animal models also demonstrated greater saturation of these drugs within the tumor compared with their normal intravenous delivery, and the compound also promoted an adaptive immune response that then successfully cleared noninjected metastases and immunized the animals against the cancer [293,294]. If successful, this innovative technology would simultaneously allow for targeted delivery of chemotherapeutics and generate an effective cancer vaccine where numerous previous attempts have failed. Nanoparticle-assisted delivery of drugs and visualization agents to hardto-target tumors are active areas of preclinical research, and excellent reviews are available on nanomedicine tumor penetration [296] and cellpenetrating peptides [297].

Conclusions Despite advances in the diagnosis and treatment of other cancers, there are still few options for identifying tumors of the pancreas although surgical resection (the only curative solution to PC) can be attempted [1,2]. There is promise as of late in characterizing serum markers that are more efficacious than CA19.9 in detecting not only early forms of PC but also in differentiating among common types of pancreatic lesions as well as other, similar diseases. However, the development of standardized marker panels has been slow, and at over 44,000 deaths per year [1], there is still a pressing need for innovation. The issue that has plagued therapeutic development for decades is the pancreatic tumor microenvironment, which is frequently ignored in the preclinical stage to the detriment of significant clinical progress. As a result, the next big discovery in PC may depend on conquering the stroma, whether through improved targeting of therapeutics via drug conjugation, the use of nanoparticles and penetration enhancers, or via stromal depletion. As always, it is still too early to know whether currently emerging technologies will be effective in the clinic. Indeed, any meaningful progress in reducing the burden of PC may require several more advances in our

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understanding of the molecular pathology of PC. Nevertheless, if the number of innovative therapeutic and diagnostic strategies that have emerged are any indication, we may soon be one step closer to diminishing the burden of PC.

Acknowledgments This work was supported by the National Institute of Health Grant R01-DK81413 (to SAS) from the National Institute of Diabetes and Digestive and Kidney Diseases and by the UCF Reach for the Stars Award (to SAS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Diabetes and Digestive and Kidney Diseases or the National Institutes of Health.

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