Pancreatic Adenocarcinoma: Unconventional Approaches for an Unconventional Disease

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Author Manuscript Published OnlineFirst on March 27, 2020; DOI: 10.1158/0008-5472.CAN-19-2731 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Christopher Gromisch,1 Motaz Qadan,2 Mariana Albuquerque Machado,1 Kebin Liu,3 Yolonda Colson,4 and Mark W. Grinstaff1*

1Departments of Pharmacology and Experimental Therapeutics, Biomedical Engineering, and Chemistry, Boston University, Boston, MA 02215. 2Division of Surgical Oncology, Massachusetts General Hospital, Boston, MA, 02114. 3Department of Biochemistry and Molecular Biology and Georgia Cancer Center, Medical College of Georgia, Augusta, GA 30912. 4Division of Thoracic Surgery, Massachusetts General Hospital, Boston, MA, 02114.

* Corresponding Author Mark W. Grinstaff Boston University Metcalf Center for Science and Engineering Room 518 590 Commonwealth Ave Boston, MA 02215 Tel: 1.617.358.3429 Fax: 1.617.358.3186 Email: [email protected]

Conflict of Interests The authors declare no conflict of interest.

Running title pancreatic cancer

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Abstract:

This review highlights current treatments, limitations, and pitfalls in the management of pancreatic cancer and discusses current research in novel targets and drug development to overcome these clinical challenges. We begin with a review of the clinical landscape of pancreatic cancer, including genetic and environmental risk factors, as well as limitations in disease diagnosis and prevention. We next discuss current treatment paradigms for pancreatic cancer and the shortcomings of targeted therapy in this disease. Targeting major driver mutations in pancreatic cancer, such as dysregulation in the KRAS and TGF-β signaling pathways, have failed to improve survival outcomes compared to non-targeted chemotherapy; thus, we describe new advances in therapy such as Ras binding pocket inhibitors. We then review next-generation approaches in nanomedicine and drug delivery, focusing on preclinical advancements in novel optical probes, antibodies, small molecule agents, and nucleic acids to improve surgical outcomes in resectable disease, augment current therapies, expand druggable targets, and minimize morbidity. We conclude by summarizing progress in current research, identifying areas for future exploration in drug development and nanotechnology, and discussing future prospects for management of this disease.

Clinical Snapshot of Pancreatic Adenocarcinoma

Pancreatic cancer is the most lethal common tumor in America. The five-year survival is estimated to be 9.3% among all cases and 2.9% among patients with metastatic disease, both lowest amongst all common tumors (1). With its rising incidence and unabated mortality, pancreatic cancer is the third leading cause of cancer related death, with a projection that it will the second by 2030 (Figure 1) (2,3). In 2019, pancreatic ductal adenocarcinoma (PDAC; 95% of patients) represented 3.2% of all new cancer cases, with an estimated 56,770 new cases and

45,750 deaths (7.5% of all cancer deaths) (1).

PDAC is a complex and clinically challenging disease, defined by multiple genetic and environmental factors. The majority of PDAC arises de novo, with hereditary or genetic factors accounting for only 5-10% of cases (4). Risk factors associated with PDAC development include: smoking (relative risk (RR): 2-3), nonhereditary or chronic pancreatitis (RR: 2-6), chronic diabetes mellitus (RR: 2), obesity and/or sedentary lifestyle (RR: 2), non-type O blood group (RR: 1-2), and age (> 97% of cases occur over the age of 45) (4,5). A few genetic syndromes and mutations correlate with higher PDAC lifetime risk. Individuals with hereditary pancreatitis, associated with Trypsin-1 (PRSS1) or serine protease inhibitor Kazal-type 1

(SPINK1) mutations, poses a lifetime PDAC risk of 50%, while patients with Peutz-Jegher syndrome and Familial Atypical Multiple Mole and Melanoma Syndrome carry lifetime risks of

30-40% and 10-20% respectively (6). Other syndromes, such as Lynch Syndrome (associated with MLH1, MSH2, and MSH6 mutations), hereditary breast and ovarian cancer syndromes

(caused by BRCA1/2 or PALB2 mutations), Ataxia-telangiectasia (caused by mutations in the

ATM), and Li-Fraumeni Syndrome (caused by germline p53 mutations), contribute to a lesser degree (6,7). Inherited germline mutations in CDKN2A, MLH1, BRCA1, BRCA2, TP53, and ATM are associated with familial PDAC history, and screening for these mutations is recommended by National Comprehensive Cancer Network guidelines (8,9).

Diagnosis of Pancreatic Adenocarcinoma

Treatment improvements in many common tumors, e.g., breast and prostate, are, in part, a consequence of advances in disease diagnosis. Unfortunately, there are no reliable or readily available screening tests for PDAC, and the majority of PDAC patients do not exhibit symptoms until advanced stage. The majority of PDAC tumors (60-70%) originate at the head of the pancreas, and this location dictates subsequent symptomatology (10,11). Head tumors typically present with pain, jaundice, pruritus, pale stools, dark urine, and gastric outlet obstruction. Body and tail tumors are largely asymptomatic and present late with distant

metastases or local disease with multivisceral and vascular invasion. Both locations are associated with anorexia, weight-loss, and generalized abdominal pain (12). A comparative case-control analysis of PDAC patients (n=120) to control patients (n=180) revealed that bile obstruction (odds ratio (OR): 20), pale stool (OR: 31), anorexia (OR: 41), abdominal pain (OR:

30), and unusual bloating/ belching (OR: 20 and 17) are the most common general pancreatic cancer symptoms (13). Non-specific early symptoms hamper early clinical diagnosis of PDAC, supporting research into non-invasive, cost-effective screening methods.

Development of accurate diagnostic tests is limited by the dearth of effective biomarkers.

Currently, carbohydrate antigen 19-9 (CA19-9), a sialyated Lewis blood group antigen, and carcinoembryonic antigen (CEA), are used as circulating biomarkers of pancreatic cancer.

CA19-9 is not sensitive nor specific, and is elevated in other pancreatic diseases, such as pancreatitis, pancreatic pseudocyst, choledocholithiasis, and cirrhosis (14). Currently, CA19-9 is used to monitor the course of patient disease, including post-surgical recurrence (15). CEA is also neither sensitive nor specific for early PDAC, and is elevated in alcoholic cirrhosis, hepatitis, and biliary disease, and, thus, its utility in screening is limited (13,16). Efforts to identify clinically relevant biomarkers are ongoing, and there are recent exciting developments in both diagnostic and predictive biomarkers. Given the wealth of new potential biomarkers and potential screening assays, the reader is referred to a recent thorough review by Hasan and colleagues (15).

Conventional Treatment of Pancreatic Adenocarcinoma

The poor 5-year survival in PDAC patients reflects the late diagnosis, limited treatment options, and molecular and biophysical properties of PDAC that contribute to resistance.

Surgical resection remains the only current curative intent therapy for pancreatic cancer.

However, surgical therapy is limited to 15-20% of all PDAC patients, and often nodal metastases and microscopically positive margins are noted following resection (1,15). Resection

typically requires a pancreaticoduodenectomy (Whipple procedure) to remove tumors localized to the pancreatic head and uncinate process (15,16). Multiple variations of the Whipple procedure (including extended lymphadenectomy, pylorus-sparing, and a variety of anastomotic techniques, as well as minimally invasive) are not associated with improved survival outcomes

(5).

Minimally invasive surgical techniques are gaining popularity in oncology as an alternative to open procedures, with the potential to reduce infection, blood loss, postoperative pain, and surgical site infection. Despite the technical challenges of a Whipple procedure, the volume of laparoscopic and robotic assisted procedures has been increasing (17). While meta- analysis comparing both laparoscopic and robotic approaches to open Whipple procedures reported lower blood loss, shorter hospital stay, and decreased morbidity without any difference in major complications, mortality, or re-operation rates, the LEOPARD-2 trial reported a higher mortality in laparoscopic procedures compared to open procedures (18,19). Robotic surgeries are also safer than laparoscopic procedures, reduce the length of surgery, decrease postoperative morbidity, and decrease the duration of hospital stay, without evidence of increased mortality (17,20-23). Furthermore, robotic Whipples afford a lower complication rate, margin positivity rate, and wound infection rate compared to open procedures (17).

To increase the rate of R0 (tumor negative margins) vs R1 (residual tumor margins) resections, neoadjuvant chemotherapy protocols are being introduced in surgery (24). Murphy and colleagues report the potential benefit of neoadjuvant fluorouracil, leucovorin, oxaliplatin, and, irinotecan (FOLFIRINOX) and losartan followed by surgical resection in a phase II clinical trial. Of the 49 enrolled patients, 42 underwent attempted surgery and an R0 (margin-negative) resection was achieved in 69% (CI 55%-82%) (25). With neoadjuvant therapy, overall median progression-free survival increases to 17.5 months and overall survival to 31.4 months, although control outcomes were not given (24). Similarly, FOLFIRINOX with gemcitabine transitions 52% patients with borderline resectable disease to R0 outcomes (25). Retrospective data analysis of

30 patients with locally advanced or borderline resectable PDAC, who received neoadjuvant gemcitabine plus nab-paclitaxel, with and without subsequent S-1 therapy reveals that eight were amenable for surgery, and six achieved R0 resection (26). Collectively, this work demonstrates the potential to improve survival outcomes in traditionally non-operable patients and to expand current curative intent therapy.

Palliative treatment of metastatic PDAC relies on systemic, non-specific chemotherapeutics (Figure 1). Current treatment options and several recent trials for metastatic

PDAC are summarized in Table 1 (27-47). First line chemotherapy in metastatic PDAC typically entails combination therapy with gemcitabine and a nanoparticle formulation of paclitaxel

(Abraxane), or the combination therapy FOLFIRINOX. Monotherapy with gemcitabine is considered for patients not amenable to more aggressive combination therapy, showing only modest improvement in overall survival (30). First line therapies, gemcitabine with or without

Abraxane and FOLFIRINOX, show marginal improvement in overall survival for patients with metastatic cancer, with overall survival falling short of one year in most trials. Lack of target specificity, dose-limiting toxicity, and poor drug penetrance underlie major limitations in PDAC therapy efficacy in metastatic disease, and highlight the discrepancy in care between PDAC and other solid tumors.

Targeted Therapies in Pancreatic Cancer

Currently, there are a dearth of targeted therapies in PDAC. Most targeted agents, such as anti-angiogenic agents (Bevacizumab), tyrosine kinase inhibitors (Cetuximab), checkpoint inhibitors (Durvalumab), and recently hyaluronic acid therapies (PEGPH20), have been inefficacious in metastatic pancreatic cancer. (Table I). Several targeted therapies afforded only marginal improvements (<2 months) in overall and progression-free survival, while others yielded worse outcomes. For example, treatment combining MEK1/2 and AKT inhibitors

Selumetinib and MK-2206 showed worse overall survival than mFOLFIRINOX alone (46).

Similarly, re-purposing successful clinical agents (such as anti-EGFR and HER2 agents) have also failed (32-38). Certain targeted therapies do show efficacy in select population groups.

Olaparib, a PARP inhibitor, improved response rates in patients with DNA damage repair deficiency (10-20% of PDAC patients). PFS was 24.7 weeks (3.9-41.1 weeks) with two partial responses and six stable disease responses (48). Similarly, the neurotropic receptor tyrosine kinase inhibitor larotrectinib, received FDA approval for NTRK fusion mutant tumors (<1% of

PDAC), based on a successful Phase III clinical trial (49). While larotrectinib may be an efficacious therapy in this subgroup, early data suggests that rapid resistance may be a problem

(50).

Several failed therapies have targeted pathways regulated by the RAS family, known to be mutated in PDAC tumors. A summary of RAS mediated signaling is detailed in Figure SI1.

The RAS family of gene (HRAS, KRAS, and NRAS) encode 21 kDA proteins with GTPase activity, responsible for regulating mitogenic cell signaling pathways (51). In PDAC, Kras mutations are observed in 95% of tumors, with codons glycine-12, glycin-13, or glutamine-61 residues frequently altered in tumor cells (51). Therapies targeting Ras regulated pathways have consistently failed in PDAC, due to the high frequency of activating mutations. Initially thought to be undruggable, there is ongoing exciting research in Ras-specific targets, which recognize transient, druggable binding pockets. The first, S-IIP, is the target of RAS inhibitors in the G12C mutant (only 1-4% of PDAC tumors) with a covalent KRAS(G12C) inhibitor AMG 510, under clinical investigation (52). Additional studies have discovered four total pockets: S-IIP pocket, Kobe site, cyclen site, and P4 site, all of which are of clinical interest (52-57).

Immunotherapy for pancreatic cancer

Immunotherapy is affording a paradigm shift in human cancer treatment. Immune checkpoint inhibitor (ICI) monoclonal antibodies that target cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or programmed death ligand 1 (PD-L1)/programmed death 1 (PD-1) have

generated durable efficacy in many types of human cancers. In 2017, the US FDA granted accelerated approval to the anti-PD-1 monoclonal antibody Keytruda for patients with solid tumors, including pancreatic tumors, which have either mismatch repair deficiency (MMR-D) or high microsatellite instability (MSI). In the first phase I trial evaluating the anti-PD-L1 antibody

MS-936559, objective responses were achieved in melanoma, non-small cell lung cancer, renal- cell cancer, and ovarian cancer, but not in the 14 pancreatic cancer patients (58). Similar disappointing results were seen in a 60-patient phase IIA trial with anti-PD-L1 antibody

Durvalumab (59). However, a robust 62% objective response rate to pembrolizumab was observed in pancreatic cancer patients with mismatch repair deficient (MMR-D) (n=8) (60).

Therefore, limited efficacy of anti-PD-L1 therapy is confounded by the low, only 0.8%, prevalence of MMR-D tumors (61). Similar short-comings were seen in response to anti-CTLA-4 mAb immunotherapy. In phase II and IIa trials, no objective response was achieved with

Ipilimumab (62). Combinational immunotherapy of anti-PD-L1 and anti-CTLA-4 mAbs showed increased efficacy, albeit at a low rate with an objective response rate of 3.1% (62).

The underlying mechanism of human pancreatic cancer unresponsiveness to ICI immunotherapy is still elusive. It is thought that pancreatic cancer is a non-immunogenic “cold” cancer type (63). Human pancreatic carcinoma, except for the MMR-D subtype, exhibits a median mutational load of 4, much lower than more immunogenic melanoma and lung cancer

(63,64). It might be expected that a lower mutational load would decrease neoantigen presentation on tumors, thereby decreasing tumor-infiltration by cytotoxic T-cells (CTL), and limiting anti-PD-L1 therapy (65,66). However, CTLs do infiltrate human pancreatic carcinoma suggesting that CTL tumor-infiltration level is unlikely a major factor that underlies human pancreatic cancer non-response to ICI immunotherapy (67-70). Furthermore, pancreatic tumor cells highly express PD-L1 and PD-L1 appears to suppress CTL tumor infiltration in the tumor microenvironment (71-74).

Yet, despite abundant PD-L1 expression and infiltrating CTLs, pancreatic cancer does not respond to anti-PD-1/PD-L1 ICI immunotherapy (60,61). Therefore, it is reasonable to suspected that a PD-L1-independent mechanism may confer immune evasion to pancreatic tumors. This interpretation is supported by data demonstrating that targeting tumor-promoting mediators in the tumor microenvironment sensitizes pancreatic cancer to ICI immunotherapy in preclinical mouse tumor models (7,74).

Treatments in Preclinical Development

Poor survival outcomes in PDAC necessitate novel, innovated approaches towards disease management. Prior therapies failed to capture the biologically relevant pathways driving

PDAC tumorigenesis. Future research must focus on addressing the biological and biophysical properties unique to PDAC. As PDAC treatment employs both surgery and chemotherapy, we will discuss new advancements in these fields including: 1) enhanced visualization of tumor margins during surgery in order to increase the incidence of complete resection; 2) better targets for biologic therapy and small molecule inhibitors; and, 3) improvements in drug delivery to overcome biophysical limitations of PDAC treatment such as delivery within the local fibrotic tumor and peri-tumoral microenvironment as well as improved systemic penetrance.

Surgical visualization aids

In order to achieve complete R0 resection, surgeons must be able to adequately visualize the entire tumor. Conventional bright-field surgery is inherently limited by the human eye, and surgeons rely on histology to evaluate their resection. To augment tumor visualization, several non-specific near-infrared (NIR) fluorescent probes are used (75-79). These dyes spread lymphatically, and aid in visualizing bulk tumor margins and sentinel lymph nodes. Such surgical visual aids (SVAs) are employed in oncologic surgery to allow NIR tumor visualization, and clinical trials are ongoing in many cancer subtypes, including: colon cancer (LUM015,

RAPIDO-TRACT, and Fluorescent lectin), lung cancer (EC17, and ICG), and breast cancer

(BLZ-100, LYM015, AVP-620, EC17), as well as in PDAC (discussed below) (77-79). While advantageous over bright-field surgery, these dyes are inherently limited by non-specific diffusion, low specificity, and poor tumor retention. Newer methods of fluorescent agent delivery, enhanced tumor specificity, and improved resolution are under development to improve primary tumor detection and identify early metastatic niches.

Polymeric nanoparticle SVAs offer an effective alternative to current, non-specific SVAs.

Relying on bulk diffusion, nanoparticles accumulate within metabolically active cells and are retained longer than lower molecular weight dyes. Responsive polymeric nanoparticles covalently linked with rhodamine dyes (HRF-eNP), designed to expand and retain dye once internalized within cells and the tumor microenvironment, readily localize to PDAC tumors in a

Panc1 rat xenograft model. HRF-eNP enable improved visualization and delineation of tumor margins compared to bright field imaging (Figure SI2a,b) (80). Targeting and retention of the

HFR-eNPs in peritoneal tumor sites is highly effective, with specificity, sensitivity, and accuracy of 0.99, 0.92, and 0.95 respectively, in the 202 normal samples and 253 histologically confirmed samples surveyed. Use of HRF-eNPs allows sub-millimeter tumor detection, a currently unmet need in image resolution. A hyaluronic acid derived nanoparticle containing indocyanine green,

NanoICG, provides better tumor visualization (2.30 + 0.67 AU in tumor vs. 0.41 + 0.10 AU in normal pancreas) compared to indocyanine green alone (0.77 + 0.12 AU in tumor vs. 0.35 +

0.12 AU in normal pancreas) in a KPC orthotopic xenograft PDAC murine model (81). While

NanoICGs are effective for delineating tumor from adjacent normal tissue, significant accumulation occurs in the stomach, liver, small intestine, and kidney. Finally, tripolymer fluorescent nanospheres (TFNS) possessing a surface coating of peanut agglutinin proteins exhibit high affinity for the tumor-associated antigen Thomsen Friedenreich (TF), and shows high affinity for PDAC, when comparing histologically fixed samples of PDAC and normal tissue

(82). TNFS nanoparticles localize primarily to PDAC tumors in an in vivo model of PDAC, using

subcutaneously implanted CRL2460 cells. The TNFS nanoparticles enable complete resection of PDAC tumors with minimal removal of normal tissue. Furthermore, signal levels in non- cancerous tissue were well below detection threshold, reducing false positives. Relying solely on bulk diffusion, these nanoparticle carriers show promise as a cost-effective, reproducible method for image-guided surgery.

In contrast to non-specific nanoparticle delivery, targeted fluorophore conjugated antibody SVAs are being evaluated. Clinical results obtained using anti-CEA (NCT02973672 and NCT0278028), anti CA19-9 (NCT02587230), anti-EGFR (NCT02736578 and

NCT03384238), and anti-VEGF (NCT02743975) based SVAs are promising with improved R0 resection rates and better surgical staging of advanced cases (83). Newer antibody conjugate systems, therefore, are of keen interest, including dual-antibody systems to increase the signal, improve specificity, and overcome the heterogeneity present in cell surface expression. For example, Zettlitz and colleagues describe a dual-labeled targeting anti-prostate stem cell antigen (PSCA) conjugated with a near IR dye and a 124I for functional PET imaging. PDAC tumors highly express the PSCA surface antigen with high sensitivity and specificity (84). Use of this dual-labeled antibody allows sequential NIRF and PET visualization of subcutaneous PDAC tumors in a murine patient derived xenograft (PDX) model (85). These bifunctional anti-PSCA antibodies retain high binding specificity, with minimal non-cancerous uptake. A dual antibody system which contains both a fluorescent dye, Alexa Fluor 700, and a quencher IRDye, QC-1, fluoresces only after proteolytic cleavage (86). This responsive system repurposes two antibodies, cetuximab and trastuzumab. Both proteolytically activated immunoconjugates provide improved signal resolution of pancreatic AsPC-1 orthotopic xenografts, as well as enhance signal detection and resolution compared to nonspecific probes. Furthermore, dual imaging improves signal resolution, maximizing signal in cancer cells which have higher

receptor expression of both targets. With the discovery of more specific PDAC targets, newer

SVA can be designed for better tumor detection and enhanced visualization.

Finally, there is ongoing activity to develop theranotics, combined visual agents and adjuvant therapy at the time of surgery. For example, gold nanoparticles functionalized with an anti-glypican-1 antibody, which targets a cell surface proteoglycan, and an NIR dye, for tumor visualization, loaded with oridonin inhibit PDAC migration and EMT (87). These nanoparticles are highly specific for PDAC tissue, and provide excellent resolution of tumor margins.

Adequate fluorescent signal remains even after 48 hours in BxPC-3 orthotopic xenografts (88).

Furthermore, these SVAs reduce tumor volume in the BxPC-3 model, affirming both a therapeutic efficacy as well as a specificity. Another PDAC gold nanoparticle theranostic targets the cell surface proteoglycan GPC1 (89). These gemcitabine-loaded nanoparticles, labeled with an NIR dye (GPC1-GEM-NPs), allow simultaneous tumor visualization and drug delivery. The combined theranostic system shows excellent uptake in PDAC BxPC-3 and Panc-1 cell lines

(>99% by flow cytometry), but minimal uptake (<1%) in control 293T cells. In an orthotopic

BxPC-3 murine model, the GPC1-GEM-NPs exhibit greater tumor retention (high signal at 48 hrs) and accumulation than GEM-NPs (lacking the GPC1 antibody) alone (loss of signal by 48 hrs), with off-target NP accumulation in the liver and spleen (89). Theranostics are an exciting surgical tool, providing adjuvant therapy while enhancing surgical visualization.

As discussed above, only 10-15% of PDAC patients are currently amenable to surgery.

Often many patients progress despite surgery, as the result of incomplete resection or undetected metastatic disease at the time of surgery (90). SVAs improve R0 resection rates and identify micrometastatic disease. In addition, the use of theranostic agents provides adjuvant treatment of micrometastases while guiding further management. It is also reassuring that several recent trials have improved R0 resection rates of previously non-surgical candidates through better neoadjuvant chemotherapy protocols (vide supra). Through better tumor visualization, improved treatment and staging is a possibility.

New antibodies and biologic targets

Identification of novel targets in pancreatic cancer is essential to improving treatment outcomes.

Available therapies for advanced and metastatic PDAC are predominately non-specific chemotherapeutics. To date few targeted therapies exist, as many have failed because they targeted KRAS-dependent pathways. Efficacious treatment of PDAC requires identification of

PDAC-specific targets, which are KRAS-independent, and which target multiple tumor hallmarks to prevent disease resistance. Several new targets have been identified which met these criteria, and preclinical advancements in these targets are exciting (Table 1).

DEspR

The dual-endothelin-1/VEGF-signal peptide receptor (DEspR) is a single transmembrane receptor, identified as an embryonic-lethal null mutation phenotype characterized by dysregulated vasculogenesis and angiogenesis, and impaired cardiac organogenesis (91,92).

DEspR binds two known ligands, ET-1 and the signaling peptide of VEGF. Both of these pathways strongly correlate with PDAC progression, but currently, there are no effective therapies targeting either one. PDAC tumor cells highly express DEspR, with minimal expression on normal pancreatic tissue. This receptor regulates multiple tumor processes: cancer stem cell (CSC) viability and anoikis resistance, angiogenesis, and cancer cell invasiveness. The exact signaling pathway is unclear, but appears to have Ras-independent signaling, through FAK, Src, and STAT3 mediated pathways (91). Treatment of a subcutaneous xenograft RNU rat model of Panc1 with a monoclonal antibody to inhibit DEspR reduces tumor volume compared to gemcitabine (Figure SI3a) (92). Inhibition of DEspR captures several key

PDAC pathways and may address the role of ET-1 and VEGF in PDAC. Further research is needed to address its safety and effect on normal cells and to interrogate its role in normal cell signaling.

GPR87

Several cancer subtypes, including squamous cell carcinomas, lung, cervical, and pancreas highly express GPR87, a recently identified G-coupled protein receptor related to the lysophosphatidic acid (LPA) receptor (93-96). GPR87 regulates multiple pathways, which influence tumor proliferation, angiogenesis, anoikis resistance, and drug resistance. GPR87 signaling involves multiple downstream mediators, with both Ras-dependent and Ras- independent signaling (97,98). In pancreatic cancer, GPR87 mRNA expression is significantly elevated and correlates with worse overall survival (98). PDAC cell lines, AsPC-1, Capan-1,

BxPC-3, MIA PaCa2, Capan-2, and PANC1 overexpress GPR87 compared to normal human pancreatic ductal epithelial cells. Inhibition of GPR87 significantly reduces tumor viability in vivo and decreases subcutaneous tumor growth compared to controls (Figure SI3b) (98). GPR87 attractive features include: it regulates multiple tumor hallmarks, correlates with worse prognosis, and is highly expressed in most PDAC. However, GPR87 is expressed on many normal tissues, albeit at lower levels. Like DEspR, GPR87 inhibition is not completely characterized, and it is unclear if GPR87 regulates survival in healthy epithelium.

CD147

CD147, a member of the immunoglobulin superfamily, regulates several key cancer hallmarks in

PDAC and is highly expressed in PDAC (87%), hepatocellular carcinoma (83%), and glioblastoma multiforme (79%) (99,100). CD147 expression positively correlates with poor survival and greater metastatic spread (100). CD147 influences multiple downstream signaling pathways, which are both Ras-dependent and independent, to include anoikis resistance, epithelial to mesenchymal transition, chemotherapeutic resistance, and CSCs pluripotency and proliferation (101-106). A monoclonal antibody targeting CD147, HAb18IgG, enhances radiation-induced cell death in CFPAC-1 and MIA PaCa-2 cells, and potentiates the effect of

gemcitabine therapy (107). Inhibition of CD147 reduces tumorsphere formation, a surrogate of

CSC-inhibition, in BxPC-3 and MIA PaCa-2 PDAC cell lines. A combination therapy of a 90Y-l radiolabeled anti-CD147 antibody and gemcitabine significantly improves the efficacy of gemcitabine therapy in a murine MIA PaCa-2 xenograft model (108). Although CD147 is an exciting target, data suggest that CD147 is a promiscuous receptor, and downstream regulation depends on cell surface density (101). Therefore, treatment responses to CD147 therapy may be highly variable.

MFAP5

Microfibril associated protein 5 (MFAP5), an extracellular matrix glycoprotein involved in elastic microfibril assembly, regulates both PDAC cells and cancer associated fibroblasts

(CAFs). Elevated MFAP5 expression correlates with worse prognosis and more aggressive disease (109,110). Murine antibodies targeting MFAP5 reduce PDAC cell viability and cell motility (109). In in vivo patient derived xenografts, inhibition of MFAP5 sufficiently arrests cancer cell growth and decreases collagen I deposition, and impairs CAF collagen production and decreases intratumoral microvessel permeability (109). Treatment with MFAP5 improves delivery of paclitaxel by decreasing biophysical barriers, such as high intratumoral pressure

(109). Given the shortcomings of hyaluronidase therapy, anti-MFAP5 therapy is an attractive alternative, reducing both tumor cell viability and CAF-induced desmoplasia.

Claudin-4

Another target of tumor barrier function is Claudin-4 (CLDN4), a main constituent protein in tight junctions. PDAC cells overexpress claudin-4 compared to normal pancreatic cells, with higher expression carrying a worse prognosis (111,112). Inhibition of CLDN4 reduces cell adhesion of

PDAC cells and enhances intracellular accumulation of chemotherapeutics (112,113). In vivo administration of anti-CLDN4 antibodies promotes intratumoral accumulation of FOLFIRINOX,

and increases efficacy in subcutaneous MIA PaCa2 murine models (112). This effect is likely a result of reducing PDAC cell adhesion and interfering with drug efflux pumps. A recent report suggests that anti-CDLN4 antibodies are safe and relatively non-toxic, but clinical data is lacking

(113). Both CLDN4 and MFAP5 are promising targets, which could disrupt natural PDAC barriers to chemotherapeutics and overcome biophysical defenses.

BAG-3

An exciting regulator of PDAC stromal cells is BCL2-associated athanogene 3 (BAG-3), a cochaperone of Hsp70. BAG-3 regulates multiple signaling pathways in PDAC, facilitating cross-talk between: tumor-associated macrophages and PDAC cells and PDAC cells and pancreatic stellate cells via an IL6 dependent manner (114,115). BAG3 expression correlates with fibrosis in PDAC tissue, and plays a key role in tumor collagen barrier regulation (116).

Administration of humanized anti-BAG-3 antibodies reduces the growth of subcutaneous MIA

PaCa-2 tumors in murine xenografts. The anti-BAG3 antibody are highly tumor specific, and do not distribute into normal parenchyma (116). While data are limited, no toxicity was observed with BAG3 administration in healthy animals. Currently, Intrepida Bio is developing an anti-

BAG3 antibody for clinical trials in PDAC.

Improvements in PDAC therapy requires highly selective targets, which regulate multiple, essential cancer pathways. The targets presented here are both novel and clinically relevant, having several advantages over previous, failed biologic targets (e.g., EGFR, IGFR, and VEGF). These include targeting multiple cancer hallmarks, inhibition of tumor cell and stromal function, KRAS-independent mechanisms, and modulation of PDAC CSC function.

Small molecule inhibitors

Small molecule inhibitors have not shown success in recent trials in PDAC. Significant resistance has been encountered in these therapies due to Ras-dependence of their targets.

Future endeavors must focus on pathways that directly regulate PDAC proliferation, metastasis, and survival. Ongoing research evaluating MEK/ERK inhibitors and autophagy, EGFR inhibitors, and re-evaluation of more classical pathways, is clinically important, but will not be discussed here (117-120). Instead we discuss several novel PDAC targets with Ras-independent signaling pathways. These inhibitors impact tumor energy regulation and chemoresistance in addition to

PDAC cell progression and metastasis. While not exhaustive, these new studies highlight recent preclinical advances demonstrating in vivo efficacy in highly expressed, biologically relevant targets.

GFAT1 (DON)

The hexosamine biosynthesis pathway (HBP) is a major regulator of cancer survival. In normal cells, the HBP is a shunt of the glycolysis pathway, acting as a sensor of energy availability in normal cells (121). HBP plays a role in cancer cell signaling, via O-

GlcNAcylation/mTOR/adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathways, tumor suppressor kinase 1/2 (LATS1/2) mediated pathways, and ER stress regulation (122). Not surprisingly, HBP regulates cancer cell metabolism and directs cellular proliferation, transcription, EMT, and self-renewal in CSCs (123). The rate limiting enzyme in

HBP is glutamine-fructose amidotransferase 1 (GFAT1), which is overexpressed in 35.7% of the

176 PDAC samples in TCGA. Inhibition of GFAT1 in vitro, using a small molecule glutamine analog (6-diazo-5-oxo-L-norleucine [DON]), decreases expression of several self-renewal genes: SOX2, OCT4, and KLF4 in MIA-PaCa2 and S2VP10 cell lines, and reduces both tumor cell proliferation and collagen matrix deposition in murine orthotopic KPC and CAF xenografts

(124). Reduction of PDAC ECM density also enhances tumor sensitivity to anti-PD1 therapy, increasing CD8+ tumor infiltration (125). GFAT1 is a promising clinical target, as its inhibition decreases tumor desmoplasia while enhancing anti-PD1 therapy. The success of anti-GFAT1 therapy in lung and breast cancer will hopefully translate to PDAC (121).

GOT1 (PF-04859989, Aspulvinone O)

Another metabolic target of interest is glutamate-oxaloacetate transaminase 1 (GOT1), which regulates amino acid metabolism and the malate-aspartate shuttle. GOT1 overexpression is common in PDAC tumors and correlates with worse disease prognosis In KRAS mutant

PDAC, alterations in GOT1 metabolism fuel biosynthesis, proliferation, and redox balance, leading to dependence on GOT1 for tumor maintenance (126,127). A novel inhibitor of GOT-1,

PF-04859989, forms a covalent adduct with the substrate of GOT1, irreversibly inhibiting enzymatic function (128). Loss of GOT1 function limits PDAC metabolism and inhibits PDAC proliferation in GOT-1 dependent cell lines. A natural inhibitor of GOT1, Aspulvinone O, exhibits similar efficacy (129). Inhibition of GOT1 by Aspulvinone O induces apoptosis in SW1990 cell lines in vitro, and modulates cellular metabolism via shifting mitochondrial respiration and increasing ROS production (129). Also, Aspulvinone O inhibits PDAC proliferation in vivo in murine subcutaneous SW1990 xenografts. These results are promising, as GOT1 inhibition could starve KRAS mutant PDAC tumors and prevent disease progression.

Creatinine transporter SLC6a8 (RGX-202)

Increased production of phosphocreatine via creatine kinase-B occurs in pancreatic cancer in order to meet the high energy demand of growing tumors (130). Importing phosphocreatine via the creatine transporter SLC6a8 allows cells in the metastatic niche to maintain ATP under high metabolic demand (131,132). Depletion of SLC6a8 in colon and pancreatic cancer reduces the ability of cancer cells to colonize the liver in murine xenografts

(133,134). The orally available small molecule RGX-202 inhibits SLC6a8 in KRAS wild-type and

KRAS mutant cell lines, as well as human PDX models (135). Inhibition of SLC6a8 correlates with decreased tumor metabolic activity, decreased proliferation, and a dramatic reduction in cell survival. Tumors treated with RGX-202 are also more susceptible to gemcitabine, likely

reflecting a change in cellular metabolism and nucleotide synthesis (135,136). Although the effect of SLC6a8 on normal cellular metabolism remains to be full understood, these results are exciting, and suggest that in phosphocreatine-dependent cells, SLC6a8 inhibition could effectively starve PDAC tumors.

CtBP (4-Cl-HIPP)

The transcriptional coregulator C-terminal Binding Proteins (CtBP) 1 and 2 regulate key tumor hallmarks: apoptosis, proliferation, and regulation of the pluripotency state (137-139).

Overexpression of CtBPs results in oncogenic transformation of human colon epithelium, similar to H-ras mutation, and may define an early activation step in GI malignancies (138,139). PDAC tumors highly overexpress both CtBP1 and 2, and both isoforms are known to regulate PDAC metastasis (140). CtBPs contain a targetable dehydrogenase domain, required for their transcriptional function. The novel anti-CtBP inhibitor (4-chloro-hydroxyimino phenylpyruvate (4-

Cl-HIPP)) targets this dehydrogenase domain (136). Loss of CtBP2 decreases metastatic capacity and reduces peritoneal PDAC seeding in a genetically engineered CKP PDAC murine model (136,140). In primary CKP tumors, treatment with 4-Cl-HIPP reduces tumor volume, equivalent to gemcitabine treatment alone. When dosed together, 4-Cl-HIPP enhances the action of gemcitabine, reducing primary CKP tumors by greater than 50% (140). While early in development, anti-CtBPs are promising adjuvants to traditional chemotherapeutics.

ABCC3 (MCI-715)

Canalicular multispecific organic anion transporter 2 (ABCC3) is a member of the ATP- binding cassette (ABC family) and is associated with chemoresistance in PDAC. ABCC3 is regulated by p53, and controls PDAC proliferation via STAT3 and HIF1α signaling (141).

ABCC3 overexpression correlates with worse prognosis after resection, greater chemoresistance, and is present in early cancerous lesions (142,143). A nonsteroidal anti-

inflammatory drug derivative, MCI-715, inhibits ABCC3 via inhibiting STAT3 and HIF1α downstream signaling, leading to apoptosis in AsPC1, HPAFII, and CFPAC-1 PDAC cell lines.

Administration of MCI-715 reduces tumor volume and improves overall survival in a HPAFII subcutaneous xenograft model, a KPC transgenic murine model, and a PDX model (144). Early data is promising that anti-ABCC3 therapy is effective alone, and further research into synergistic potential between MCI-715 and conventional chemotherapeutics would be of considerable interest.

SRC-3 (bufalin)

The steroid receptor coactivator family (SRCs) are nuclear receptors, which act as scaffolds for assembly of coactivator homocomplexes. Of the three SRC members, SRC-3 is more frequently overexpressed in human tumors and SRC-3 levels increase from low-grade

PanIN to malignant PDAC (145). Using bufalin, a 14-β-hydroxy steroid, inhibition of SRC3 significantly reduces PDAC cell viability in Panc-1 and Capan-2 cell lines (146). In vivo, treatment with bufalin reduces tumor proliferation and increases animal survival in orthotopic

Panc-1-Luc cell line xenografts. Several studies report SRC-3 as a potent oncogene, which may contribute to the genetic complexity of PDAC (147-149).

The small molecule inhibitors described here possess significant advantages over previously tested, and failed, agents in PDAC. Several of these agents target PDAC cellular metabolism, starving metastatic niches and limiting primary tumor growth. Other targets strip

PDAC cells of protective chemoresistance defenses, making tumors vulnerable to conventional chemotherapeutics. These inhibitors target proteins which regulate multiple tumor hallmarks, influencing proliferation, metastasis, and CSC potentiation. All of the agents demonstrate efficacy in vivo and have potential to be translated to clinical trials. Although unlikely to be

sufficient by themselves, several show a benefit when combined with conventional chemotherapeutics.

Nanoparticle treatments

As an alternative to the delivery of antibodies and small molecule inhibitors, nanoparticle-based strategies deliver high pay-loads of cytotoxic agents to the tumor as a concentrated package (Figure 1). The targeting or localization of the drug-loaded nanoparticle typically relies on the greater metabolic activity and leakier vasculature of tumors compared to normal tissue, a property termed the enhanced permeability and retention effect (EPR)

(150,151). While the differences in biochemical and biomechanical properties between normal and cancerous tissue can be useful in aiding drug delivery, the EPR effect is often exaggerated in many preclinical models, where murine tumor proliferation rates exceed those of human tumors (152). This higher proliferation rate produces less developed, leakier blood vessels, which are not reflective of most human tumor vessels. Therefore, modifications to nanoparticle materials to improve tumor specificity, such as including tumor specific proteins in the polymeric structure, is likely necessary (153).

Resistance to cancer therapies is both molecular and biophysical in nature. Molecular resistance includes the redundancy of pro-oncogenic pathways, modification or alteration of tumor specific proteins and receptors, and survival of target negative cells. The challenge of significant tumor heterogeneity is reflected in the common failure of targeted therapies in pancreatic cancer patients. Biophysical mechanisms of resistance encompass environmental factors within the tumor microenvironment that pose barriers to drug delivery (154). The composition of the tumor extracellular matrix, a dense collagen and elastic network, interspersed with glycosaminoglycans and proteoglycans, produces a hydrophilic gel, thereby increasing local hydrostatic pressure. In PDAC, spare blood vessel density and thick desmoplastic matrix further inhibit drug penetrance (153,154). Additionally, the tumor

microenvironment tends to be more acidic, contains numerous proteases, and is a nidus of reactive oxygen species, all of which can de-activate the drug, degrade protein therapies, and alter drug kinetics (152-154). Nanoparticle based drug delivery vehicles are designed to overcome the above challenges with local accumulation of drug payload (e.g., conventional chemotherapeutics, nucleic acids, or immunomodulatory agents). Various nanoparticle formulations are being investigated, ranging from polymeric, protein, or lipid assembly, and we next discuss recent preclinical advances in nanoparticles for the treatment of PDAC.

Polymeric nanoparticles

Biocompatible polymers, such as poly lactic acid, PLA, and copolymer derivatives, such as poly lactic-co-glycolic acid (PLGA) are commonly employed in nanoparticle development. A paclitaxel-loaded PLGA nanoparticle (NP) with an anti-MUC1 antibody, TAB004, on the surface readily internalizes in MUC1 positive cells, with MUC1 positive cells exhibiting decreased viability when treated with the nanoparticle vs. paclitaxel alone (155). In vivo imaging studies of isocyanine green encapsulated TAB004 conjugated NPs shows localization to KCM pancreatic cell line orthotopic tumors following a single IP administration of 50 mg/kg of the nanoparticle. In another study, α-mangostin, a sonic hedgehog inhibitor, encapsulated PLGA NPs reduce growth in CSC-like cells and prevent EMT by upregulating E-cadherin and inhibiting N-cadherin and Slug, Nanog, c-Myc, and Oct4 (Figure SI4a) (156). In the genetically engineered KPC mouse model of PDAC, this NP formulation prevents progression of pancreatic intraneoplasia to adenocarcinoma and liver metastasis. As with in vitro studies, the in vivo efficacy of this nanoparticle stems from inhibition of pluripotency factors, Gli targets, and the Shh pathway, preventing crucial steps in tumor progression. Using a rat model of metastatic PDAC, a paclitaxel loaded responsive polymeric NP accumulates in pancreatic tumor cells following intraperitoneal injection and specifically delivers drug to these tumors (157). The NPs maintain tight encapsulation of paclitaxel prior to reaching the tumor but then responsively swell and

release a high dose of paclitaxel when in the acidic tumor environment. Although treatment with the NPs does not significantly improve survival outcomes compared to free paclitaxel; substantially fewer side-effects are present, suggesting that NP delivery under this method may reduce dose-limiting toxicities and enable completion of treatment regimens.

Polymeric nanoparticles also provide a means to deliver poorly soluble immunomodulatory or tumor suppressive agents that cannot otherwise be delivered intravenously (158). Curcumin, a suppressor of carcinogenesis in several cancer subtypes: breast, colon, pancreatic, prostate, and ovarian, acts through several pathways, regulating cell cycle arrest through upregulation of p16, p53, and p14ARF, induction of apoptosis through Bax,

Bad, and Bcl proteins, and modulating NFkB signaling (158). Curcumin inhibits metastasis and angiogenesis in numerous xenograft cancer models, particularly in orthotopic xenograft models of PDAC, suggesting that its benefits extend beyond cancer prevention. Encapsulation sidesteps curcumin’s poor aqueous solubility and stability and offers a potentially viable method for delivery at sufficient levels. Curcumin-loaded PLGA-chitosan nanoparticles internalize and retain in Panc1 and MIA PaCa2 cells at a higher level than curcumin itself, with greater in vitro potency and reduced tumor migration via a scratch assay (Figure SI4b) (158). In vivo studies are needed to assess the potential benefit of curcumin.

In order to improve the local delivery and penetrance of polymeric NPs, which typically rely on EPR and bulk diffusion, modifications to the polymer backbone are being explored. By changing the specific characteristics of the polymer (e.g., charge, hydrophilicity, lipophilicity) or through the addition of surface aptamers, peptides, or even antibodies (which improve selective internalization) better pharmacokinetic profiles are achieved. For example, a sequentially triggered NP relies on an aptamer conjugated to a cell-penetrating peptide-campothecin prodrug

(Apt/CPP-CPTD) and amphiphilic PEG copolymer. The NP utilizes a GBI-10 aptamer

“camouflage”, relying on GBI-10’s negative charge and tenascin-C’s targeting ability to reduce non-specific uptake of a cell-penetrating peptide nanoparticle (Figure SI4c) (159). The NP

polymer backbone contains a reductively cleaved disulfide linkage, taking advantage of the greater glutathione concentration within the tumor environment, to release camptothecin following NP internalization. In vitro, the NP performs equivalently to free campothecin in MIA

PaCa2 cells but shows improved drug penetration, as measured by spheroid penetrance. After intravenous administration, the NPs localize primarily in the tumors of a subcutaneous xenograft

MIA PaCa2 mouse, and the NP treatment group, containing an equivalent 10 mg/kg of camptothecin, affords a significant decrease in tumor volumes compared to free camptothecin alone, or saline control, demonstrating improved efficacy.

Protein based nanoparticles offer potentially greater biocompatibility, improved stability, and alternative functionalization capability compared to polymeric nanoparticles (160).

Abraxane®, a paclitaxel loaded albumin-based nanoparticle, is the prime example and is currently used clinically (27,161,162). More recently, the tumor suppressor gene, hMDA-7, has been loaded into a bovine serum albumin (BSA) NPs. Successful gene delivery occurs in Panc1 and BxPC-3 cancer cells, with a marked reduction in cell proliferation and an increase in apoptosis associated with elevation of vascular endothelial growth factor (VEGF) expression

(163). Treatment of subcutaneous BxPC-3 and Panc-1 xenografts with the NPs significantly decreases tumor volume relative to controls. Encapsulation of Parviflorin D, an anti-proliferative agent derived from the Plectranthus genus, in an albumin-based nanoparticle significantly improves drug delivery by increasing drug solubility in vitro, and demonstrates improved potency in BxPc-3 and Panc-1 PDAC cell lines (164). To improve the specificity of albumin- based nanoparticles, Ji and colleagues surface-conjugated the arginine-glycine-aspartic acid

(RGD) peptide to their nanoparticles. These RGD modified BSA nanoparticles show greater internalization and retention in BxPC-3 cells compared to BSA nanoparticles alone, and are safe and tolerated in vitro and in vivo (165). The greater specificity of these RGD-albumin nanoparticles improves internalization beyond the EPR effect. Encapsulation of the nanoparticles with gemcitabine significantly improves the efficacy of gemcitabine treatment in

BxPC-3 cells, suggesting that this formulation may improve overall success of BSA derived nanoparticles and offer an advancement over Abraxane®.

Investigating whether tumor derived exosome-based nanoparticles repress tumor cell growth is another active research area and an exciting alternative to polymer-based approaches

(166). Protein characterization of pancreatic cancer cell line, SOJ, exosome derived NPs reveals a diverse packaging of proteins, including glucose-6-phosphate isomerase, Hsp90, and

BSDL (166). Treatment of SOJ cells with the SOJ-derived exosome NPs reduces proliferation in a dose dependent response, with similar, but variable response in other cell lines (167).

Evidence of nanoparticle efficacy includes: a decrease in overall PI3K/Akt/GSK-3β activity, an increase in phosphatase and tensin homolog protein (PTEN) dephosphorylation, an elevation of

Bax, and a decrease in Bcl-2 expression. The viability of the control human umbilical vein endothelial cells is not affected by the SOJ NPs.

Currently, two nanoparticle formulations, Abraxane® (discussed above) and Onivyde, are FDA approved for treatment of advanced PDAC. Onivyde is a liposomal formulation of irinotecan approved for metastatic PDAC patients having failed gemcitabine therapy. Onivyde extends survival in combination with fluorouracil and leucovorin, compared to these drugs alone

(mOS 6.1 months vs 4.2 months) (168). A Phase I/II trial is evaluating Onivyde with fluorouracil, leucovorin, and oxaliplatin as a first line therapy in PDAC. These clinical successes hold promise for nanoparticle research as part of cancer therapeutic development.

Nucleic acid treatments via gene delivery and aptamer conjugates

The direct modification of pro-oncogenic genes and re-activation of tumor suppressor genes, through gene therapy, offers a potentially more specific approach to treating pancreatic cancer. Known genetic mutations have been discussed, and represent key targets for drug developers. For example, the loss of crucial tumor suppressive genes can result in acquired drug resistance in cancer cells, limiting the efficacy of many therapies (169). Although gene

delivery, considered a holy grail in cancer therapy, is advancing in the clinic, many successful preclinical studies have failed to show efficacy in clinical trials (169,170). In this section, we describe several recent nucleic acid treatments in PDAC (Figure 1).

Delivery of silencing RNAs (siRNAs) that target oncogenes offers a therapeutic potential to abrogate tumor progression. siRNAs rapidly degrade before reaching the tumor tissue, therefore, a protective carrier is needed to enable selective delivery of the siRNA to the tumor.

For example, programmable DNA nanoparticles (DNPs), containing a nucleolin targeting aptamer, deliver Bcl2 targeting siRNA and reduce tumor volume in DMS53 subcutaneous xenografts (171). An alternative strategy uses a peptide-based nanoparticle to deliver siRNA for

Ras inhibition. This peptide-based siRNA loaded nanoparticle successfully reduces KP1 cell viability and KPCC pancreatic tumors in mice (172). The former approach, through a specific targeting aptamer, offers the advantage of selective targeting to the tumor and reduces off- targeting effects.

Rather than relying on delivery of siRNA through a nanoparticle carrier, an exciting alternative approach involves reprograming mesenchymal stroma/stem cells (AD-MSC) to release trimeric and multimeric sTRAIL (173). These modified AD-MSCs produce stable multimeric, soluble sTRAIL structures, via lentiviral transduction, capable of inducing apoptosis in BxPC-3, MIA PaCa-2, and primary PDAC cell lines with greater cytotoxicity than rhTrail

(Figure SI5a). Modified AD-MSCs transplanted into subcutaneous tumor bearing BxPC-3 mice produce sufficient levels of sTRAIL to decrease tumor volume. However, this technique will be technically challenging and expensive compared to nanoparticle delivery, and may not be amenable to metastatic disease treatment.

MicroRNA delivery provides an alternative to direct gene modification, relying on pan- suppressive effects. A pentablock copolymer of poly(ethylene glycol) diacrylate (PEG-DA) and miR-345 coblock polymer with encapsulated gemcitabine delivers both miR-345 and gemcitabine to significantly reduce Capan-1 cell viability in vitro and tumor burden in vivo in

subcutaneous xenograft models (Figure SI5b) (174). Human umbilical cord mesenchymal stromal cells derived exosomes, containing miR-145-5p, rapidly internalize in human Panc-1 cell lines affording an increase in apoptosis and cell cycle arrest (175). Overexpression of miR-145-

5p significantly reduces tumor volume in a subcutaneous murine xenograft model of Panc1. Cell derived exosomes offer better potential biocompatibility than polymeric nanoparticles; however, polymeric nanoparticles are better characterized and readily functionalized compared to the native systems.

Aptamer based approaches

An alternative approach to nucleic acid therapy is the use of aptamers-single stranded oligonucleotides- for targeted delivery (176,177) Aptamers are a practical alternative due to ease of chemical synthesis, chemical modification, and high stability; however, limited success in aptamer targeted therapies have been seen in cancer (178-181). A novel aptamer-drug conjugate (ApDC) utilizing AS1411 binds selectively to the nucleolin, a cellular protein highly expressed in several PDAC subtypes (182). Capan-1, MIA PaCa2, and AsPC-1 cancer cell lines, but not normal H6c7 cell lines, internalize and retain stable conjugates of AS1411 and gemcitabine (Figure SI5c) (182). The AS1411-gemcitabine conjugate shows a slight decrease in potency, but greater target specificity than gemcitabine alone, and demonstrates reduced tumor volumes in a subcutaneous xenograft model of Capan-1 in mice. An alternative approach utilizes a pancreatic cancer specific aptamer, 2’-Fluoropyrimidine RNA aptamers (2’F-RNAs) conjugate designed to deliver a small activating RNA (saRNA) for upregulating C/EBPα, an inhibitor of p21 (183). Downregulation of C/EBPα occurs from loss of KDM6B, leading to increased metastatic drive in pancreatic cancer. C/EBPα-based aptamers are highly specific to cancer cell lines, demonstrating good internalization and retention in Panc1, AsPC-1, MIA

PaCa2, and Capan-1 cell lines. Restoration of C/EBPα in Panc-1 cell lines correlates with decreased cell proliferation. Treatment of subcutaneous models of Panc1 with C/EBPα-aptamer

affords a modest decrease in tumor size, without significant toxicity, confirming target specificity and relevance of C/EBPα in PDAC progression. Expanding on this concept, researchers are investigating more potent chemotherapeutics, typically reserved for antibody drug conjugates, for the development of aptamer drug conjugates. For example, an auristatin bound EGFR targeting aptamer is cytotoxic to Panc1, MIA PaCa2, and BxPC-3 cell lines, offering the potential to create highly specific anti-tumor agents with potency exceeding conventional chemotherapeutics, with a greater therapeutic window (184).

Identification of more selective aptamers along with target validation is an active area of research. For example, cell-SELEX screening of human pancreatic cancer (HPAC)-derived spheroids to target potential cancer stem cell (CSC)-initiating or CSC-related genes yielded seven aptamers exhibiting high affinity binding, and target CD133, CD244, IHH, Nanog, and

ALDH1 expressing cells (185). Excitingly, these aptamers bind to circulating tumor cells derived from patients. Further validation of these targets is necessary, but may provide an additional method for aptameric drug delivery.

Advances in nucleic acid delivery have progressed significantly since their inception.

With the development of more stable carriers and application of aptamers as drug delivery vectors, nucleic acid therapy shows exciting progress in PDAC therapy. However, relative to other therapies discussed here, nucleic acid therapies are not widely used in the clinic. It is unknown how stable these therapies will be in clinical practice or how well tolerated – although the recent approvals of nucleic acid therapies provide impetus for continued discovery, research, and translation.

Conclusion

Outcomes in PDAC are limited by the dearth of available, successful treatment options.

Nonspecific early symptoms, tumor aggressiveness, and diagnosis of late stage disease result in clinical outcomes that are significantly worse than those of other cancers. For early stage

disease, surgical resection with curative intent is still the preferred option, but the utilization of new imaging modalities may be able to improve surgical outcomes through improved detection of microscopic disease and delineation of tumor margins to assure complete resection. FDA- approved surgical cameras with NIR detection already enhance visualization of the tumor beyond conventional surgical approaches. Coupled with new developments in systemic tumor detection, visual contrast agents, and combined theranostic materials, the cure rates in specific patient subsets will likely be improved.

Current treatment modalities in PDAC are disappointing. To date, there is no targeted therapy that shows superior outcomes compared to current systemic chemotherapy even though these regimens can cause unacceptable toxicity. Developing successful targeted therapies for PDAC is complicated by tumor heterogeneity, biophysical limitations in the tumor microenvironment, and redundancy of pro-oncogenic pathways. Furthermore, while cancer immunotherapy has been a boon to many cancer patients, pancreatic cancer stands out as one of the few human cancers that does not respond to the current immunotherapy protocols. The failure in implementing therapies used to treat other cancers underscores these unique challenges in PDAC. To address these issues, advances in identifying novel targets and improving the implementation and delivery of therapeutics specifically in PDAC are being employed. These approaches address specific patterns and pathways unique to PDAC with the goal of preventing cancer cells from escaping therapy and host immunosurveillance, as well as avoiding the development of chemoresistance. Through specific targeting of PDAC cancer cells and known biochemical processes, advanced disease can be treated and its progression stopped or significantly delayed.

Several discussed agents augment conventional chemotherapeutics and inhibit pathways that confer resistance. Novel methods of drug delivery are affording both safer and targeted delivery of conventional chemotherapeutics, with the goals of improving their therapeutic window, providing specific delivery to the tumor, or avoiding the systemic toxicity

responsible for limiting doses in clinic. In concert with other treatment modalities, improved and multimodal targeting will prevent drug evasion and tumor progression characteristic of late-stage

PDAC. Continuing advancements in tumor-omics, as well as development of novel delivery and imaging systems, as described herein, must translate to better patient outcomes in the coming decade. PDAC outcomes are dismal and this is particularly evident in light of the success in significantly reducing cancer related deaths in breast, prostate, colon, and lung. Failures in

PDAC therapy and disparities in outcomes relative to other cancers outline a unique clinical challenge, one which will require a multidisciplinary approach and rely on innovative, evidence- based treatments.

Acknowledgements This work was supported in part by National Institutes of Health (F30 CA220843, C.G.; R01 CA227433 & R01 CA232056 M.W.G., Y.L.C.; CA133085, K.L.), Veterans Administration (CX001364, K.L.), and Boston University.

Keywords: pancreatic cancer, chemotherapy, fluorescence guided surgery, antibody therapies, nucleic acid therapies, nanoparticles, drug delivery

References 1. Surveillance, Epidemiology, and End Results (SEER) Program (www.seer.cancer.gov) Research Data (1973-2015), National Cancer Institute, DCCPS, Surveillance Research Program, released April 2019, based on the November 2018 submission. 2. Wu W, He X, Yang L, Wang Q, Bian X, Ye J, et al. Rising trends in pancreatic cancer incidence and mortality in 2000-2014. Clinical Epidemiology. 2018;10:789–97. 3. Rahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM, Matrisian LM. Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Research. 2014;74:2913– 21 4. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA: A Cancer Journal for Clinicians. 2011;61:212–236. 5. Ryan DP, Hong TS, Bardeesy,N. Pancreatic Adenocarcinoma. New England Journal of Medicine. 2014;371:1039–1049. 6. Amundadottir LT. Pancreatic Cancer Genetics. International Journal of Biological Sciences. 2016;12:314-25.

7. Han H, Von Hoff DD. SnapShot: Pancreatic Cancer. Cancer Cell. 2013;23:424–424. 8. Hu C, Hart SN, Polley EC, Gnanaolivu R, Shimelis H, Lee KY, et al. Association Between Inherited Germline Mutations in Cancer Predisposition Genes and Risk of Pancreatic Cancer. The Journal of the American Medical Association. 2018;319(23):2401-2409 9. Tempero MA. NCCN Guidelines Updates: Pancreatic Cancer. Journal of the National Comprehensive Cancer Network. 2019;17:603-605. 10. Modolell I, Guarner L, Malagelada JR. Vagaries of clinical presentation of pancreatic and biliary tract cancer. Annals of Oncology. 1999;10 Suppl 4:82–4. 11. Artinyan A, Soriano PA, Prendergast C, Low T, Ellenhorn JD, Kim J. The anatomic location of pancreatic cancer is a prognostic factor for survival. HPB (Oxford). 2008;10:371–6. 12. Deshwar AB, Sugar E, Torto D, De Jesus-Acosta A, Weiss MJ, Wolfgang CL. Diagnostic intervals and pancreatic ductal adenocarcinoma (PDAC) resectability: a single-center retrospective analysis. Annals of Pancreatic Cancer. 2018;1:13. 13. Holly EA., Chaliha I, Bracci PM, Gautam M. Signs and symptoms of pancreatic cancer: a population-based case-control study in the San Francisco Bay area. Clinical Gastroenterology and Hepatology. 2004;2:510-517. 14. Lee KJ, Yi SW, Chung MJ, Park SW, Song SY, Chung JB. Serum CA 19-9 and CEA levels as a prognostic factor in pancreatic adenocarcinoma. Yonsei Medical Journal. 2013;54:643–9. 15. Strobel O, Neoptolemos J, Jäger D, Büchler MW. Optimizing the outcomes of pancreatic cancer surgery. Nature Reviews Clinical Oncology. 2019;16:11-26. 16. Oberstein PE, Olive KP. Pancreatic cancer: why is it so hard to treat? Therapeutic Advances in Gastroenterology. 2013;6:321-37. 17. Ricci C, Casadei R, Taffurelli G, Pacilio CA, Ricciardiello M, Minni F. Minimally Invasive Pancreaticoduodenectomy: What is the Best "Choice"? A Systematic Review and Network Meta-analysis of Non-randomized Comparative Studies. World Journal of Surgery. 2018;42(3):788-805 18. Yan JF, Pan Y, Chen K, Zhu HP, Chen QL. Minimally invasive pancreatoduodenectomy is associated with lower morbidity compared to open pancreatoduodenectomy: An updated meta-analysis of randomized controlled trials and high-quality nonrandomized studies. Medicine (Baltimore). 2019;98(32):e16730. 19. van Hilst J, de Rooij T, Bosscha K, Brinkman DJ, van Dieren S, Dijkgraaf MG, et al. Laparoscopic versus open pancreatoduodenectomy for pancreatic or periampullary tumours (LEOPARD-2): a multicentre, patient-blinded, randomised controlled phase 2/3 trial. The Lancet: Gastroenterology & Hepatology. 2019;4(3):199-207. 20. Lai EC, Yang GP, Tang CN. Robot-assisted laparoscopic pancreaticoduodenectomy versus open pancreaticoduodenectomy--a comparative study. Internal Journal of Surgery. 2012;10(9):475-9. 21. Buchs NC, Addeo P, Bianco FM, Ayloo S, Benedetti E, Giulianotti PC. Robotic versus open pancreaticoduodenectomy: a comparative study at a single institution. World Journal of Surgery. 2011;35(12):2739-46 22. Peng L, Lin S, Li Y, Xiao W. Systematic review and meta-analysis of robotic versus open pancreaticoduodenectomy. Surgical Endoscopy. 2017;31(8):3085-97.

23. Kamarajah SK, Bundred JR, Marc OS, Jiao LR, Hilal MA, Manas DM, et al. A systematic review and network meta-analysis of different surgical approaches for pancreaticoduodenectomy. HPB. 2019; S1365-182X(19):30728-2. 24. Murphy JE, Wo JY, Ryan DP, Clark JW, Jiang W, Yeap BY, et al. Total Neoadjuvant Therapy With FOLFIRINOX in Combination With Losartan Followed by Chemoradiotherapy for Locally Advanced Pancreatic Cancer: A Phase 2 Clinical Trial. The Journal of the American Medical Association- Oncology. 2019;5(7):1020-7. 25. Tran NH, Sahai V, Griffith KA, Nathan H, Kaza R, Cuneo KC, et al. Phase 2 Trial of Neoadjuvant FOLFIRINOX and Intensity Modulated Radiation Therapy Concurrent With Fixed-Dose Rate-Gemcitabine in Patients With Borderline Resectable Pancreatic Cancer. International Journal of Radiation Oncology, Biology, Physics. 2020;106(1):124-33. 26. Tsujimoto A, Sudo 2, Nakamura K, Kita E, Hara R, Takayama W, et al. Gemcitabine plus nab-paclitaxel for locally advanced or borderline resectable pancreatic cancer. Scientific Reports. 2019;9(1):16187. 27. Von Hoff DD, Ervin T, Arena FP, Chiorean EG, Infante J, Moore M, Seay T, et al. Increased Survival in Pancreatic Cancer with nab-Paclitaxel plus Gemcitabine. New England Journal of Medicine 2013;369:1691–703.. 28. Lee HS, Chung MJ, Park JY, Bang S, Park SW, Kim HG, et al. A randomized, multicenter, phase III study of gemcitabine combined with capecitabine versus gemcitabine alone as first-line chemotherapy for advanced pancreatic cancer in South Korea. Medicine (Baltimore). 2018;96:e5702. 29. Makielski RJ, Lubner SJ, Mulkerin DL, Traynor AM, Groteluschen D, Eickhoff J, et al. A phase II study of sorafenib, oxaliplatin, and 2 days of high-dose capecitabine in advanced pancreas cancer. Cancer Chemotherapy and Pharmacology. 2015;76:317-23. 30. Conroy T, Desseigne F, Ychou M, Bouché O, Guimbaud R, Bécouarn Y, et al. FOLFIRINOX versus Gemcitabine for Metastatic Pancreatic Cancer. New England Journal of Medicine. 2011;364:1817-1825. 31. Chung MJ, Kang H, Kim HG, Hyun JJ, Lee JK, Lee KH, et al. Multicenter phase II trial of modified FOLFIRINOX in gemcitabine-refractory pancreatic cancer. World Journal of Gastrointestinal Oncology. 2018;10:505–15. 32. Philip PA, Benedetti J, Corless CL, Wong R, O'Reilly EM, Flynn PJ, et al. Phase III study comparing gemcitabine plus cetuximab versus gemcitabine in patients with advanced pancreatic adenocarcinoma: Southwest Oncology Group-directed intergroup trial S0205. Journal of Clinical Oncology. 2010;28:3605–10. 33. Wang JP, Wu CY, Yeh YC, Shyr YM, Wu YY, Kuo CY. Erlotinib is effective in pancreatic cancer with epidermal growth factor receptor mutations: a randomized, open-label, prospective trial. Oncotarget. 2015;6:18162–73. 34. Moore MJ, Goldstein D, Hamm J, Figer A, Hecht JR, Gallinger S, et al. Erlotinib Plus Gemcitabine Compared With Gemcitabine Alone in Patients With Advanced Pancreatic Cancer: A Phase III Trial of the National Cancer Institute of Canada Clinical Trials Group. Journal of Clinical Oncology. 2007;25:1960–66 35. Schultheis B, Reuter D, Ebert MP, Siveke J, Kerkhoff A, Berdel WE, et al. Gemcitabine combined with the monoclonal antibody nimotuzumab is an active first- line regimen in KRAS wildtype patients with locally advanced or metastatic

pancreatic cancer: a multicenter, randomized phase IIb study. Annals of Oncology. 2017;28:2429–35. 36. Assenat E, Azria D, Mollevi C, Guimbaud R, Tubiana-Mathieu N, Smith D, et al. Dual targeting of HER1/EGFR and HER2 with cetuximab and trastuzumab in patients with metastatic pancreatic cancer after gemcitabine failure: results of the “THERAPY” phase 1-2 trial. Oncotarget. 2015;5:12796–808.. 37. Ko AH, Bekaii-Saab T, Van Ziffle J, Mirzoeva OM, Joseph NM, Talasaz A, et al. A Multicenter, Open-Label Phase II Clinical Trial of Combined MEK plus EGFR Inhibition for Chemotherapy-Refractory Advanced Pancreatic Adenocarcinoma. Clinical Cancer Research. 2016;22: 61–8. 38. Philip PA, Goldman B, Ramanathan RK, Lenz HJ, Lowy AM, Whitehead RP, et al. Dual blockade of epidermal growth factor receptor and insulin-like growth factor receptor-1 signaling in metastatic pancreatic cancer: phase Ib and randomized phase II trial of gemcitabine, erlotinib, and cixutumumab versus gemcitabine plus erlotinib (SWOG S0727). Cancer. 2014; 120(19): 2980-5. 39. Ko A, Murrary J, Horgan KE, Dauer J, Curley M, Baum J et al.. A multicenter phase II study of istiratumab (MM-141) plus nab-paclitaxel (A) and gemcitabine (G) in metastatic pancreatic cancer (MPC). Journal of Clinical Oncology. 2016;34:TPS481- TPS481. 40. Bergmann L, Maute L, Heil G, Rüssel J, Weidmann E, Köberle D. A prospective randomised phase-II trial with gemcitabine versus gemcitabine plus sunitinib in advanced pancreatic cancer. European Journal of Cancer. 2015;51:27-36. 41. Gonçalves A, Gilabert M, François E, Dahan L, Perrier H, Lamy R. BAYPAN study: a double-blind phase III randomized trial comparing gemcitabine plus sorafenib and gemcitabine plus placebo in patients with advanced pancreatic cancer. Annals of Oncology. 2012;23:2799–2805. 42. Van Cutsem E, van de Velde H, Karasek P, Oettle H, Vervenne WL, Szawlowski A. Phase III Trial of Gemcitabine Plus Tipifarnib Compared With Gemcitabine Plus Placebo in Advanced Pancreatic Cancer. Journal of Clinical Oncology. 2004;22:1430–38. 43. Furuse J, Kurata T, Okano N, Fujisaka Y, Naruge D, Shimizu T. An early clinical trial of Salirasib, an oral RAS inhibitor, in Japanese patients with relapsed/refractory solid tumors. Cancer Chemotherapy and Pharmacology. 2018;82:511–19. 44. Bodoky G, Timcheva C, Spigel DR, La Stella PJ, Ciuleanu TE, Pover G, et al. A phase II open-label randomized study to assess the efficacy and safety of selumetinib (AZD6244 [ARRY-142886]) versus capecitabine in patients with advanced or metastatic pancreatic cancer who have failed first-line gemcitabine therapy. Investigational New Drugs. 2012:30:1216–23. 45. Infante JR, Somer BG, Park JO, Li CP, Scheulen ME, Kasubhai SM, et al. A randomised, double-blind, placebo-controlled trial of trametinib, an oral MEK inhibitor, in combination with gemcitabine for patients with untreated metastatic adenocarcinoma of the pancreas. European Journal of Cancer. 2014;50:2072-81. 46. Chung V, McDonough S, Philip PA, Cardin D, Wang-Gillam A, Hui L, et al Effect of Selumetinib and MK-2206 vs Oxaliplatin and Fluorouracil in Patients With Metastatic Pancreatic Cancer After Prior Therapy. Journal of the American Medical Association- Oncology. 2017;3:516.

47. Melisi D, Garcia-Carbonero R, Macarulla T, Pezet D, Deplanque G, Fuchs M, et al. Galunisertib plus gemcitabine vs. gemcitabine for first-line treatment of patients with unresectable pancreatic cancer. British Journal of Cancer. 2018;119:1208–1214. 48. Golan T, Hammel P, Reni M, Van Cutsem E, Macarulla T, Hall MJ, et al. Maintenance Olaparib for Germline BRCA-Mutated Metastatic Pancreatic Cancer. New England Journal of Medicine. 2019;281(4):317-27. 49. Drilon A, Laetsch TW, Kummar S, DuBois SG, Lassen UN, Demetri GD, et al. Efficacy of Larotrectinib in TRK Fusion-Positive Cancers in Adults and Children. New England Journal of Medicine. 2018;378(8):731-39. 50. O'Reilly EM, Hechtman JF. Tumour response to TRK inhibition in a patient with pancreatic adenocarcinoma harbouring an NTRK gene fusion. Annals of Oncology. 2019;30(Supplement_9):36-40. 51. Zeitouni D, Pylayeva-Gupta Y, Der CJ, Bryant KL. KRAS Mutant Pancreatic Cancer: No Lone Path to an Effective Treatment. Cancers (Basel). 2016;8(4):E45. 52. Lito P, Solomon M, Li LS, Hansen R, Rosen N. Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism. Science. 2016;351(6273):604-8 53. Ostrem JM, Peters U, Sos ML, Wells JA, Shokat KM. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature. 2013;503(7477):548-51 54. Patricelli MP, Janes MR, Li LS, Hansen R, Peters U, Kessler LV, et al. Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State. Cancer Discovery. 2016;6(3):316-29. 55. Gentile LF, Plitas G, Zabor EC, Stempel M, Morrow M, Barrio AV. Tumor Biology Predicts Pathologic Complete Response to Neoadjuvant Chemotherapy in Patients Presenting with Locally Advanced Breast Cancer. Annals of Surgical Oncology. 2017;24(13):3896-3902 56. Shima F, Yoshikawa Y, Ye M, Araki M, Matsumoto S, Liao J, et al. In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras- effector interaction. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(20):8182-7. Crystal Structures of Wild-Type and F448A Mutant Citrobacter freundii Tyrosine Phenol-Lyase Complexed with a Substrate and Inhibitors: Implications for the Reaction Mechanism. Biochemistry. 2018;57(43):6166-79 57. Lu S, Ni D, Wang C, He X, Lin H, Wang Z, et al. Deactivation Pathway of Ras GTPase Underlies Conformational Substates as Target for Drug Design. ACS Catalysis. 2019;9:7188-96. 58. Brahmer, JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and Activity of Anti–PD-L1 Antibody in Patients with Advanced Cancer. New England Journal of Medicine. 2012;366, 2455–65. 59. O’Reilly EM, Oh DY, Dhani N, Renouf DJ, Lee MA, Sun W, et al. Durvalumab With or Without Tremelimumab for Patients With Metastatic Pancreatic Ductal Adenocarcinoma. JAMA Oncology. 2019. 60. Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science.2017;357:409-13.

61. Hu C, Hart SN, Polley EC, Gnanaolivu R, Shimelis H, Lee KY, et al. Association Between Inherited Germline Mutations in Cancer Predisposition Genes and Risk of Pancreatic Cancer. JAMA. 2019;319:2401-09.. 62. Royal RE, Levy C, Turner K, Mathur A, Hughes M, Kammula US, et al. Phase 2 Trial of Single Agent Ipilimumab (Anti-CTLA-4) for Locally Advanced or Metastatic Pancreatic Adenocarcinoma. Journal of Immunotherapy. 2010;33:828–33. 63. Lutz ER, Wu AA, Bigelow E, Sharma R, Mo G, Soares K, et al. Immunotherapy Converts Nonimmunogenic Pancreatic Tumors into Immunogenic Foci of Immune Regulation. Cancer Immunology Research. 2014;2:616–31. 64. Waddell N, Pajic M, Patch AM, Chang DK, Kassahn KS, Bailey P, et al. Whole genomes redefine the mutational landscape of pancreatic cancer. Nature. 2015;518:495–501. 65. Wang X, Wang G, Wang Z, Liu B, Han N, Li J, et al. PD-1-expressing B cells suppress CD4+ and CD8+ T cells via PD-1/PD-L1-dependent pathway. Molecular Immunology. 2019;109:20-6. 66. Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK, et al. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. New England Journal of Medicine. 2015;372:2509-20. 67. Fukunaga A, Miyamoto M, Cho Y, Murakami S, Kawarada Y, Oshikiri T et al. CD8+ tumor-infiltrating lymphocytes together with CD4+ tumor-infiltrating lymphocytes and dendritic cells improve the prognosis of patients with pancreatic adenocarcinoma. Pancreas. 2004;28(1):e26-31. 68. Schmitz-Winnenthal, FH, Volk C, Z'graggen K, Galindo L, Nummer D, Ziouta Y, et al. High Frequencies of Functional Tumor-Reactive T Cells in Bone Marrow and Blood of Pancreatic Cancer Patients. Cancer Research. 2005;65:10079–87. 69. Lu C, Talukder A, Savage NM, Singh N, Liu K. STAT-mediated chronic inflammation impairs cytotoxic T lymphocyte activation to decrease anti-PD-1 immunotherapy efficacy in pancreatic cancer. OncoImmunology. 2017;6:e1291106. 70. Nomi T, Sho M, Akahori T, Hamada K, Kubo A, Kanehiro H, et al. Clinical Significance and Therapeutic Potential of the Programmed Death-1 Ligand/Programmed Death-1 Pathway in Human Pancreatic Cancer. Clinical Cancer Research. 2007;13:2151–57. 71. Birnbaum DJ, Finetti P, Lopresti A, Gilabert M, Poizat F, Turrini O, et al. Prognostic value of PDL1 expression in pancreatic cancer. Oncotarget. 2016;7:71198–210. 72. Lu C, Redd PS, Lee JR, Savage N, Liu K., The expression profiles and regulation of PD-L1 in tumor-induced myeloid-derived suppressor cells OncoImmunology. 2016;5:e1247135. 73. Lu C, Paschall AV, Shi H, Savage N, Waller JL, Sabbatini ME. The MLL1-H3K4me3 Axis-Mediated PD-L1 Expression and Pancreatic Cancer Immune Evasion. JNCI. 2017;109(6). 74. Mace TA, Shakya R, Pitarresi JR, Swanson B, McQuinn CW, Loftus S, et al. IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer. Gut. 2018;67:320–32. 75. Donahue TR, Reber HA. Surgical Management of Pancreatic Cancer— Pancreaticoduodenectomy. Seminars in Oncology. 2015;42:98-109. 76. Buchs NC, Hagen ME, Pugin F, Volonte F, Bucher P, Schiffer E, et al. Intra-operative fluorescent cholangiography using indocyanin green during robotic single site

cholecystectomy. International Journal of Medical Robotics and Computer Assisted Surgery. 2012;8:436–40. 77. Tummers QR, Boonstra MC, Frangioni JV, van de Velde CJ, Vahrmeijer AL, Bonsing BA. Intraoperative near-infrared fluorescence imaging of a paraganglioma using methylene blue: A case report. International Journal of Surgical Case Reports. 2015;6:150–3 (2015). 78. Sherwinter DA. Identification of Anomolous Biliary Anatomy Using Near-Infrared Cholangiography. Journal of Gastrointestinal Surgery. 2012;16:1814–5. 79. Tringale KR, Pang J, Nguyen QT. Image-guided surgery in cancer: A strategy to reduce incidence of positive surgical margins. Wiley Interdisciplinary Reviews: Systems Biology and Medicine. 2018:10:e1412. 80. Colby AH, Berry SM, Moran AM, Pasion KA, Liu R, Colson YL, et al. Highly Specific and Sensitive Fluorescent Nanoprobes for Image-Guided Resection of Sub- Millimeter Peritoneal Tumors. ACS Nano. 2017;11:1466–77. 81. Qi B, Crawford AJ, Wojtynek NE, Holmes MB, Souchek JJ, Almeida-Porada G, et al. Indocyanine green loaded hyaluronan-derived nanoparticles for fluorescence- enhanced surgical imaging of pancreatic cancer. Nanomedicine. 2018;14:769-80. 82. Barton S, Li B, Siuta M, Vaibhav J, Song J, Holt CM, et al. Specific Molecular Recognition as a Strategy to Delineate Tumor Margin Using Topically Applied Fluorescence Embedded Nanoparticles. Precision Medicine. Precision Nanomedicine. 2018;1(3):194-207. 83. Vuijk FA, Hilling DE, Mieog JSD, Vahrmeijer AL. Fluorescent-guided surgery for sentinel lymph node detection in gastric cancer and carcinoembryonic antigen targeted fluorescent-guided surgery in colorectal and pancreatic cancer. Journal of Surgical Oncology. 2018;118(2):315-323. 84. Wente MN, Jain A, Kono E, Berberat PO, Giese T, Reber HA, et al. Prostate stem cell antigen is a putative target for immunotherapy in pancreatic cancer. 2005;31(2):119-25. 85. Zettlitz KA, Tsai WK, Knowles SM, Kobayashi N, Donahue TR, Reiter RE, et al. Dual-Modality Immuno-PET and Near-Infrared Fluorescence Imaging of Pancreatic Cancer Using an Anti-Prostate Stem Cell Antigen Cys-Diabody. Journal of Nuclear Medicine. 2018;59(9):1398-1405. 86. Obaid G, Spring BQ, Bano S, Hasan T. Activatable clinical fluorophore-quencher antibody pairs as dual molecular probes for the enhanced specificity of image-guided surgery. Journal of Biomedical Optics. 2017;22(12)1-6. 87. Liu QQ, Chen K, Ye Q, Jiang XH, Sun YW. Oridonin inhibits pancreatic cancer cell migration and epithelial-mesenchymal transition by suppressing Wnt/β-catenin signaling pathway. Cancer Cell International. 2016;16:57. 88. Qiu W, Chen R, Chen X, Zhang H, Song L, Cui W, et al. Oridonin-loaded and GPC1- targeted gold nanoparticles for multimodal imaging and therapy in pancreatic cancer. International Journal of Nanomedicine. 2018;13:6809–27. 89. Qiu W, Zhang H, Chen X, Song L, Cui W, Ren S, et al. A GPC1-targeted and gemcitabine-loaded biocompatible nanoplatform for pancreatic cancer multimodal imaging and therapy. Nanomedicine. 2019;14:17 90. Ghaneh P, Hanson R, Titman A, Lancaster G, Plumpton C, Lloyd-Williams H, et al. PET-PANC: multicentre prospective diagnostic accuracy and health economic analysis study of the impact of combined modality 18fluorine-2-fluoro-2-deoxy-d-

glucose positron emission tomography with computed tomography scanning in the diagnosis and management of pancreatic cancer. Health Technology Assessment. 2018;22(7):1-114. 91. Herrera VL, Steffen M, Moran AM, Tan GA, Pasion KA, Rivera K, et al. Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein. BMC Molecular Biology. 2016;17:15. 92. Herrera VL, Decano JL, Tan GA, Moran AM, Pasion KA, Matsubara Y, et al.. DEspR Roles in Tumor Vasculo-Angiogenesis, Invasiveness, CSC-Survival and Anoikis Resistance: A ‘Common Receptor Coordinator’ Paradigm. PLoS One. 2014;9:e85821. 93. Gugger M, White R, Song S, Waser B, Cescato R, Rivière P, et al. GPR87 is an overexpressed G-protein coupled receptor in squamous cell carcinoma of the lung. Disease Markers. 2008;24:41–50. 94. Nii K, Tokunaga Y, Liu D, Zhang X, Nakano J, Ishikawa S, et al. Overexpression of G protein-coupled receptor 87 correlates with poorer tumor differentiation and higher tumor proliferation in non-small-cell lung cancer. Molecular and Clinical Oncology. 2014;2:539–44. 95. Zhang Y, Qian Y, Lu W, Chen X. The G protein-coupled receptor 87 is necessary for p53-dependent cell survival in response to genotoxic stress. Cancer Research. 2009;69:6049–56. 96. Zhang X, Liu D, Hayashida Y, Okazoe H, Hashimoto T, Ueda N, et al. G Protein- Coupled Receptor 87 (GPR87) Promotes Cell Proliferation in Human Bladder Cancer Cells. International Journal of Molecular Sciences. 2015;16:24319–31. 97. Yan M, Li H, Zhu M, Zhao F, Zhang L, Chen T, et al. G protein-coupled receptor 87 (GPR87) promotes the growth and metastasis of CD133(+) cancer stem-like cells in hepatocellular carcinoma. PLoS ONE. 2013;8:e61056. 98. Wang L, Zhou W, Zhong Y, Huo Y, Fan P, Zhan S, et al. Overexpression of G protein-coupled receptor GPR87 promotes pancreatic cancer aggressiveness and activates NF-κB signaling pathway. Molecular Cancer. 2017;16:61. 99. Riethdorf S, Reimers N, Assmann V, Kornfeld JW, Terracciano L, Sauter G, et al. High incidence of EMMPRIN expression in human tumors. International Journal of Cancer. 2006;119:1800-10. 100. Zhang Z, Zhang Y, Chen R, Luo D, Chen ZN. Clinical impact and prognostic value of CD147 and MMP-7 expression in patients with pancreatic ductal adenocarcinoma. International Journal of Clinical and Experimental Pathology. 2016;9(9):9175-83. 101. Li L, Tang W, Wu X, Karnak D, Meng X, Thompson R, et al. HAb18G/CD147 promotes pSTAT3-mediated pancreatic cancer development via CD44s. Clinical Cancer Research. 2013;19(24):6703-15 102. Grass GD, Toole BP. How, with whom and when: an overview of CD147- mediated regulatory networks influencing matrix metalloproteinase activity. Bioscience Reports. 2015;36:e00283. 103. Ke X, Li L, Dong HL, Chen ZN. Acquisition of anoikis resistance through CD147 upregulation: A new mechanism underlying metastasis of hepatocellular carcinoma cells. Oncology Letters. 2012;3(6):1249-54

104. Wu J, Ru NY, Zhang Y, Li Y, Wei D, Ren Z, et al. HAb18G/CD147 promotes epithelial-mesenchymal transition through TGF-β signaling and is transcriptionally regulated by Slug. 2011;30:4410-27. 105. Wu J, Li Y, Dang YZ, Gao HX, Jiang JL, Chen ZN. HAb18G/CD147 promotes radioresistance in hepatocellular carcinoma cells: a potential role for integrin β1 signaling. Molecular Cancer Therapeutics. 2015;14:553-63. 106. Landras A, Reger de Moura C, Jouenne F, Lebbe C, Menashi S, Mourah S. CD147 Is a Promising Target of Tumor Progression and a Prognostic Biomarker. Cancers (Basel). 2019;11:E1803. 107. Fan XY, He D, Sheng CB, Wang B, Wang LJ, Wu XQ, et al. Therapeutic anti- CD147 antibody sensitizes cells to chemoradiotherapy via targeting pancreatic cancer stem cells. American Journal of Translational Research. 2019;11:3543-54. 108. Sugyo A, Tsuji AB, Sudo H, Koizumi M, Ukai Y, Kurosawa G, et al. Efficacy Evaluation of Combination Treatment Using Gemcitabine and Radioimmunotherapy with 90Y-Labeled Fully Human Anti-CD147 Monoclonal Antibody 059-053 in a BxPC- 3 Xenograft Mouse Model of Refractory Pancreatic Cancer. International Journal of Molecular Sciences. 2018;19:E2979. 109. Yeung TL, Leung CS, Yip KP, Sheng J, Vien L, Bover LC, et al. Anticancer Immunotherapy by MFAP5 Blockade Inhibits Fibrosis and Enhances Chemosensitivity in Ovarian and Pancreatic Cancer. Clinical Cancer Research. 2019;35:6417-28. 110. Michl P, Buchholz M, Rolke M, Kunsch S, Löhr M, McClane B, et al. Claudin-4: a new target for pancreatic cancer treatment using Clostridium perfringens enterotoxin. Gastroenterology. 2001;121:678-84. 111. Sato N, Fukushima N, Maitra A, Iacobuzio-Donahue CA, van Heek NT, Cameron JL, et al. Gene expression profiling identifies genes associated with invasive intraductal papillary mucinous neoplasms of the pancreas. The American Journal of Pathology. 2004;164:903-14. 112. Hashimoto Y, Kawahigashi Y, Hata T, Li X, Watari A, Tada M, et al. Efficacy and safety evaluation of claudin-4-targeted antitumor therapy using a human and mouse cross-reactive monoclonal antibody. Pharmacology Research and Perspectives. 2016;4:e00266. 113. Sasaki T, Fujiwara-Tani R, Kishi S, Mori S, Luo Y, Ohmori H, et al. Targeting claudin-4 enhances chemosensitivity of pancreatic ductal carcinomas. Cancer Medicine. 2019;8:6700-08. 114. Rosati A, Basile A, D’Auria R, d’Avenia M, De Marco M, Falco A, et al. BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages. Nature Communications. 2015;6:8695 115. Li C, An MX, Jiang JY, Yao HB, Li S, Yan J, et al. BAG3 Suppresses Loading of Ago2 to IL6 mRNA in Pancreatic Ductal Adenocarcinoma. Frontiers Oncology. 2019;9:225. 116. Basile A, De Marco M, Festa M, Falco A, Iorio V, Guerriero L, et al. Development of an anti-BAG3 humanized antibody for treatment of pancreatic cancer. Molecular Oncology. 2019;13:1388-99. 117. Bryant K, Channing D. Blocking autophagy to starve pancreatic cancer. Nature Reviews Molecular Cell Biology. 2019;20:265.

118. Kinsey C, Camolotto S, Boespflug A, Guillen K, Foth M, Truong A, et al. Protective autophagy elicited by Raf-MEK-ERK inhibition suggests a treatment strategy for Ras-drive cancers. Nature Medicine. 2019;25:620-7. 119. Chiramel J, Backen A, Pihlak R, Lamarca A, Frizziero M, Tariq N, et al. Targeting the Epidermal Growth Factor Receptor in Addition to Chemotherapy in Patients with Advanced Pancreatic Cancer: A Systematic Review and Meta-Analysis. International Journal of Molecular Sciences. 2017;18(5):909 120. Venkata B, Lakkakula KS, Farran B, Lakkakula S, Peela S, Yarla NS, et al. Small molecule tyrosine kinase inhibitors and pancreatic cancer-Trials and troubles. Seminars in Cancer Biology. 2019;56:149-167. 121. Akella NM, Ciraku L, Reginato MJ. Fueling the fire: emerging role of the hexosamine biosynthetic pathway in cancer. BMC Biology. 2019;17:52. 122. Sharma NS, Gupta VK, Garrido VT, Hadad R, Durden BC, Kesh K, et al. Targeting tumor-intrinsic hexosamine biosynthesis sensitizes pancreatic cancer to anti-PD1 therapy. The Journal of Clinical Investigation. 2020;130(1):451-65 123. Cervantes-Madrid D, Romero Y, Dueñas-González A. Reviving Lonidamine and 6-Diazo-5-oxo-L-norleucine to Be Used in Combination for Metabolic Cancer Therapy. Biomedical Research International. 2015;2015:690492. 124. Feld FM, Nagel PD, Weissinger SE, Welke C, Stenzinger A, Möller P. GOT1/AST1 expression status as a prognostic biomarker in pancreatic ductal adenocarcinoma. Oncotarget. 2015;6(6):4516-26. 125. Son J, Lyssiotis CA, Ying H, Wang X, Hua S, Ligorio M, et al. Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway. Nature. 2013:496:101-5. 126. Abrego J, Gunda V, Vernucci E, Shukla SK, King RJ, Dasgupta A, et al. GOT1- mediated anaplerotic glutamine metabolism regulates chronic acidosis stress in pancreatic cancer cells. Cancer Letters. 2017;400:37-46. 127. Zhou X, Curbo S, Li F, Krishnan S, Karlsson A. Inhibition of glutamate oxaloacetate transaminase 1 in cancer cell lines results in altered metabolism with increased dependency of glucose. BMC Cancer. 2018;18:559. 128. Yoshida T, Yamasaki S, Kaneko O, Taoka N, Tomimoto Y, Namatame I, et al. A covalent small molecule inhibitor of glutamate-oxaloacetate transaminase 1 impairs pancreatic cancer growth. Biochemical and Biophysical Research Communications. 2020;522:633-38. 129. Sun W, Luan S, Qi C, Tong Q, Yan S, Li H, et al. Aspulvinone O, a natural inhibitor of GOT1 suppresses pancreatic ductal adenocarcinoma cells growth by interfering glutamine metabolism. Cell Communications and Signaling. 2019;17:111. 130. Sullivan WJ, Christofk HR. The metabolic milieu of metastases. Cell. 2015;160: 363–4. 131. Kovi RC, Paliwal S, Pande S, Grossman SR. An ARF/CtBP2 complex regulates BH3-only gene expression and p53-independent apoptosis. Cell Death and Differentiation. 2010;17(3):513-21. 132. Dai F, Xuan Y, Jin JJ, Yu S, Long ZW, Cai H, et al. CtBP2 overexpression promotes tumor cell proliferation and invasion in gastric cancer and is associated with poor prognosis. Oncotarget. 2017;8(17):28736-49. 133. Kim TW, Kang BH, Jang H, Kwak S, Shin J, Kim H, et al. Ctbp2 Modulates NuRD-Mediated Deacetylation of H3K27 and Facilitates PRC2-Mediated H3K27me3

in Active Embryonic Stem Cell Genes During Exit from Pluripotency. Stem Cells. 2015;33(8):2442-55. 134. Sumner ET, Chawla AT, Cororaton AD, Koblinski JE, Kovi RC, Love IM, et al. Transforming activity and therapeutic targeting of C-terminal-binding protein 2 in Apc-mutated neoplasia. 2017;36(33):4810-6. 135. Kurth I, Andreu C, Takeda S, Tian H, Gonsalves F, Leites K, et al. RGX-202, a first-in-class small-molecule inhibitor of the creatine transporter SLC6a8, is a robust suppressor of cancer growth and metastatic progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5863. 136. Kovi RC, Paliwal S, Pande S, Grossman SR. An ARF/CtBP2 complex regulates BH3-only gene expression and p53-independent apoptosis. Cell Death Differ 2010; 17:513-21 137. Zhang C, Gao C, Xu Y, Zhang Z. CtBP2 could promote prostate cancer cell proliferation through c-myc signaling. Gene 2014; 546:73-9 138. Patel J, Baranwal S, Love IM, Patel NJ, Grossman SR, Patel BB. Inhibition of C- terminal binding protein attenuates transcription factor 4 signaling to selectively target colon cancer stem cells. Cell Cycle. 2014; 13:3506-18 139. Blevins M, Huang M, Zhao R. The role of CtBP1 in oncogenic processes and its potential as a therapeutic target. Molecular Cancer Therapy. 2018;16(6):981-90. 140. Chawla AT, Chougoni KK, Joshi P2, Cororaton AD, Memari P, Stansfield JC, et al. CtBP-a targetable dependency for tumor-initiating cell activity and metastasis in pancreatic adenocarcinoma. Oncogenesis. 2019;8(10):55. 141. Adamska A, Ferro R, Lattanzio R, Capone E, Domenichini A, Damiani V, et al. ABCC3 is a novel target for the treatment of pancreatic cancer. Advances in Biological Regulation. 2019;73:100634. 142. Adamska A, Falasca M. ATP-binding cassette transporters in progression and clinical outcome of pancreatic cancer: What is the way forward? World Journal of Gastroenterology. 2018;24(29):3222-38. 143. Mohelnikova-Duchonova B, Brynychova V, Oliverius M, Honsova E, Kala Z, Muckova K, Soucek P. Differences in transcript levels of ABC transporters between pancreatic adenocarcinoma and nonneoplastic tissues. Pancreas. 2013;42(4):707- 16. 144. Adamska A, Domenichini A, Capone E, Damiani V, Akkaya BG, Linton KJ, et al. Pharmacological inhibition of ABCC3 slows tumour progression in animal models of pancreatic cancer. Journal of Experimental & Clinical Cancer Research. 2019;38:312. 145. Henke RT, Haddad BR, Kim SE, Rone JD, Mani A, Jessup JM, et al. Overexpression of the nuclear receptor coactivator AIB1 (SRC-3) during progression of pancreatic adenocarcinoma. 2004; 10(18):6134-42 146. Wang Y, Lonard D, Yu Y, Chow DC, Palzkill T, Wang J, et al. Bufalin is a potent small molecule inhibitor of the steroid receptor coactivators SRC-3 and SRC-1. Cancer Research. 2014;75(5):1506-17. 147. Song X, Chen H, Zhang C, Yu Y, Chen Z, Liang H, et al. SRC-3 inhibition blocks tumor growth of pancreatic ductal adenocarcinoma. Cancer Letters. 2019;442:310- 319

148. Ma G, Ren Y, Wang K, He J. SRC-3 has a role in cancer other than as a nuclear receptor coactivator. International Journal of Biological Sciences. 2011;7(5):664-72 149. Irby RB, Yeatman TJ. Role of Src expression and activation in human cancer. Oncogene. 2000;19(49):5636-42. 150. Brigger I, Dubernet C, Couvreur P. Nanoparticles in Cancer Therapy and Diagnosis. Advanced Drug Delivery Review. 2002;54(5):631–51.. 151. Petros RA, DeSimone, JM. Strategies in the Design of Nanoparticles for Therapeutic Applications. Nature Reviews Drug Discovery. 2010;9(8):615–27. 152. Lammers T, Kiessling F, Hennink WE, Storm G. Drug Targeting to Tumors: Principles, Pitfalls and (Pre-) Clinical Progress. Journal of Controlled Release. 2012;161(2):175–87. 153. Ding Y, Li S, Nie G. Nanotechnological Strategies for Therapeutic Targeting of Tumor Vasculature. Nanomedicine. 2013;8(7), 1209–22. 154. Nie S, Xing Y, Kim GJ, Simons JW. Nanotechnology Applications in Cancer. Annual Review of Biomedical Engineering. 2007;9(1):257–88. 155. Wu ST, Fowler AJ, Garmon CB, Fessler AB, Ogle JD, Grover KR, et al. Treatment of pancreatic ductal adenocarcinoma with tumor antigen specific-targeted delivery of paclitaxel loaded PLGA nanoparticles. BMC Cancer. 2018;18:457. 156. Verma RK, Yu W, Shrivastava A, Srivastava RK, Shankar S. Abstract 3728: α- Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in KC and KPC mice. Cancer Research. 2018;78:3728. 157. Herrera VL, Colby AH, Tan GA, Moran AM, O'Brien MJ, Colson YL, et al. Evaluation of expansile nanoparticle tumor localization and efficacy in a cancer stem cell-derived model of pancreatic peritoneal carcinomatosis. Nanomedicine. 2016;11:1001–15. 158. Arya G, Das M, Sahoo, SK. Evaluation of curcumin loaded chitosan/PEG blended PLGA nanoparticles for effective treatment of pancreatic cancer. Biomedicine and Pharmacotherapy. 2018;102:555–66. 159. He X, Chen X, Liu L, Zhang Y, Lu Y, Zhang Y, et al. Sequentially Triggered Nanoparticles with Tumor Penetration and Intelligent Drug Release for Pancreatic Cancer Therapy. Advanced Science. 2018;5:1701070. 160. Jain A, Singh SK, Arya SK, Kundu SC, Kapoor S. Protein Nanoparticles: Promising Platforms for Drug Delivery Applications. ACS Biomaterials Science & Engineering. 2018;4:3939–61. 161. Grinstaff M, Soon-Shiong P, Wong M, Sandford P, Suslick K, Desai N. (1995). Composition useful for in vivo delivery of biologics and methods employing same. US. Patent Number US549421A. 162. Suslick KS, Grinstaff, MW. Protein microencapsulation of nonaqueous liquids. Journal of the American Chemical Society. 1990;112:7807–9 (1990). 163. Zhu Q, Pan X, Sun Y, Wang Z, Liu F, Li A et al. Biological nanoparticles carrying the Hmda-7 gene are effective in inhibiting pancreatic cancer in vitro and in vivo. PLoS One. 2017;12:e0185507. 164. Santos-Rebelo A, Garcia C, Eleutério C, Bastos A, Coelho SC, Coelho MAN, et al. Development of Parvifloron D-loaded Smart Nanoparticles to Target Pancreatic Cancer. Pharmaceutics. 2018;10:216.

165. Ji S, Xu J, Zhang B, Yao W, Xu W, Wu W, et al. RGD-conjugated albumin nanoparticles as a novel delivery vehicle in pancreatic cancer therapy. Cancer Biology & Therapeutics. 2012;13:206–15 166. Ristorcelli E, Beraud E, Verrando P, Villard C, Lafitte D, Sbarra V, et al. Human tumor nanoparticles induce apoptosis of pancreatic cancer cells. FASEB Journal. 2008;22: 3358–69. 167. Nayerossadat N, Maedeh T, Ali PA. Viral and nonviral delivery systems for gene delivery. Advanced Biomedical Research. 2012;1:27. 168. Wang-Gilliam A, Hubner RA, Siveke JT, Von Hoff DD, Belanger B, de Jong FA, et al. NAPOLI-1 phase 3 study of liposomal irinotecan in metastatic pancreatic cancer: Final overall survival analysis and characteristics of long-term survivors. European Journal of Cancer. 2019;108:78-87 169. Touchefeu Y, Harrington KJ, Galmiche JP, Vassaux G. Review article: Gene therapy, recent developments and future prospects in gastrointestinal oncology. Alimentary Pharmacology & Therapeutics. 2010;32:953–68. 170. Liu SX, Xia ZS, Zhong YQ. Gene therapy in pancreatic cancer. World Journal of Gastroenterology. 2014;20:13343–68. 171. Rahman MA, Wang P, Wang D, Nannapaneni S, Hurwitz S, Chen Z, et al. Abstract 3701: Targeted Bcl2 siRNA delivery using DNA nanoparticles in cancer therapy. Cancer Research. 2018;78:3701 172. Strand MS, Krasnick BA, Pan H, Zhang X, Bi Y, Brooks C. Precision delivery of RAS-inhibiting siRNA to pancreatic cancer via peptide-based nanoparticles. Oncotarget. 2019;10(46):4761-75. 173. Spano C, Grisendi G, Golinelli G, Rossignoli F, Prapa M, Bestagno M. Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer. Scientific Reports. 2019;9:1788. 174. Uz M, Kalaga M, Pothuraju R, Ju J, Junker WM, Batra SK. Dual delivery nanoscale device for miR-345 and gemcitabine co-delivery to treat pancreatic cancer. Journal of Controlled Release. 2019;294:237-46. 175. Ding Y, Cao F, Sun H, Wang Y, Liu S, Wu Y. Exosomes derived from human umbilical cord mesenchymal stromal cells deliver exogenous miR-145-5p to inhibit pancreatic ductal adenocarcinoma progression. Cancer Letters. 2019;442:351–61. 176. Kruspe S, Giangrande PH. Aptamer-siRNA chimeras: Discovery, progress, and future prospects. Biomedicines. 2017;5:45. 177. Xu F, Sun Y, Yang S, Zhou T, Jhala N, McDonald J, et al. Cytoplasmic PARP-1 promotes pancreatic cancer tumorigenesis and resistance. International Journal of Cancer. 2019;145(2):474-83. 178. Poolsup S, Kim CY. Therapeutic applications of synthetic nucleic acid aptamers. Current Opinion in Biotechnology. 2017;48:180–6. 179. Arab R, Ramezani AM, Abnous K, and Taghdisi, SM. Application of aptamers in treatment and diagnosis of leukemia. International Journal of Pharmacology. 2017;529:44–54. 180. Zhou G, Wilson G, Hebbard L, Duan W, Liddle C, George J, et al. Aptamers: A promising chemical antibody for cancer therapy. Oncotarget. 2016;7:13446–63. 181. Zhu H, Li J, Zhang XB, Ye M, Tan W. Nucleic acid aptamer-mediated drug delivery for targeted cancer therapy. ChemMedChem. 2015;10:39–45. Morita Y,

Leslie M, Kameyama H, Volk D, Tanaka T. Aptamer Therapeutics in Cancer: Current and Future. Cancers (Basel). 2018;10:E80. 182. Park JY, Cho Y2, Chae JR, Moon SH, Cho WG, Choi YJ, Lee SJ, et al. Gemcitabine-Incorporated G-Quadruplex Aptamer for Targeted Drug Delivery into Pancreas Cancer. Molecular Therapeutics- Nucleic Acid. 2018;12:543–53. 183. Yoon S, Rossi, JJ. Treatment of Pancreatic Cancer by Aptamer Conjugated C/EBPα-saRNA. Advances in Experimental Medicine and Biology. 2017;983:173–88. 184. Kratschmer C, Levy M. Targeted Delivery of Auristatin-Modified Toxins to Pancreatic Cancer Using Aptamers. Molecular Therapy- Nucleic Acids. 2018;10:227–36. 185. Kim YJ, Lee HS, Jung DE, Kim JM, Song SY. The DNA aptamer binds stemness-enriched cancer cells in pancreatic cancer. Journal of Molecular Recognition. 2017;30:e2591.

Table I: Current Chemotherapy Regimens used, Previous targeted therapy trials, and Selected Targets for Antibody Therapy in PDAC Current Chemotherapy Regimens Treatment Trial OS/PFS Study Size (n) P Value Ref Gemcitabine + Phase III MPACT 8.7 vs. 6.6 mo 861 p < 0.001 (27) nab-paclitaxel vs. gemcitabine Gemcitabine + Phase III 10.3 vs. 7.6 214 p < 0.06 (28) capecitabine vs. mo; 6.2 vs. 5.3 p < 0.08 gemcitabine mo Gemcitabine, Phase II GEMOXEL 11.9 vs. 7.1 67 p < 0.001 (29) Oxaliplatin, mo capecitabine vs. 6.8 vs. 7.1 mo gemcitabine FOLFIRINOX vs. Phase III 11.1 vs. 6.8 342 p < 0.001 (30) gemcitabine PRODIGE/ACCORD 11 mo p < 0.001 6.4 vs. 3.3 mo mFOLFIRINOX Phase II 10.2 vs. 37 (31) 6.1 mo Previous Targeted Therapy Trials Target Drug Trial OS PFS Ref EGFR Cetuximab Phase III 6.3 vs. 5.9 mo 3.4 vs 3.0 mo (32-35) Erlotinib Phase III 6.2 vs. 5.9 mo 3.8 vs. 3.6 mo Phase II 7.2 vs. 4.4 mo 3.8 vs. 2.4 mo Nimotuzumab Phase IIb 8.6 vs. 6.0 mo 5.3 vs. 3.6 mo EGFR/HER2 Cetuximab+ Phase I/II 4.6 mo 1.8 mo (36) Trastuzumab EGFR + MEK1/2 Erlotinib + Phase II 7.3 mo 1.9 mo (37) Selumetinib IGFR Cixutumumab Phase II 7.0 vs. 6.7 mo 3.6 vs 3.6 mo (38,39) MM-141 Phase II - - RTK inhibitor Sunitinib Phase II 7.6 vs. 9.1 mo 2.9 vs. 3.3 mo (40,41) Sorafenib Phase II 8.1 mo 6.0 mo Ras Tipifarnib Phase III 6.4 vs. 6.1 mo 3.7 vs 3.6 mo (42,43) Salirasib Phase I 6.2 mo 3.9 mo MEK1/2 Selumetinib Phase II 5.4 vs 5.0 mo 2.1 vs. 2.2 mo (44,45) Trametinib Phase II 8.4 vs. 6.7 mo 4.0 vs. 3.8 mo MEK1/2 + AKT Selumetinib + MK- Phase II 3.9 vs. 6.7 mo 1.9 vs. 2.0 mo (46) 2206 TGF-β Galunisertib Phase II 8.9 vs. 7.1 mo - (47) Selected Targets for Antibody Therapy Targets Function Downstream Signaling Ref DEspR Angiogenesis, anoikis resistance STAT1/3, FAK, ERK1/2 (91, 92) GPR87 Tumor proliferation, angiogenesis, PI3K, AKT (93, 96, 97, 98) anoikis resistance, drug resistance CD147 Anoikis resistance, EMT, FAK/Src/Stat3, PI3K, AKT/ (100, 101, 102, chemotherapy resistance, ECM mTOR 103, 104, 105, regulation 106, 107) MFAP5 ECM regulation, tumor proliferation, FAK, ERK1/2 (109,110) microvessel regulation CLDN4 Cell barrier function, chemoresistance Tight Junction Protein (111, 112,113) BAG-3 ECM regulation and stromal cell IL-6 mediated tumor-stromal (114, 115, 116) regulation signaling

Figure Caption

Figure 1. Pancreatic Cancer. The incidence and mortality continue to increase with pancreatic cancer despite the use of conventional chemotherapeutics and surgery. New procedures (e.g., fluorescence guided surgery) and therapies (e.g., antibodies, nucleic acid, nanoparticles) and are being pursued to improve patient outcomes.

Conventional Nanoparticles Chemotherapy

Fluorescence Nucleic Acid Guided Surgery Therapies

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Pancreatic Adenocarcinoma: Unconventional Approaches for an Unconventional Disease

Christopher Gromisch, Motaz Qadan, Mariana Albuquerque Machado, et al.

Cancer Res Published OnlineFirst March 27, 2020.

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