Pancreatic Adenocarcinoma

Published OnlineFirst March 27, 2020; DOI: 10.1158/0008-5472.CAN-19-2731

CANCER RESEARCH | REVIEW

Pancreatic Adenocarcinoma: Unconventional Approaches for an Unconventional Disease Christopher Gromisch1, Motaz Qadan2, Mariana Albuquerque Machado1, Kebin Liu3, Yolonda Colson4, and Mark W. Grinstaff1

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

Clinical Snapshot of Pancreatic few genetic syndromes and mutations correlate with higher PDAC Adenocarcinoma lifetime risk. Individuals with hereditary pancreatitis, associated with Trypsin-1 (PRSS1) or serine protease inhibitor Kazal-type 1 (SPINK1) Pancreatic cancer is the most lethal common tumor in America. The mutations, poses a lifetime PDAC risk of 50%, while patients with ﬁve-year survival is estimated to be 9.3% among all cases and 2.9% Peutz–Jegher syndrome and familial atypical multiple mole and among patients with metastatic disease, both lowest among all com- melanoma syndrome carry lifetime risks of 30%–40% and 10%– mon tumors (1). With its rising incidence and unabated mortality, 20%, respectively (6). Other syndromes, such as Lynch syndrome pancreatic cancer is the third leading cause of cancer-related death, (associated with MLH1, MSH2, and MSH6 mutations), hereditary with a projection that it will the second by 2030 (Fig. 1; refs. 2, 3). In breast and ovarian cancer syndromes (caused by BRCA1/2 or PALB2 2019, pancreatic ductal adenocarcinoma (PDAC; 95% of patients) mutations), Ataxia-telangiectasia (caused by mutations in the ATM), represented 3.2% of all new cancer cases, with an estimated 56,770 new and Li-Fraumeni Syndrome (caused by germline p53 mutations), cases and 45,750 deaths (7.5% of all cancer-related deaths; ref. 1). contribute to a lesser degree (6, 7). Inherited germline mutations in PDAC is a complex and clinically challenging disease, deﬁned CDKN2A, MLH1, BRCA1, BRCA2, TP53, and ATM are associated with by multiple genetic and environmental factors. The majority of familial PDAC history, and screening for these mutations is recom- PDAC arises de novo, with hereditary or genetic factors accounting mended by National Comprehensive Cancer Network for only 5–10% of cases (4). Risk factors associated with PDAC guidelines (8, 9). development include: smoking (RR: 2–3), nonhereditary or chronic pancreatitis (RR: 2–6), chronic diabetes mellitus (RR: 2), obesity and/or sedentary lifestyle (RR: 2), nontype O blood group (RR: Diagnosis of Pancreatic 1–2), and age (≥97% of cases occur over the age of 45; refs. 4, 5). A Adenocarcinoma Treatment improvements in many common tumors, for example, breast and prostate, are, in part, a consequence of advances in disease 1Departments of Pharmacology and Experimental Therapeutics, Biomedical diagnosis. Unfortunately, there are no reliable or readily available Engineering, and Chemistry, Boston University, Boston, Massachusetts. screening tests for PDAC, and the majority of patients with PDAC do 2Division of Surgical Oncology, Massachusetts General Hospital, Boston, 3 not exhibit symptoms until advanced stage. The majority of PDAC Massachusetts. Department of Biochemistry and Molecular Biology and – Georgia Cancer Center, Medical College of Georgia, Augusta, Georgia. 4Division tumors (60% 70%) originate at the head of the pancreas, and this of Thoracic Surgery, Massachusetts General Hospital, Boston, Massachusetts. location dictates subsequent symptomatology (10, 11). Head tumors typically present with pain, jaundice, pruritus, pale stools, dark urine, Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). and gastric outlet obstruction. Body and tail tumors are largely asymptomatic and present late with distant metastases or local disease Corresponding Author: Mark W. Grinstaff, Boston University, 590 Common- with multivisceral and vascular invasion. Both locations are associated wealth Ave, Boston, MA 02215. Phone: 617-358-3429; Fax: 617-353-6466; E-mail: [email protected] with anorexia, weight loss, and generalized abdominal pain (12). A comparative case–control analysis of patients with PDAC (n ¼ 120) to Cancer Res 2020;80:3179–92 control patients (n ¼ 180) revealed that bile obstruction (OR: 20), pale doi: 10.1158/0008-5472.CAN-19-2731 stool (OR: 31), anorexia (OR: 41), abdominal pain (OR: 30), and 2020 American Association for Cancer Research. unusual bloating/belching (OR: 20 and 17) are the most common

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Conventional Nanoparticles Chemotherapy

Fluorescence Nucleic Acid Guided Surgery Therapies

Antibody Small Molecule Therapies Inhibitors

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) are being pursued to improve patient outcomes.

general pancreatic cancer symptoms (13). Nonspecific early symptoms tumors localized to the pancreatic head and uncinate process (15, 16). hamper early clinical diagnosis of PDAC, supporting research into Multiple variations of the Whipple procedure (including extended noninvasive, cost-effective screening methods. lymphadenectomy, pylorus-sparing, and a variety of anastomotic Development of accurate diagnostic tests is limited by the dearth of techniques, as well as minimally invasive) are not associated with effective biomarkers. Currently, carbohydrate antigen 19-9 (CA19-9), improved survival outcomes (5). a sialyated Lewis blood group antigen, and carcinoembryonic antigen Minimally invasive surgical techniques are gaining popularity in (CEA), are used as circulating biomarkers of pancreatic cancer. oncology as an alternative to open procedures, with the potential to CA19-9 is not sensitive nor specific, and is elevated in other pancreatic reduce infection, blood loss, postoperative pain, and surgical site diseases, such as pancreatitis, pancreatic pseudocyst, choledocho- infection. Despite the technical challenges of a Whipple procedure, lithiasis, and cirrhosis (14). Currently, CA19-9 is used to monitor the the volume of laparoscopic and robotic assisted procedures has been course of patient disease, including postsurgical recurrence (15). CEA increasing (17). While meta-analysis comparing both laparoscopic and is also neither sensitive nor specific for early PDAC, and is elevated in robotic approaches to open Whipple procedures reported lower blood alcoholic cirrhosis, hepatitis, and biliary disease, and, thus, its utility loss, shorter hospital stay, and decreased morbidity without any in screening is limited (13, 16). Efforts to identify clinically relevant difference in major complications, mortality, or reoperation rates, the biomarkers are ongoing, and there are recent exciting developments in LEOPARD-2 trial reported a higher mortality in laparoscopic proce- both diagnostic and predictive biomarkers. Given the wealth of new dures compared with open procedures (18, 19). Robotic surgeries are potential biomarkers and potential screening assays, the reader is also safer than laparoscopic procedures, reduce the length of surgery, referred to a recent thorough review by Hasan and colleagues (15). decrease postoperative morbidity, and decrease the duration of hospital stay, without evidence of increased mortality (17, 20–23). Fur- Conventional Treatment of Pancreatic thermore, robotic Whipples afford a lower complication rate, margin Adenocarcinoma positivity rate, and wound infection rate compared with open procedures (17). The poor 5-year survival in patients with PDAC reflects the late To increase the rate of R0 (tumor negative margins) versus R1 diagnosis, limited treatment options, and molecular and biophysical (residual tumor margins) resections, neoadjuvant chemotherapy pro- properties of PDAC that contribute to resistance. Surgical resection tocols are being introduced in surgery (24). Murphy and colleagues remains the only current curative intent therapy for pancreatic cancer. report the potential benefit of neoadjuvant fluorouracil, leucovorin, However, surgical therapy is limited to 15%–20% of all patients with oxaliplatin, and irinotecan (FOLFIRINOX) and losartan followed by PDAC, and often nodal metastases and microscopically positive surgical resection in a phase II clinical trial. Of the 49 enrolled patients, margins are noted following resection (1, 15). Resection typically 42 underwent attempted surgery and an R0 (margin-negative) resec- requires a pancreaticoduodenectomy (Whipple procedure) to remove tion was achieved in 69% (CI, 55%-82%; ref. 25). With neoadjuvant

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therapy, overall median progression-free survival increases to particle formulation of paclitaxel (Abraxane), or the combination 17.5 months and overall survival to 31.4 months, although control therapy FOLFIRINOX. Monotherapy with gemcitabine is considered outcomes were not given (24). Similarly, FOLFIRINOX with gemci- for patients not amenable to more aggressive combination therapy, tabine transitions 52% patients with borderline resectable disease to R0 showing only modest improvement in overall survival (30). First-line outcomes (25). Retrospective data analysis of 30 patients with locally therapies, gemcitabine with or without Abraxane and FOLFIRINOX, advanced or borderline resectable PDAC, who received neoadjuvant show marginal improvement in overall survival for patients with gemcitabine plus nab-paclitaxel, with and without subsequent S-1 metastatic cancer, with overall survival falling short of one year in therapy reveals that eight were amenable for surgery, and six achieved most trials. Lack of target specificity, dose-limiting toxicity, and poor R0 resection (26). Collectively, this work demonstrates the potential to drug penetrance underlie major limitations in PDAC therapy efficacy improve survival outcomes in traditionally nonoperable patients and in metastatic disease, and highlight the discrepancy in care between to expand current curative intent therapy. PDAC and other solid tumors. Palliative treatment of metastatic PDAC relies on systemic, nonspecific chemotherapeutics (Fig. 1). Current treatment options and several recent trials for metastatic PDAC are summarized Targeted Therapies in Pancreatic Cancer in Table 1 (27–47). First-line chemotherapy in metastatic PDAC Currently, there is a dearth of targeted therapies in PDAC. Most typically entails combination therapy with gemcitabine and a nano- targeted agents, such as antiangiogenic agents (bevacizumab), tyrosine

Table 1. 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 Refs.

Gemcitabine þ nab-paclitaxel vs. Phase III MPACT 8.7 vs. 6.6 mo 861 P < 0.001 27 gemcitabine Gemcitabine þ capecitabine vs. Phase III 10.3 vs. 7.6 mo; 6.2 vs. 5.3 mo 214 P < 0.06; P < 0.08 28 gemcitabine Gemcitabine, oxaliplatin, Phase II GEMOXEL 11.9 vs. 7.1 mo 6.8 vs. 7.1 mo 67 P < 0.001 29 capecitabine vs. gemcitabine FOLFIRINOX vs. gemcitabine Phase III PRODIGE/ 11.1 vs. 6.8 mo 6.4 vs. 3.3 mo 342 P < 0.001; P < 0.001 30 ACCORD 11 mFOLFIRINOX Phase II 10.2 vs. 6.1 mo 37 31

Previous targeted therapy trials Target Drug Trial OS PFS Refs. 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 Cetuximabþ Phase I/II 4.6 mo 1.8 mo 36 trastuzumab EGFR þ MEK1/2 Erlotinib þ 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 þ AKT Selumetinib þ MK- Phase II 3.9 vs. 6.7 mo 1.9 vs. 2.0 mo 46 2206 TGF-b Galunisertib Phase II 8.9 vs. 7.1 mo — 47

Selected targets for antibody therapy Targets Function Downstream signaling Refs. DEspR Angiogenesis, anoikis resistance STAT1/3, FAK, ERK1/2 90, 91 GPR87 Tumor proliferation, angiogenesis, anoikis PI3K, AKT 92, 95, 96, 97 resistance, drug resistance CD147 Anoikis resistance, EMT, chemotherapy resistance, FAK/Src/Stat3, PI3K, AKT/mTOR 99, 100, 101, 102, ECM regulation 103, 104, 105, 106 MFAP5 ECM regulation, tumor proliferation, microvessel FAK, ERK1/2 108, 109 regulation CLDN4 Cell barrier function, chemoresistance Tight junction protein 110, 111, 112 BAG-3 ECM regulation and stromal cell regulation IL6 mediated tumor–stromal signaling 113, 114, 115

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kinase inhibitors (cetuximab), checkpoint inhibitors (durvalumab), increased efficacy, albeit at a low rate with an objective response rate and recently hyaluronic acid therapies (PEGPH20), have been inef- of 3.1% (61). ficacious in metastatic pancreatic cancer. (Table 1). Several targeted The underlying mechanism of human pancreatic cancer unrespon- therapies afforded only marginal improvements (≤2 months) in siveness to ICI immunotherapy is still elusive. It is thought that overall and progression-free survival, while others yielded worse pancreatic cancer is a nonimmunogenic “cold” cancer type (62). outcomes. For example, treatment combining MEK1/2 and AKT Human pancreatic carcinoma, except for the MMR-D subtype, exhi- inhibitors selumetinib and MK-2206 showed worse overall survival bits a median mutational load of 4, much lower than more immuno- than mFOLFIRINOX alone (46). Similarly, repurposing successful genic melanoma and lung cancer (62, 63). It might be expected that a clinical agents (such as anti-EGFR and HER2 agents) have also lower mutational load would decrease neoantigen presentation on failed (32–38). Certain targeted therapies do show efficacy in select tumors, thereby decreasing tumor infiltration by CTLs, and limiting population groups. Olaparib, a PARP inhibitor, improved response anti-PD-L1 therapy (64, 65). However, CTLs do infiltrate human rates in patients with DNA damage repair deficiency (10–20% of pancreatic carcinoma, suggesting that CTL tumor infiltration level is patients with PDAC). PFS was 24.7 weeks (3.9–41.1 weeks) with two unlikely a major factor that underlies human pancreatic cancer partial responses and six stable disease responses (48). Similarly, the nonresponse to ICI immunotherapy (66–69). Furthermore, pancreatic neurotropic receptor tyrosine kinase inhibitor larotrectinib, received tumor cells highly express PD-L1 and PD-L1 appears to suppress CTL FDA approval for NTRK fusion–mutant tumors (<1% of PDAC), tumor infiltration in the tumor microenvironment (70–73). based on a successful phase III clinical trial (49). While larotrectinib Yet, despite abundant PD-L1 expression and infiltrating CTLs, may be an efficacious therapy in this subgroup, early data suggests that pancreatic cancer does not respond to anti-PD-1/PD-L1 ICI immu- rapid resistance may be a problem (50). notherapy (60, 8). Therefore, it is reasonable to suspect that a PD-L1– Several failed therapies have targeted pathways regulated by independent mechanism may confer immune evasion to pancreatic the RAS family, known to be mutated in PDAC tumors. A summary tumors. This interpretation is supported by data demonstrating that of RAS mediated signaling is detailed in Supplementary Fig. S1. targeting tumor-promoting mediators in the tumor microenviron- The RAS family of gene (HRAS, KRAS,andNRAS)encode ment sensitizes pancreatic cancer to ICI immunotherapy in preclinical 21 kDA proteins with GTPase activity, responsible for regulating mouse tumor models (7, 73). 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). Treatments in Preclinical Development Therapies targeting RAS-regulated pathways have consistently Poor survival outcomes in PDAC necessitate novel, innovated failed in PDAC, due to the high frequency of activating mutations. approaches towards disease management. Prior therapies failed to Initially thought to be undruggable, there is ongoing exciting capture the biologically relevant pathways driving PDAC tumorigen- research in RAS-specific targets, which recognize transient, drug- esis. Future research must focus on addressing the biological and gable binding pockets. The first, S-IIP, is the target of RAS inhi- biophysical properties unique to PDAC. As PDAC treatment employs bitors in the G12C mutant (only 1%–4% of PDAC tumors) with both surgery and chemotherapy, we will discuss new advancements in a covalent KRAS(G12C) inhibitor AMG 510, under clinical investi- these fields including: (i) enhanced visualization of tumor margins gation (52). Additional studies have discovered four total pockets: during surgery to increase the incidence of complete resection; (ii) S-IIP pocket, Kobe site, cyclen site, and P4 site, all of which are of better targets for biologic therapy and small-molecule inhibitors; and clinical interest (52–57). (iii) improvements in drug delivery to overcome biophysical limitations of PDAC treatment such as delivery within the local fibrotic tumor and peritumoral microenvironment as well as improved sys- Immunotherapy for Pancreatic Cancer temic penetrance. Immunotherapy is affording a paradigm shift in human cancer treatment. Immune checkpoint inhibitor (ICI) mAbs that target Surgical visualization aids CTL-associated protein 4 (CTLA-4) or programmed death ligand 1 To achieve complete R0 resection, surgeons must be able to (PD-L1)/programmed death 1 (PD-1) have generated durable efficacy adequately visualize the entire tumor. Conventional brightfield surgery in many types of human cancers. In 2017, the FDA granted accelerated is inherently limited by the human eye, and surgeons rely on histology approval to the anti-PD-1 mAb Keytruda for patients with solid to evaluate their resection. To augment tumor visualization, several tumors, including pancreatic tumors, which have either mismatch nonspecific near-infrared (NIR) fluorescent probes are used (74–78). repair deficiency (MMR-D) or high microsatellite instability (MSI). In These dyes spread lymphatically, and aid in visualizing bulk tumor the first phase I trial evaluating the anti-PD-L1 antibody MS-936559, margins and sentinel lymph nodes. Such surgical visual aids (SVA) are objective responses were achieved in melanoma, non–small cell lung employed in oncologic surgery to allow NIR tumor visualization, and cancer, renal cell cancer, and ovarian cancer, but not in the 14 patients clinical trials are ongoing in many cancer subtypes, including: colon with pancreatic cancer (58). Similar disappointing results were seen in cancer (LUM015, RAPIDO-TRACT, and Fluorescent lectin), lung a 60-patient phase IIA trial with anti-PD-L1 antibody durvalu- cancer (EC17 and ICG), and breast cancer (BLZ-100, LYM015, mab (59). However, a robust 62% objective response rate to pembro- AVP-620, EC17), as well as in PDAC (discussed below; refs. 76–78). lizumab was observed in pancreatic cancer patients with mismatch While advantageous over bright-field surgery, these dyes are inher- repair deficient (MMR-D; n ¼ 8) (60). Therefore, limited efficacy of ently limited by nonspecific diffusion, low specificity, and poor tumor anti-PD-L1 therapy is confounded by the low, only 0.8%, prevalence of retention. Newer methods of fluorescent agent delivery, enhanced MMR-D tumors (8). Similar shortcomings were seen in response to tumor specificity, and improved resolution are under development to anti-CTLA-4 mAb immunotherapy. In phase II and IIa trials, no improve primary tumor detection and identify early metastatic niches. objective response was achieved with Ipilimumab (61). Combinational Polymeric nanoparticle SVAs offer an effective alternative to cur- immunotherapy of anti-PD-L1 and anti-CTLA-4 mAbs showed rent, nonspecific SVAs. Relying on bulk diffusion, nanoparticles

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accumulate within metabolically active cells and are retained longer Finally, there is ongoing activity to develop theranotics, combined than lower molecular weight dyes. Responsive polymeric nanoparticles visual agents and adjuvant therapy at the time of surgery. For example, covalently linked with rhodamine dyes (HRF-eNP), designed to gold nanoparticles functionalized with an anti-glypican-1 antibody, expand and retain dye once internalized within cells and the tumor which targets a cell surface proteoglycan, and an NIR dye, for tumor microenvironment, readily localize to PDAC tumors in a Panc1 rat visualization, loaded with oridonin inhibit PDAC migration and xenograft model. HRF-eNP enable improved visualization and delin- EMT (86). These nanoparticles are highly specific for PDAC tissue, eation of tumor margins compared with bright-field imaging (Sup- and provide excellent resolution of tumor margins. Adequate fluo- plementary Fig. SI2A and S12B; ref. 79). Targeting and retention of the rescent signal remains even after 48 hours in BxPC-3 orthotopic HFR-eNPs in peritoneal tumor sites is highly effective, with specificity, xenografts (87). Furthermore, these SVAs reduce tumor volume in sensitivity, and accuracy of 0.99, 0.92, and 0.95, respectively, in the 202 the BxPC-3 model, affirming both a therapeutic efficacy as well as a normal samples and 253 histologically confirmed samples surveyed. specificity. Another PDAC gold nanoparticle theranostic targets Use of HRF-eNPs allows submillimeter tumor detection, a currently the cell surface proteoglycan GPC1 (88). These gemcitabine-loaded unmet need in image resolution. A hyaluronic acid derived nanopar- nanoparticles, labeled with an NIR dye (GPC1-GEM-NP), allow ticle containing indocyanine green, NanoICG, provides better tumor simultaneous tumor visualization and drug delivery. The combined visualization (2.30 0.67 AU in tumor vs. 0.41 0.10 AU in normal theranostic system shows excellent uptake in PDAC BxPC-3 and pancreas) compared with indocyanine green alone (0.77 0.12 AU in Panc-1 cell lines (>99% by flow cytometry), but minimal uptake tumor vs. 0.35 0.12 AU in normal pancreas) in a KPC orthotopic (<1%) in control 293T cells. In an orthotopic BxPC-3 murine model, xenograft PDAC murine model (80). While NanoICGs are effective for the GPC1-GEM-NPs exhibit greater tumor retention (high signal at delineating tumor from adjacent normal tissue, significant accumu- 48 hours) and accumulation than GEM-NPs (lacking the GPC1 lation occurs in the stomach, liver, small intestine, and kidney. Finally, antibody) alone (loss of signal by 48 hours), with off-target NP tripolymer fluorescent nanospheres (TFN) possessing a surface coat- accumulation in the liver and spleen (88). Theranostics are an exciting ing of peanut agglutinin proteins exhibit high affinity for the tumor- surgical tool, providing adjuvant therapy while enhancing surgical associated antigen Thomsen Friedenreich, and shows high affinity for visualization. PDAC, when comparing histologically fixed samples of PDAC and As discussed above, only 10%–15% of patients with PDAC are normal tissue (81). TNFs nanoparticles localize primarily to PDAC currently amenable to surgery. Often many patients progress despite tumors in an in vivo model of PDAC, using subcutaneously implanted surgery, as the result of incomplete resection or undetected metastatic CRL2460 cells. The TNFs nanoparticles enable complete resection of disease at the time of surgery (89). SVAs improve R0 resection rates PDAC tumors with minimal removal of normal tissue. Furthermore, and identify micrometastatic disease. In addition, the use of thera- signal levels in noncancerous tissue were well below detection thresh- nostic agents provides adjuvant treatment of micrometastases while old, reducing false positives. Relying solely on bulk diffusion, these guiding further management. It is also reassuring that several recent nanoparticle carriers show promise as a cost-effective, reproducible trials have improved R0 resection rates of previously nonsurgical method for image-guided surgery. candidates through better neoadjuvant chemotherapy protocols (vide In contrast to nonspecific nanoparticle delivery, targeted fluor- supra). Through better tumor visualization, improved treatment and ophore-conjugated antibody SVAs are being evaluated. Clinical staging is a possibility. results obtained using anti-CEA (NCT02973672 and NCT0278028), anti CA19-9 (NCT02587230), anti-EGFR (NCT02736578 and New antibodies and biologic targets NCT03384238), and anti-VEGF (NCT02743975) based SVAs are Identification of novel targets in pancreatic cancer is essential to promising with improved R0 resection rates and better surgical improving treatment outcomes. Available therapies for advanced and staging of advanced cases (82). Newer antibody conjugate systems, metastatic PDAC are predominately nonspecific chemotherapeutics. therefore, are of keen interest, including dual-antibody systems to To date few targeted therapies exist, as many have failed because they increase the signal, improve specificity, and overcome the hetero- targeted KRAS-dependent pathways. Efficacious treatment of PDAC geneity present in cell surface expression. For example, Zettlitz and requires identification of PDAC-specific targets, which are KRAS- colleagues describe a dual-labeled targeting anti-prostate stem cell independent, and which target multiple tumor hallmarks to prevent antigen (PSCA) conjugated with a near IR dye and a 124Iforfunc- disease resistance. Several new targets have been identified that met tional PET imaging. PDAC tumors highly express the PSCA surface these criteria, and preclinical advancements in these targets are antigen with high sensitivity and specificity (83). Use of this dual- exciting (Table 1). labeled antibody allows sequential NIRF and PET visualization of subcutaneous PDAC tumors in a murine patient-derived xenograft DEspR (PDX) model (84). These bifunctional anti-PSCA antibodies retain The dual-endothelin-1/VEGF-signal peptide receptor (DEspR) is a high binding specificity, with minimal noncancerous uptake. A dual single transmembrane receptor, identified as an embryonic-lethal null antibody system that contains both a fluorescent dye, Alexa Fluor mutation phenotype characterized by dysregulated vasculogenesis and 700, and a quencher IRDye, QC-1, fluoresces only after proteolytic angiogenesis, and impaired cardiac organogenesis (90, 91). DEspR cleavage (85). This responsive system repurposes two antibodies, binds two known ligands, ET-1 and the signaling peptide of VEGF. cetuximab and trastuzumab. Both proteolytically activated immu- Both of these pathways strongly correlate with PDAC progression, but noconjugates provide improved signal resolution of pancreatic currently, there are no effective therapies targeting either one. PDAC AsPC-1 orthotopic xenografts, as well as enhance signal detection tumor cells highly express DEspR, with minimal expression on normal and resolution compared with nonspecific probes. Furthermore, dual pancreatic tissue. This receptor regulates multiple tumor processes: imaging improves signal resolution, maximizing signal in cancer cells cancer stem cell (CSC) viability and anoikis resistance, angiogenesis, that have higher receptor expression of both targets. With the dis- and cancer cell invasiveness. The exact signaling pathway is unclear, covery of more specific PDAC targets, newer SVA can be designed for but appears to have RAS-independent signaling, through FAK, SRC, better tumor detection and enhanced visualization. and STAT3-mediated pathways (90). Treatment of a subcutaneous

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xenograft RNU rat model of Panc1 with a mAb to inhibit DEspR as high intratumoral pressure (108). Given the shortcomings of reduces tumor volume compared with gemcitabine (Supplementary hyaluronidase therapy, anti-MFAP5 therapy is an attractive alterna- Fig. SI3A; ref. 91). Inhibition of DEspR captures several key PDAC tive, reducing both tumor cell viability and CAF-induced desmoplasia. 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 Claudin-4 cells and to interrogate its role in normal cell signaling. Another target of tumor barrier function is Claudin-4 (CLDN4), a main constituent protein in tight junctions. PDAC cells overexpress GPR87 claudin-4 compared with normal pancreatic cells, with higher expres- Several cancer subtypes, including squamous cell carcinomas, lung, sion carrying a worse prognosis (110, 111). Inhibition of CLDN4 cervical, and pancreas highly express GPR87, a recently identified reduces cell adhesion of PDAC cells and enhances intracellular G-coupled protein receptor related to the lysophosphatidic acid (LPA) accumulation of chemotherapeutics (111, 112). In vivo administration receptor (92–95). GPR87 regulates multiple pathways, which influence of anti-CLDN4 antibodies promotes intratumoral accumulation of tumor proliferation, angiogenesis, anoikis resistance, and drug resis- FOLFIRINOX, and increases efficacy in subcutaneous MIA PaCa2 tance. GPR87 signaling involves multiple downstream mediators, murine models (111). This effect is likely a result of reducing PDAC with both RAS-dependent and RAS-independent signaling (96, 97). cell adhesion and interfering with drug efflux pumps. A recent report In pancreatic cancer, GPR87 mRNA expression is significantly ele- suggests that anti-CDLN4 antibodies are safe and relatively nontoxic, vated and correlates with worse overall survival (97). PDAC cell lines, but clinical data is lacking (112). Both CLDN4 and MFAP5 are AsPC-1, Capan-1, BxPC-3, MIA PaCa2, Capan-2, and PANC1 over- promising targets, which could disrupt natural PDAC barriers to express GPR87 compared with normal human pancreatic ductal chemotherapeutics and overcome biophysical defenses. epithelial cells. Inhibition of GPR87 significantly reduces tumor viability in vivo and decreases subcutaneous tumor growth compared BAG-3 with controls (Supplementary Fig. S3B; ref. 97). GPR87 attractive An exciting regulator of PDAC stromal cells is BCL2-associated features include: it regulates multiple tumor hallmarks, correlates with athanogene 3 (BAG-3), a cochaperone of HSP70. BAG-3 regulates worse prognosis, and is highly expressed in most PDAC. However, multiple signaling pathways in PDAC, facilitating cross-talk GPR87 is expressed on many normal tissues, albeit at lower levels. Like between: tumor-associated macrophages and PDAC cells and PDAC DEspR, GPR87 inhibition is not completely characterized, and it is cells and pancreatic stellate cells via an IL6-dependent man- unclear if GPR87 regulates survival in healthy epithelium. ner (113, 114). BAG3 expression correlates with fibrosis in PDAC tissue, and plays a key role in tumor collagen barrier regulation (115). CD147 Administration of humanized anti-BAG-3 antibodies reduces the CD147, a member of the immunoglobulin superfamily, regulates growth of subcutaneous MIA PaCa-2 tumors in murine xenografts. several key cancer hallmarks in PDAC and is highly expressed in The anti-BAG3 antibody are highly tumor specific, and do not PDAC (87%), hepatocellular carcinoma (83%), and glioblastoma distribute into normal parenchyma (115). While data are limited, multiforme (79%) (98, 99). CD147 expression positively correlates no toxicity was observed with BAG3 administration in healthy with poor survival and greater metastatic spread (99). CD147 influ- animals. Currently, Intrepida Bio is developing an anti-BAG3 anti- ences multiple downstream signaling pathways, which are both RAS- body for clinical trials in PDAC. dependent and independent, to include anoikis resistance, epithelial- Improvements in PDAC therapy requires highly selective targets, to-mesenchymal transition, chemotherapeutic resistance, and CSCs which regulate multiple, essential cancer pathways. The targets pre- pluripotency and proliferation (100–105). A mAb targeting CD147, sented here are both novel and clinically relevant, having several HAb18IgG, enhances radiation-induced cell death in CFPAC-1 and advantages over previous, failed biologic targets (e.g., EGFR, IGFR, MIA PaCa-2 cells, and potentiates the effect of gemcitabine thera- and VEGF). These include targeting multiple cancer hallmarks, inhi- py (106). Inhibition of CD147 reduces tumorsphere formation, a bition of tumor cell and stromal function, KRAS-independent surrogate of CSC-inhibition, in BxPC-3 and MIA PaCa-2 PDAC cell mechanisms, and modulation of PDAC CSC function. lines. A combination therapy of a 90Y-l radiolabeled anti-CD147 antibody and gemcitabine significantly improves the efficacy of gem- Small-molecule inhibitors citabine therapy in a murine MIA PaCa-2 xenograft model (107). Small-molecule inhibitors have not shown success in recent trials in Although CD147 is an exciting target, data suggest that CD147 is a PDAC. Significant resistance has been encountered in these therapies promiscuous receptor, and downstream regulation depends on cell due to RAS-dependence of their targets. Future endeavors must focus surface density (100). Therefore, treatment responses to CD147 on pathways that directly regulate PDAC proliferation, metastasis, and therapy may be highly variable. survival. Ongoing research evaluating MEK/ERK inhibitors and autophagy, EGFR inhibitors, and reevaluation of more classical path- MFAP5 ways, is clinically important, but will not be discussed here (116–119). Microfibril associated protein 5 (MFAP5), an extracellular matrix Instead we discuss several novel PDAC targets with RAS-independent glycoprotein involved in elastic microfibril assembly, regulates both signaling pathways. These inhibitors impact tumor energy regulation PDAC cells and cancer-associated fibroblasts (CAF). Elevated MFAP5 and chemoresistance in addition to PDAC cell progression and expression correlates with worse prognosis and more aggressive metastasis. While not exhaustive, these new studies highlight recent disease (108, 109). Murine antibodies targeting MFAP5 reduce PDAC preclinical advances demonstrating in vivo efficacy in highly cell viability and cell motility (108). In in vivo PDXs, inhibition of expressed, biologically relevant targets. MFAP5 sufficiently arrests cancer cell growth and decreases collagen I deposition, and impairs CAF collagen production and decreases GFAT1 (DON) intratumoral microvessel permeability (108). Treatment with MFAP5 The hexosamine biosynthesis pathway (HBP) is a major regulator of improves delivery of paclitaxel by decreasing biophysical barriers, such cancer survival. In normal cells, the HBP is a shunt of the glycolysis

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pathway, acting as a sensor of energy availability in normal cells (120). CtBP (4-Cl-HIPP) HBP plays a role in cancer cell signaling, via O-GlcNAcylation/mTOR/ The transcriptional coregulator C-terminal Binding Proteins adenosine monophosphate (AMP)-activated protein kinase (AMPK) (CtBP) 1 and 2 regulate key tumor hallmarks: apoptosis, pro- pathways, tumor suppressor kinase 1/2 (LATS1/2)-mediated path- liferation, and regulation of the pluripotency state (135–137). ways, and ER stress regulation (121). Not surprisingly, HBP regulates Overexpression of CtBPs results in oncogenic transformation of cancer cell metabolism and directs cellular proliferation, transcription, human colon epithelium, similar to HRAS mutation, and may deﬁne EMT, and self-renewal in CSCs (122). The rate limiting enzyme in an early activation step in gastrointestinal malignancies (136, 137). HBP is glutamine-fructose amidotransferase 1 (GFAT1), which is PDAC tumors highly overexpress both CtBP1 and 2, and both iso- overexpressed in 35.7% of the 176 PDAC samples in TCGA. Inhibition forms are known to regulate PDAC metastasis (138). CtBPs contain a of GFAT1 in vitro, using a small-molecule glutamine analogue targetable dehydrogenase domain, required for their transcriptional (6-diazo-5-oxo-L-norleucine, DON), decreases expression of several function. The novel anti-CtBP inhibitor [4-chloro-hydroxyimino self-renewal genes: SOX2, OCT4, and KLF4 in MIA-PaCa2 and phenylpyruvate (4-Cl-HIPP)] targets this dehydrogenase domain S2VP10 cell lines, and reduces both tumor cell proliferation and (131). Loss of CtBP2 decreases metastatic capacity and reduces peri- collagen matrix deposition in murine orthotopic KPC and CAF toneal PDAC seeding in a genetically engineered CKP PDAC murine xenografts (123). Reduction of PDAC ECM density also enhances model (131, 138). In primary CKP tumors, treatment with 4-Cl-HIPP tumor sensitivity to anti-PD1 therapy, increasing CD8þ tumor inﬁl- reduces tumor volume, equivalent to gemcitabine treatment alone. tration (124). GFAT1 is a promising clinical target, as its inhibition When dosed together, 4-Cl-HIPP enhances the action of gemcitabine, decreases tumor desmoplasia while enhancing anti-PD1 therapy. The reducing primary CKP tumors by greater than 50% (138). While early success of anti-GFAT1 therapy in lung and breast cancer will hopefully in development, anti-CtBPs are promising adjuvants to traditional translate to PDAC (120). chemotherapeutics.

GOT1 (PF-04859989, Aspulvinone O) ABCC3 (MCI-715) Another metabolic target of interest is glutamate-oxaloacetate Canalicular multispecific organic anion transporter 2 (ABCC3) is transaminase 1 (GOT1), which regulates amino acid metabolism and a member of the ATP-binding cassette (ABC family) and is asso- the malate-aspartate shuttle. GOT1 overexpression is common in ciated with chemoresistance in PDAC. ABCC3 is regulated by p53, PDAC tumors and correlates with worse disease prognosis In and controls PDAC proliferation via STAT3 and HIF1a signaling KRAS-mutant PDAC, alterations in GOT1 metabolism fuel biosyn- (139). ABCC3 overexpression correlates with worse prognosis after thesis, proliferation, and redox balance, leading to dependence on resection, greater chemoresistance, and is present in early cancerous GOT1 for tumor maintenance (125, 126). A novel inhibitor of GOT-1, lesions (140, 141). A nonsteroidal anti-inflammatory drug derivative, PF-04859989, forms a covalent adduct with the substrate of GOT1, MCI-715, inhibits ABCC3 via inhibiting STAT3 and HIF1a down- irreversibly inhibiting enzymatic function (127). Loss of GOT1 func- stream signaling, leading to apoptosis in AsPC1, HPAFII, and tion limits PDAC metabolism and inhibits PDAC proliferation CFPAC-1 PDAC cell lines. Administration of MCI-715 reduces tumor in GOT-1 dependent cell lines. A natural inhibitor of GOT1, volume and improves overall survival in a HPAFII subcutaneous Aspulvinone O, exhibits similar efficacy (128). Inhibition of GOT1 xenograft model, a KPC transgenic murine model, and a PDX model by Aspulvinone O induces apoptosis in SW1990 cell lines in vitro, (142). Early data is promising that anti-ABCC3 therapy is effective and modulates cellular metabolism via shifting mitochondrial re- alone and further research into synergistic potential between MCI-715 spiration and increasing ROS production (128). Also, Aspulvinone and conventional chemotherapeutics would be of considerable O inhibits PDAC proliferation in vivo in murine subcutaneous interest. SW1990 xenografts. These results are promising, as GOT1 inhibition could starve KRAS-mutant PDAC tumors and prevent disease SRC-3 (bufalin) progression. The steroid receptor coactivator family (SRC) are nuclear receptors, which act as scaffolds for assembly of coactivator homocom- Creatinine transporter SLC6A8 (RGX-202) plexes. Of the three SRC members, SRC-3 is more frequently Increased production of phosphocreatine via creatine kinase-B overexpressed in human tumors and SRC-3 levels increase from occurs in pancreatic cancer to meet the high energy demand of low-grade PanIN to malignant PDAC (143). Using bufalin, a 14-b- growing tumors (129). Importing phosphocreatine via the creatine hydroxy steroid, inhibition of SRC3 significantly reduces PDAC transporter SLC6A8 allows cells in the metastatic niche to maintain cell viability in Panc-1 and Capan-2 cell lines (144). In vivo, ATP under high metabolic demand (130, 131). Depletion of treatment with bufalin reduces tumor proliferation and increases SLC6A8 in colon and pancreatic cancer reduces the ability of cancer animal survival in orthotopic Panc-1-Luc cell line xenografts. cells to colonize the liver in murine xenografts (132, 133). The orally Several studies report SRC-3 as a potent oncogene, which may con- available small-molecule RGX-202 inhibits SLC6a8 in KRAS tribute to the genetic complexity of PDAC (145–147). wild-type and KRAS-mutant cell lines, as well as human PDX The small-molecule inhibitors described here possess signi- models (134). Inhibition of SLC6A8 correlates with decreased ficant advantages over previously tested, and failed, agents in tumor metabolic activity, decreased proliferation, and a dramatic PDAC. Several of these agents target PDAC cellular metabolism, reduction in cell survival. Tumors treated with RGX-202 are also starving metastatic niches and limiting primary tumor growth. more susceptible to gemcitabine, likely reflecting a change in cellular Other targets strip PDAC cells of protective chemoresistance metabolism and nucleotide synthesis (134, 131). Although the effect of defenses, making tumors vulnerable to conventional chemothera- SLC6A8 on normal cellular metabolism remains to be full understood, peutics. These inhibitors target proteins that regulate multiple these results are exciting, and suggest that in phosphocreatine- tumor hallmarks, influencing proliferation, metastasis, and CSC dependent cells, SLC6A8 inhibition could effectively starve PDAC potentiation. All of the agents demonstrate efficacy in vivo and have tumors. potential to be translated to clinical trials. Although unlikely to be

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sufficient by themselves, several show a benefitwhencombinedwith cinoma and liver metastasis. As with in vitro studies, the in vivo efficacy conventional chemotherapeutics. of this nanoparticle stems from inhibition of pluripotency factors, Gli targets, and the Shh pathway, preventing crucial steps in tumor Nanoparticle treatments progression. Using a rat model of metastatic PDAC, a paclitaxel- As an alternative to the delivery of antibodies and small-molecule loaded responsive polymeric NP accumulates in pancreatic tumor cells inhibitors, nanoparticle-based strategies deliver high pay-loads of following intraperitoneal injection and specifically delivers drug to cytotoxic agents to the tumor as a concentrated package (Fig. 1). The these tumors (155). The NPs maintain tight encapsulation of paclitaxel targeting or localization of the drug-loaded nanoparticle typically prior to reaching the tumor but then responsively swell and release a relies on the greater metabolic activity and leakier vasculature of high dose of paclitaxel when in the acidic tumor environment. tumors compared with normal tissue, a property termed the enhanced Although treatment with the NPs does not significantly improve permeability and retention effect (EPR; refs. 148, 149). While the survival outcomes compared to free paclitaxel; substantially fewer differences in biochemical and biomechanical properties between side-effects are present, suggesting that NP delivery under this method normal and cancerous tissue can be useful in aiding drug delivery, may reduce dose-limiting toxicities and enable completion of treat- the EPR effect is often exaggerated in many preclinical models, where ment regimens. murine tumor proliferation rates exceed those of human tumors (150). Polymeric nanoparticles also provide a means to deliver poorly This higher proliferation rate produces less developed, leakier blood soluble immunomodulatory or tumor-suppressive agents that cannot vessels, which are not reflective of most human tumor vessels. There- otherwise be delivered intravenously (156). Curcumin, a suppressor of fore, modifications to nanoparticle materials to improve tumor spec- carcinogenesis in several cancer subtypes: breast, colon, pancreatic, ificity, such as including tumor specific proteins in the polymeric prostate, and ovarian, acts through several pathways, regulating cell- structure, is likely necessary (151). cycle arrest through upregulation of p16, p53, and p14ARF, induction Resistance to cancer therapies is both molecular and biophysical in of apoptosis through BAX, BAD, and BCL2 proteins, and modulating nature. Molecular resistance includes the redundancy of pro- NFkB signaling (156). Curcumin inhibits metastasis and angiogenesis oncogenic pathways, modification or alteration of tumor-specific in numerous xenograft cancer models, particularly in orthotopic proteins and receptors, and survival of target negative cells. The xenograft models of PDAC, suggesting that its benefits extend beyond challenge of significant tumor heterogeneity is reflected in the com- cancer prevention. Encapsulation sidesteps curcumin's poor aqueous mon failure of targeted therapies in patients with pancreatic cancer. solubility and stability and offers a potentially viable method for Biophysical mechanisms of resistance encompass environmental fac- delivery at sufficient levels. Curcumin-loaded PLGA-chitosan nano- tors within the tumor microenvironment that pose barriers to drug particles internalize and retain in Panc1 and MIA PaCa2 cells at a delivery (152). The composition of the tumor extracellular matrix, a higher level than curcumin itself, with greater in vitro potency and dense collagen and elastic network, interspersed with glycosamino- reduced tumor migration via a scratch assay (Supplementary Fig. SI4B; glycans and proteoglycans, produces a hydrophilic gel, thereby ref. 156). In vivo studies are needed to assess the potential benefitof increasing local hydrostatic pressure. In PDAC, spare blood vessel curcumin. density and thick desmoplastic matrix further inhibit drug pene- To improve the local delivery and penetrance of polymeric NPs, trance (151, 152). In addition, the tumor microenvironment tends to which typically rely on EPR and bulk diffusion, modifications to be more acidic, contains numerous proteases, and is a nidus of reactive the polymer backbone are being explored. By changing the specific oxygen species, all of which can deactivate the drug, degrade protein characteristics of the polymer (e.g., charge, hydrophilicity, lipo- therapies, and alter drug kinetics (150–152). Nanoparticle-based drug philicity) or through the addition of surface aptamers, peptides, or delivery vehicles are designed to overcome the above challenges with even antibodies (which improve selective internalization) better local accumulation of drug payload (e.g., conventional chemothera- pharmacokinetic profiles are achieved. For example, a sequentially peutics, nucleic acids, or immunomodulatory agents). Various nano- triggered NP relies on an aptamer conjugated to a cell-penetrating particle formulations are being investigated, ranging from polymeric, peptide-campothecin prodrug (Apt/CPP-CPTD) and amphiphilic protein, or lipid assembly, and we next discuss recent preclinical PEG copolymer. The NP utilizes a GBI-10 aptamer “camouflage,” advances in nanoparticles for the treatment of PDAC. relying on GBI-100s negative charge and tenascin-C's targeting ability to reduce nonspecific uptake of a cell-penetrating peptide Polymeric nanoparticles nanoparticle (Supplementary Fig. S4C; ref. 157). The NP polymer Biocompatible polymers, such as poly lactic acid, PLA, and copol- backbone contains a reductively cleaved disulfide linkage, taking ymer derivatives, such as poly lactic-co-glycolic acid (PLGA) are advantage of the greater glutathione concentration within the commonly employed in nanoparticle development. A paclitaxel- tumor environment, to release camptothecin following NP inter- loaded PLGA nanoparticle (NP) with an anti-MUC1 antibody, nalization. In vitro, the NP performs equivalently to free campothe- TAB004, on the surface readily internalizes in MUC1-positive cells, cin in MIA PaCa2 cells, but shows improved drug penetration, as with MUC1-positive cells exhibiting decreased viability when treated measured by spheroid penetrance. After intravenous administra- with the nanoparticle versus paclitaxel alone (153). In vivo imaging tion, the NPs localize primarily in the tumors of a subcutaneous studies of isocyanine green encapsulated TAB004 conjugated NPs xenograft MIA PaCa2 mouse, and the NP treatment group, contain- shows localization to KCM pancreatic cell line orthotopic tumors ing an equivalent 10 mg/kg of camptothecin, affords a significant following a single IP administration of 50 mg/kg of the nanoparticle. In decrease in tumor volumes compared with free camptothecin alone, another study, a-mangostin, a sonic hedgehog inhibitor, encapsulated or saline control, demonstrating improved efficacy. PLGA NPs reduce growth in CSC-like cells and prevent EMT by Protein-based nanoparticles offer potentially greater biocompati- upregulating E-cadherin and inhibiting N-cadherin and Slug, SNAI2, bility, improved stability, and alternative functionalization capability NANOG, c-MYC, and Oct4 (Supplementary Fig. SI4A; ref. 154). In the compared with polymeric nanoparticles (158). Abraxane, a paclitaxel genetically engineered KPC mouse model of PDAC, this NP formu- loaded albumin-based nanoparticle, is the prime example and is lation prevents progression of pancreatic intraneoplasia to adenocar- currently used clinically (27, 159, 160). More recently, the tumor

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suppressor gene, IL24, has been loaded into a bovine serum albumin is needed to enable selective delivery of the siRNA to the tumor. For (BSA) NPs. Successful gene delivery occurs in Panc1 and BxPC-3 example, programmable DNA nanoparticles (DNP), containing a cancer cells, with a marked reduction in cell proliferation and an nucleolin targeting aptamer, deliver BCL2 targeting siRNA and reduce increase in apoptosis associated with elevation of VEGF expres- tumor volume in DMS53 subcutaneous xenografts (169). An alterna- sion (161). Treatment of subcutaneous BxPC-3 and Panc-1 xenografts tive strategy uses a peptide-based nanoparticle to deliver siRNA for Ras with the NPs significantly decreases tumor volume relative to controls. inhibition. This peptide-based siRNA loaded nanoparticle successfully Encapsulation of Parviflorin D, an anti-proliferative agent derived reduces KP1 cell viability and KPCC pancreatic tumors in mice (170). from the Plectranthus genus, in an albumin-based NP significantly The former approach, through a specific targeting aptamer, offers the improves drug delivery by increasing drug solubility in vitro, and advantage of selective targeting to the tumor, and reduces off-targeting demonstrates improved potency in BxPc-3 and Panc-1 PDAC cell effects. lines (162). To improve the specificity of albumin-based nanoparticles, Rather than relying on delivery of siRNA through a nanoparticle Ji and colleagues surface-conjugated the arginine-glycine-aspartic acid carrier, an exciting alternative approach involves reprograming mes- (RGD) peptide to their nanoparticles. These RGD modified BSA enchymal stroma/stem cells (AD-MSC) to release trimeric and multi- nanoparticles show greater internalization and retention in BxPC-3 meric sTRAIL (171). These modified AD-MSCs produce stable multi- cells compared with BSA nanoparticles alone, and are safe and meric, soluble sTRAIL structures, via lentiviral transduction, capable tolerated in vitro and in vivo (163). The greater specificity of these of inducing apoptosis in BxPC-3, MIA PaCa-2, and primary PDAC cell RGD-albumin nanoparticles improves internalization beyond the EPR lines with greater cytotoxicity than rhTrail (Supplementary Fig. SI5A). effect. Encapsulation of the nanoparticles with gemcitabine signifi- Modified AD-MSCs transplanted into subcutaneous tumor bearing cantly improves the efficacy of gemcitabine treatment in BxPC-3 cells, BxPC-3 mice produce sufficient levels of sTRAIL to decrease tumor suggesting that this formulation may improve overall success of BSA volume. However, this technique will be technically challenging and derived nanoparticles and offer an advancement over Abraxane. expensive compared with nanoparticle delivery, and may not be Investigating whether tumor derived exosome-based nanoparticles amenable to metastatic disease treatment. repress tumor cell growth is another active research area and an miRNA delivery provides an alternative to direct gene modification, exciting alternative to polymer-based approaches (164). Protein char- relying on pan-suppressive effects. A pentablock copolymer of poly acterization of pancreatic cancer cell line, SOJ, exosome-derived NPs (ethylene glycol) diacrylate (PEG-DA) and miR-345 coblock polymer reveals a diverse packaging of proteins, including glucose-6-phosphate with encapsulated gemcitabine delivers both miR-345 and gemcitabine isomerase, Hsp90, and BSDL (164). Treatment of SOJ cells with the to significantly reduce Capan-1 cell viability in vitro and tumor burden SOJ-derived exosome NPs reduces proliferation in a dose-dependent in vivo in subcutaneous xenograft models (Supplementary Fig. S5B; response, with similar, but variable response in other cell lines (165). ref. 172). Human umbilical cord mesenchymal stromal cell–derived Evidence of nanoparticle efficacy includes: a decrease in overall exosomes, containing miR-145-5p, rapidly internalize in human PI3K/Akt/GSK-3b activity, an increase in PTEN dephosphorylation, Panc-1 cell lines affording an increase in apoptosis and cell-cycle an elevation of BAX, and a decrease in BCL2 expression. The viability arrest (173). Overexpression of miR-145-5p significantly reduces of the control human umbilical vein endothelial cells is not affected tumor volume in a subcutaneous murine xenograft model of Panc1. by the SOJ NPs. Cell-derived exosomes offer better potential biocompatibility than Currently, two nanoparticle formulations, Abraxane (discussed polymeric nanoparticles; however, polymeric nanoparticles are above) and Onivyde, are FDA approved for treatment of advanced better characterized and readily functionalized compared with the PDAC. Onivyde is a liposomal formulation of irinotecan approved for native systems. patients with metastatic PDAC having failed gemcitabine therapy. Onivyde extends survival in combination with fluorouracil and leu- Aptamer-based approaches covorin, compared with these drugs alone (mOS 6.1 months vs. An alternative approach to nucleic acid therapy is the use of 4.2 months; ref. 166). A phase I/II trial is evaluating Onivyde with aptamers (single-stranded oligonucleotides) for targeted deliv- fluorouracil, leucovorin, and oxaliplatin as a first line therapy in ery (174, 175) Aptamers are a practical alternative due to ease of PDAC. These clinical successes hold promise for nanoparticle research chemical synthesis, chemical modification, and high stability; however, as part of cancer therapeutic development. limited success in aptamer-targeted therapies have been seen in cancer (176–179). A novel aptamer–drug conjugate (ApDC) utilizing Nucleic acid treatments via gene delivery and aptamer AS1411 binds selectively to the nucleolin, a cellular protein highly conjugates expressed in several PDAC subtypes (180). Capan-1, MIA PaCa2, and The direct modification of pro-oncogenic genes and reactivation of AsPC-1 cancer cell lines, but not normal H6c7 cell lines, internalize tumor suppressor genes, through gene therapy, offers a potentially and retain stable conjugates of AS1411 and gemcitabine (Supplemen- more specific approach to treating pancreatic cancer. Known genetic tary Fig. S5C; ref. 181). The AS1411-gemcitabine conjugate shows a mutations have been discussed, and represent key targets for drug slight decrease in potency, but greater target specificity than gemci- developers. For example, the loss of crucial tumor-suppressive genes tabine alone, and demonstrates reduced tumor volumes in a subcu- can result in acquired drug resistance in cancer cells, limiting the taneous xenograft model of Capan-1 in mice. An alternative approach efficacy of many therapies (167). Although gene delivery, considered a utilizes a pancreatic cancer–specific aptamer, 20-Fluoropyrimidine holy grail in cancer therapy, is advancing in the clinic, many success- RNA aptamers (20F-RNA) conjugate designed to deliver a small ful preclinical studies have failed to show efficacy in clinical trials activating RNA (saRNA) for upregulating C/EBPa, an inhibitor of (167, 168). In this section, we describe several recent nucleic acid p21 (181). Downregulation of C/EBPa occurs from loss of KDM6B, treatments in PDAC (Fig. 1). leading to increased metastatic drive in pancreatic cancer. C/EBPa- Delivery of silencing RNAs (siRNA) that target oncogenes offers a based aptamers are highly specific to cancer cell lines, demonstrating therapeutic potential to abrogate tumor progression. siRNAs rapidly good internalization and retention in Panc1, AsPC-1, MIA PaCa2, and degrade before reaching the tumor tissue, therefore, a protective carrier Capan-1 cell lines. Restoration of C/EBPa in Panc-1 cell lines

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correlates with decreased cell proliferation. Treatment of subcutane- cause unacceptable toxicity. Developing successful targeted therapies ous models of Panc1 with C/EBPa-aptamer affords a modest decrease for PDAC is complicated by tumor heterogeneity, biophysical limita- in tumor size, without significant toxicity, confirming target specificity tions in the tumor microenvironment, and redundancy of pro- and relevance of C/EBPa in PDAC progression. Expanding on this oncogenic pathways. Furthermore, while cancer immunotherapy has concept, researchers are investigating more potent chemotherapeutics, been a boon to many patients with cancer, pancreatic cancer stands out typically reserved for antibody–drug conjugates, for the development as one of the few human cancers that does not respond to the current of aptamer–drug conjugates. For example, an auristatin-bound EGFR- immunotherapy protocols. The failure in implementing therapies used targeting aptamer is cytotoxic to Panc1, MIA PaCa2, and BxPC-3 cell to treat other cancers underscores these unique challenges in PDAC. lines, offering the potential to create highly specific antitumor agents To address these issues, advances in identifying novel targets and with potency exceeding conventional chemotherapeutics, with a improving the implementation and delivery of therapeutics specifically greater therapeutic window (182). in PDAC are being employed. These approaches address specific Identification of more selective aptamers along with target valida- patterns and pathways unique to PDAC with the goal of preventing tion is an active area of research. For example, cell-SELEX screening of cancer cells from escaping therapy and host immunosurveillance, as human pancreatic cancer (HPAC)-derived spheroids to target poten- well as avoiding the development of chemoresistance. Through specific tial cancer stem cell (CSC)-initiating or CSC-related genes yielded targeting of PDAC cancer cells and known biochemical processes, seven aptamers exhibiting high affinity binding, and target CD133, advanced disease can be treated and its progression stopped or CD244, IHH, NANOG, and ALDH1-expressing cells (183). Excitingly, significantly delayed. these aptamers bind to circulating tumor cells derived from patients. Several discussed agents augment conventional chemotherapeutics Further validation of these targets is necessary, but may provide an and inhibit pathways that confer resistance. Novel methods of drug additional method for aptameric drug delivery. delivery are affording both safer and targeted delivery of conventional Advances in nucleic acid delivery have progressed significantly since chemotherapeutics, with the goals of improving their therapeutic their inception. With the development of more stable carriers and window, providing specific delivery to the tumor, or avoiding the application of aptamers as drug delivery vectors, nucleic acid therapy systemic toxicity responsible for limiting doses in clinic. In concert shows exciting progress in PDAC therapy. However, relative to other with other treatment modalities, improved and multimodal targeting therapies discussed here, nucleic acid therapies are not widely used in will prevent drug evasion and tumor progression characteristic of late- the clinic. It is unknown how stable these therapies will be in clinical stage PDAC. Continuing advancements in tumor-omics, as well as practice or how well tolerated, although the recent approvals of nucleic development of novel delivery and imaging systems, as described acid therapies provide impetus for continued discovery, research, and herein, must translate to better patient outcomes in the coming decade. translation. 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 Conclusion outcomes relative to other cancers outline a unique clinical challenge, Outcomes in PDAC are limited by the dearth of available, successful one that will require a multidisciplinary approach and relies on treatment options. Nonspecific early symptoms, tumor aggressiveness, innovative, evidence-based treatments. and diagnosis of late-stage disease result in clinical outcomes that are significantly worse than those of other cancers. For early-stage disease, Disclosure of Potential Conflicts of Interest surgical resection with curative intent is still the preferred option, but Y. Colson has ownership interest (including patents) in Patent US 7,671,095 the utilization of new imaging modalities may be able to improve B2/US 8,334,324 B2/US 8,338,492 B2 (available for license) and Patent US 8,795,707 surgical outcomes through improved detection of microscopic disease B2 (available for license). M.W. Grinstaff has ownership interest (including patents) and delineation of tumor margins to assure complete resection. FDA- in patent application by BU (available for licensing). No potential conflicts of interest approved surgical cameras with NIR detection already enhance visu- were disclosed by the other authors. alization of the tumor beyond conventional surgical approaches. Acknowledgments Coupled with new developments in systemic tumor detection, visual This work was supported, in part, by NIH (F30 CA220843 to C. Gromisch; R01 contrast agents, and combined theranostic materials, the cure rates in CA227433 and R01 CA232056 to M.W. Grinstaff and Y. Colson; CA133085 to fi speci c patient subsets will likely be improved. K. Liu), Veterans Administration (CX001364 to K. Liu), and Boston University. Current treatment modalities in PDAC are disappointing. To date, there is no targeted therapy that shows superior outcomes compared Received September 7, 2019; revised February 8, 2020; accepted March 24, 2020; with current systemic chemotherapy even though these regimens can published first March 27, 2020.

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3192 Cancer Res; 80(16) August 15, 2020 CANCER RESEARCH

Pancreatic Adenocarcinoma: Unconventional Approaches for an Unconventional Disease

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

Cancer Res 2020;80:3179-3192. Published OnlineFirst March 27, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-2731

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