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Emerging Evidence for the Clinical Relevance of Pancreatic Cancer Exosomes

Massoumi, Roxanne L., MD; Hines, O. Joe, MD; Eibl, Guido, MD; King, Jonathan C., MD

doi: 10.1097/MPA.0000000000001203
Reviews

The last 5 years have seen a dramatic increased interest in the field of exosome biology. Although much is unknown about the role of exosomes in human health and disease, disparate scientific disciplines are recognizing the highly conserved role that exosomes play in fundamental biological processes. Recently, there have been intriguing discoveries defining the role of exosomes in cancer biology. We performed a structured review of the English-language literature using the PubMed database searching for articles relating to exosomes and pancreatic ductal adenocarcinoma (PDAC). Articles were screened for relevance and content to judge for inclusion. Evidence implicates exosomes in the pathogenesis, local progression, metastasis, immune evasion, and intercellular communication of PDAC. Basic science discoveries in exosome biology have the potential to change the clinical management of PDAC, where, despite advances in early detection, diagnosis, staging, chemotherapy, and surgery, survival rates have been stagnant for decades and PDAC remains the most deadly human gastrointestinal malignancy.

From the Department of Surgery, David Geffen School of Medicine at the University of California Los Angeles, Los Angeles, CA.

Received for publication April 13, 2018; accepted October 23, 2018.

Address correspondence to: Jonathan C. King, MD, Department of Surgery, David Geffen School of Medicine at the University of California Los Angeles,1304 15th St, Suite 102, Santa Monica, CA 90404 (e-mail: joking@mednet.ucla.edu).

Funding was received from Hirshberg Foundation for Pancreatic Cancer Research Seed Grant.

The authors declare no conflict of interest.

Pancreatic ductal adenocarcinoma (PDAC) is nearly universally fatal despite even the most aggressive treatments. It is the fourth leading cancer killer in men and women in the United States and is projected to be the second leading cause of cancer death by 2030.1,2 Traditional methods of detection by cross-sectional imaging and treatment with surgery and/or cytotoxic chemotherapy are mostly ineffective; new methods for prevention, early detection, and treatment are urgently needed. Herein we present evidence for exosomes as diagnostic and therapeutic targets in PDAC.

Exosomes are lipid bilayer–bound nanovesicles produced by nearly every human cell type.3 While exosomes are often described in terms of their size (40–100 nm), presence of surface markers, and molecular contents, it is their endosomal origin that most rigorously defines them (Table 1).4 Pan and Johnstone5 first described exosomes following in vitro experiments performed on sheep reticulocytes where they observed budding of intracellular endosomes. The resulting multivesicular bodies (MVBs) fused with the plasma membrane to release exosomes extracellularly (Fig. 1). This biogenesis pathway distinguishes exosomes from other members of the extracellular vesicle family.6 Exosomes are assembled under control of the endosomal sorting complex required for transport (ESCRT).7 Careful research into the mechanics of ESCRT-dependent and ESCRT-independent models demonstrate exosome biosynthesis is tightly regulated and deliberate, not random. Furthermore, the molecular cargo packaged within exosomes appears to be similarly regulated: RNA, protein, and lipid contents of exosomes are typically distinct from the parent cells, supporting their origin from the endosomal pathway and distinct physiologic function(s).8–10 The final steps of exosome biosynthesis involve Rab GTPases, which regulate the transport of MVBs to the plasma membrane and soluble NSF-attachment protein receptors, which facilitate fusion of MVBs to the plasma membrane, thereby releasing exosomes.3 Endosomal sorting complex required for transport, Rab, and soluble NSF-attachment protein receptor machinery represent potential therapeutic targets to modulate exosome production and release.

TABLE 1

TABLE 1

FIGURE 1

FIGURE 1

Cancer cells have abnormal exosome physiology, giving rise to interest in the role of targeting exosomes in cancer therapy.11 When compared with nonmalignant tissues, malignant cells secrete greater quantities of exosomes, contain elevated quantities of miRNAs, and contain miRNAs associated with the RNA induced silencing complex, a necessary component for miRNA silencing of mRNAs in target cells.12–14 These findings imply a powerful mechanism for cancer cells to influence target cell behaviors both locally in the tumor microenvironment and in distant tissues. Recent findings have confirmed roles for exosomes in local invasion and migration, angiogenesis, metastatic niche formation, immune evasion, and therapeutic resistance.15–20

These mechanisms are not demonstrated in all malignancies, and there may be heterogeneity in the degree to which different tumors and histologic subtypes utilize the mechanisms listed above.21 However, it is becoming clear that exosomes and the biologic processes they control are potentially important in multiple human malignancies. This article is a review of the current literature relating to the various roles of exosomes in PDAC.

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EXOSOMES AND THE BIOLOGY OF PANCREATIC CANCER

Angiogenesis

Angiogenesis is a requisite for neoplastic growth and metastasis.22 Despite initially encouraging results in preclinical experiments, attempts to translate pharmacologic angiogenesis inhibitors into clinical use consistently failed to show benefit.23–25 The dense fibroblastic stroma of PDAC creates high intratumoral interstitial pressures and poor drug delivery. There are multiple and parallel molecular pathways governing angiogenesis in vivo leading to heterogeneous clinical responses seen in large clinical trials.26 Angiogenesis researchers in PDAC have begun looking beyond vascular endothelial growth factor (VEGF) for additional mediators of angiogenesis.

There is evidence that exosomes are contributors to tumor neoangiogenesis via protein mediators. Tspan8 is found in rat PDAC-derived exosomes and is a potent stimulator of VEGF-independent angiogenesis.27 Exosomes expressing Tspan8 bind endothelial cells through CD49d leading to stimulated expression of multiple angiogenesis-related genes, chemokines, and receptors including von Willebrand factor, Tspan8, CXCL5, macrophage migration inhibitory factor (MIF), and CCR1. Tspan8-positive exosomes applied to human endothelial cells in vitro induce angiogenic behaviors such as proliferation, tubule formation, and increased motility.27

Another peptide hormone, myoferlin, is a transmembrane protein involved in exocytosis of VEGF and is overexpressed in pancreatic tumors and associated with increased tumorigenesis and tumor vascularity. Depleting myoferlin expression in PDAC cells using shRNA silencing stimulates PDAC cells to produce smaller exosomes with altered protein content.28 In particular, regulators of vesicular transport RAB7A and CD63 are down-regulated, leading to functionally defective exosomes. Pancreatic ductal adenocarcinoma cells treated with myoferlin knockdown produce exosomes that are not internalized by human endothelial cells and fail to induce proliferation and migration, implying reduced angiogenic capacity.28

Exosome-mediated, VEGF-independent mechanisms of angiogenesis require further exploration in in vivo PDAC models as well as validation in human subjects. However, these findings highlight the potential for synergistic therapeutics combined with existing anti-VEGF therapies in the future.

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Local Invasion, Migration

A fundamental hallmark of malignancy is the ability of tumor cells to invade and migrate to lymphatic stations and through blood vessels, ultimately leading to widely disseminated disease, which is the fate of a vast majority of PDAC patients. Cancer-initiating cells (CICs) may initiate the migratory process, and identification of CIC cell markers may allow early detection of CICs as well as serve as a therapeutic target aimed at preventing tumor progression.29,30

The CIC marker CD44v6 is a splice variant of CD44 that is carried by exosomes and has been observed in multiple human malignancies, including PDAC.31 CD44v6 augments Wnt/β-catenin signaling and promotes cancer cell motility and invasion. CD44v6 knockdown in PDAC cells leads to a loss of local invasion and metastatic capacity via altered expression of proteases and associated factors such as plasminogen activator inhibitor 1 and tissue inhibitor of metalloproteases 1.31 Interestingly, the addition of PDAC exosomes expressing CD44v6 to knockdown cells reconstitutes their invasive capacity both in vitro and in vivo. Exosomes expressing CD44v6 also stimulates the expression of matrix metalloproteinases, plasminogen activator inhibitor 1, and tissue inhibitor of metalloproteases 1 that is inhibited by CD44v6 knockdown.31

These findings attribute fundamental invasive behaviors such as tumor cell motility, invasion, anchorage-independent growth, and apoptosis resistance to exosomes. Perhaps most intriguing is evidence that these are transferable qualities, implying a mechanism by which stepwise genetic mutations could generate tumor clones that drive invasiveness of the entire tumor. Therapeutic targeting of CD44v6 is underway in metastatic breast cancer, although no human trials in PDAC exist.

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Immune Evasion

Early in the process of tumorigenesis, neoplastic cells develop mechanisms to evade the immune system. These mechanisms are increasingly understood as the field of tumor immunology has expanded, and recent clinical advances in malignancies such as melanoma have demonstrated the effectiveness of therapeutic immune modulation. There are no immunotherapies for PDAC currently. Exosomes appear to have a role in immune evasion through multiple mechanisms including impaired lymphocyte activation, survival, and function. The most profound effects of PDAC exosomes are on dendritic cells (DCs), which represent a key intermediary in activating the adaptive immune system. Rat PDAC-derived exosomes (ASML exosomes) are internalized by DCs and macrophages, interfere with interleukin 2–stimulated lymphocyte activation, and induce lymphocyte apoptosis via inhibition of the AKT/PI3K pathway, which can be abrogated by stimulatory signals from DCs.32 miRNAs carried by exosomes may be the effector molecules responsible for DC inhibition: exosome miRNA profiling and genomic database analysis revealed miR-212-3p specifically down-regulates regulatory factor X–associated protein in DCs.33 Regulatory factor X–associated protein is a key transcription factor in the production of MHCII (major histocompatibility complex II), and down-regulation leads to decreased transcription/translation of MHCII itself, which directly impairs the ability of DCs to present antigens to T cells.33 Exosome-derived miR-203 also decreases Toll-like receptor 4 expression and decreases expression of cytokines such as tumor necrosis factor α and interleukin 12 from DCs.34 These effects lead to decreased DC function, which is reversed by exposure to exosomes depleted of miR-203.

Realizing the immunosuppressive properties of PDAC exosome miRNAs on DCs, Que et al35 manipulated PDAC-derived exosomes to activate DCs against PDAC cells as a therapeutic strategy to reverse tumor-associated immune tolerance. They depleted PDAC exosomes of their miRNAs while retaining protein components such as complement peptides. miRNA-depleted exosomes were effective DC activators with increased proliferation, tumor necrosis factor α production, and perforin secretion, which led to improved killing of PDAC cells in vitro, demonstrating exosomes have the potential to be useful immune-modulating tools in PDAC.35

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Metastasis

Metastases to the liver, peritoneum, and lungs are a nearly universal occurrence in PDAC and the most common cause of death. Metastases appear early in the course of disease through unknown mechanisms. For more than 100 years, Paget's “seed and soil” hypothesis has been the most plausible explanation for the nonrandom distribution of metastases.36 Originally postulated in 1889, theories explaining the molecular events of this phenomenon have only recently been elucidated. The premetastatic niche is the microenvironment found at future metastatic sites prior to the arrival of the cancer cells. Structural and molecular changes in the tissue allow tumor cell colonization and development of macroscopic metastases. Exosomes and their cargo are mediators of the premetastatic niche.37

Pancreatic ductal adenocarcinoma cells release exosomes containing MIF, a protein that is important in creating a liver premetastatic niche.18 The cascade of events starts with exosome uptake by Kupffer cells, inducing transforming growth factor β (TGF-β) expression, hepatic macrophage migration, stellate cell activation, and fibronectin deposition. These molecular and cellular changes constitute the liver premetastatic niche, and experimental pretreatment of tumor-naive mice with PDAC exosomes leads to improved efficiency of metastasis, which is abrogated by knockdown of MIF in exosomes. Human PDAC subjects with liver metastases have plasma exosomes with elevated levels of MIF compared with both healthy controls and patients who remain disease-free 5 years following diagnosis, supporting the clinical relevance of MIF-containing exosomes in PDAC.

Our laboratory has examined peritoneal tissue of human subjects with metastatic PDAC and found elevated TGF-β and increased fibronectin deposition compared with peritoneal tissues from subjects with nonmetastatic PDAC (Fig. 2A, unpublished data) supporting a peritoneal premetastatic niche similar to that found in the liver. In vitro, we demonstrated that human mesothelial cells internalize PDAC exosomes and subsequently proliferate and migrate—markers of mesothelial-mesenchymal transition and a potential peritoneal correlate of the premetastatic niche. Labeled exosomes injected into the peritoneum of mice also traffic to mesothelial cells in vivo (Fig. 2B, unpublished data). We continue work to elucidate the cellular and molecular components of the peritoneal premetastatic niche as well as strategies to interrupt its development to prevent metastasis.

FIGURE 2

FIGURE 2

Another observation that has defied scientific explanation has been the preference of tumors for certain metastatic sites. Recent work attributes the tissue specificity of metastases to exosomes. Specifically, integrin expression on the exosome surface directs the uptake of exosomes to future metastatic sites.38 Pancreatic ductal adenocarcinoma exosomes express the ITGαvβ5 heterodimer, which confers specificity to liver macrophages. In contrast, lung- and brain-trophic exosomes from sarcoma and melanoma cell lines express exosomes with distinct integrin isoforms that bind to type II pneumocytes (lung) and endothelial cells (brain), thereby directing metastatic tumor cells to these tissues preferentially. This is compelling evidence for exosome-mediated metastatic trophism as the mechanism behind Paget's observations and represents another potential target of therapy for preventing PDAC metastases.

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Metabolism

Cancer-associated fibroblasts (CAFs) are prominent components of PDAC tumors, and stromal cells can make up the majority of the tumor mass.39,40 Cross-talk between PDAC cells and CAFs has been a topic of investigation, and some chemotherapeutic strategies to target stromal cells have been tested with mixed results.41–43 Recent work demonstrates CAF exosomes can reprogram PDAC cells to increase mitochondrial oxidative phosphorylation, leading to increased glycolysis and glutamine-dependent reductive carboxylation.44 Cancer-associated fibroblast exosomes can also directly supply amino acids, lipids, and TCA intermediates to nutrient-deprived PDAC cells, allowing them to thrive in nutrient-poor and stressful conditions.44

Additional evidence of cross-talk between peripancreatic adipose tissue and pancreatic ductal cells is emerging. Our group has studied the role of adipose tissue inflammation in PDAC tumorigenesis and found evidence that humoral factors derived from the mesenteric adipose tissue accelerate the development of precancerous pancreatic intraepithelial neoplasia lesions and PDAC in the conditional Kras G12D model of mouse PDAC. Adipose tissue–derived exosomes are a potential source of these signals and are a direction of ongoing investigation.45,46

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Systemic Metabolic Effects: Cancer Cachexia, Cancer-Induced Diabetes (Type IIIc)

Most cancers promote a systemic hypermetabolic state resulting in cachexia, but type IIIc diabetes is a paraneoplastic syndrome unique to PDAC, which is distinguished by its presentation in association with pancreatic exocrine insufficiency.47,48 Emerging evidence suggests that the development of cachexia and diabetes in PDAC is related to exosome signaling.

Adrenomedullin (AM) is a potential mediator of paraneoplastic diabetes in PDAC patients, although initial reports lacked a mechanism by which AM could affect β cells.49 Subsequent investigations linked AM-containing exosomes to diabetogenic effects both in vitro and in vivo. Additional investigations establish that exosomes containing AM induce lipolysis in adipocytes, potentially explaining the cancer cachexia syndrome seen in PDAC patients.49 Mediating the lipolysis effect of AM exosomes is a signal transduction cascade involving p38 and ERK-1/2, which is abrogated by blockade of the AM receptor.50 There have been no studies to date investigating whether inhibition of exosome production or uptake produces similar results to AM receptor blockade, although this is theoretically plausible.

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EXOSOMES AS BIOMARKERS IN PANCREATIC CANCER

Early Detection

Despite a potential latency period in the progression of preinvasive lesions to invasive cancer, there are no effective screening tests for PDAC. Most patients are diagnosed after locally advanced or metastatic disease is already established, and there is no chance for cure. A noninvasive screening modality to detect early invasive PDAC is a major unmet need.

Exosomes transport intracellular biomolecules throughout the body in a stable configuration, whereas free molecules are rapidly degraded in the extracellular space. As a result, exosomes are a window into the unique molecular machinery of cancer cells and carry a multitude of potential biomarkers for screening and early detection. Whole-genome sequencing (WGS) of exosome DNA by a so-called liquid biopsy is one such approach.51–56 Serum and pleural fluid exosomes contain mutant KRAS and TP53 as well as amplified ERBB2 and MYC. A study utilizing WGS of exosome DNA found mutant KRAS in 7.4% of controls, 66.7% of localized, 80% of locally advanced, and 85% of metastatic PDAC subjects.52 Whole-genome sequencing performance is markedly improved when applied to exosome DNA as opposed to cell-free DNA. However, the validation cohort showed more than 20% of healthy controls harbored mutant KRAS, limiting its utility for diagnostic purposes.52 Another similar study detected KRAS G12D mutation in 39.6% of PDAC patients, 28.6% of IPMN patients, and 55.6% of chronic pancreatitis patients as compared with 2.6% of healthy volunteers.51

As stated above, exosomes exist in essentially all bodily fluids, making noninvasive sampling cheap, easy, and essentially risk-free. This is a particularly attractive prospect for screening PDAC because imaging and biopsy are typically expensive and invasive. Salivary exosomes isolated from mice with orthotopic xenograft PDAC tumors harbor mRNA transcripts with diagnostic potential.53 In proof-of-concept studies, murine PDAC cells with impaired exosome expression through a dominant negative RAB11 construct have reduced expression of candidate PDAC biomarkers and diminished exosome mRNAs in the saliva. These findings illustrate the feasibility of assaying exosome mRNA from the saliva, although confirmation in a human study is needed.53

Exosome proteins can also function as PDAC biomarkers. Pancreatic ductal adenocarcinoma CIC markers CD44v6, Tspan8, and EpCAM, as well as CD104 and GPC-1, are potential candidates.53,56 GPC-1 is a proteoglycan on the surface of PDAC-derived exosomes, and remarkably, GPC-1+ exosomes identified subjects with PDAC with 100% sensitivity and specificity in mice and humans. GPC-1 also distinguished healthy patients from those with benign diseases and early- or late-stage PDAC.56 Although promising, this last study awaits confirmation in a larger cohort of human patients, although it illustrates the potential of exosome-derived biomarkers for screening and early detection of PDAC.

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Prognosis

The best available PDAC prognostic marker is serum carbohydrate antigen 19-9, which is useless in many individuals because of lack of expression of the sialyl Lewis-A antigen. Monitoring effectiveness of therapy is likewise difficult using computed tomography or magnetic resonance imaging, given the inability to differentiate subtle differences in tumor size or discriminate between live tumor, inflammation, desmoplastic tissue, and necrotic tumor.

As mentioned previously, WGS of exosome-derived DNA identified mutant KRAS in the serum of PDAC subjects but is limited by poor specificity. However, as a prognostic biomarker, the mutational load of KRAS in exosome-derived DNA is strongly correlated with disease-free survival.52 The average size of exosomes and overall concentration in peripheral blood also correlates to PDAC disease extent and underscores the concept of disordered exosome biosynthesis in PDAC.

Finally, miR-196a expression is correlated to pathologic stage and survival, making it a potential prognostic biomarker. miR-196a is elevated in a higher proportion of stages III and IV PDAC patients, as compared with stages I and II, and increased levels of miR-196a in exosomes of PDAC patients are associated with survival of roughly one-half that of individuals with lower levels (6 vs 12 months).57 It is not known whether these prognostic biomarkers have utility in determining the efficacy of therapy.

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EXOSOMES AND PANCREATIC CANCER CHEMOTHERAPY

Chemotherapeutic Resistance

Despite decades of research, there has not been much improvement in systemic chemotherapy options for PDAC—particularly in the adjuvant setting. Until recently, standard of care has been gemcitabine or 5-fluorouracil single-agent chemotherapy, with multiagent regimens only recently coming to the forefront.58–60 Multiagent regimens for locally advanced and metastatic disease have improved efficacy but are associated with substantial toxicity.44,61 Molecular targeted therapies have been largely ineffective.

One theory explaining the source of chemotherapeutic resistance in PDAC implicates the dense fibrous stroma found in most tumors where fibroblasts often outnumber epithelial cells. The tumor-stroma interaction may depend on exosomes to allow cross-talk between epithelial and mesodermal components of PDAC tumors and contribute to chemotherapeutic resistance. Cancer-associated fibroblasts protect PDAC cells from cytotoxic chemotherapy and harbor an intrinsic resistance to the cytotoxic effects of gemcitabine.62 Cancer-associated fibroblasts respond to gemcitabine treatment with increased production of exosomes containing miR-146a, which stimulates expression of the gemcitabine resistance protein Snail, thus conferring resistance to PDAC cells.62 Cancer-associated fibroblasts treated with an inhibitor of exosome release along with gemcitabine are unable to induce gemcitabine resistance in PDAC cells resulting in reduced PDAC cell survival in vitro.62

Similarly, gemcitabine-treated PDAC cells produce exosomes with elevated SOD2 and CAT mRNAs (both detoxify reactive oxidative species that protects cells from oxidative cell death).63 Exosomes also contain miR-155, which suppresses expression of an enzyme required to create the active metabolites of gemcitabine—deoxycytidine kinase. Suppressing miR-155 expression in exosomes reverses transferrable gemcitabine resistance in PDAC cells in vitro.63 Furthermore, miR-155 expression in pretreatment human PDAC patient samples correlates with gemcitabine resistance and early disease progression following R0 resection, establishing the clinical relevance of this mechanism.64

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Exosomes as Therapeutics

For all the evidence of roles exosomes fill to make PDAC more aggressive and treatment resistant, there are now efforts to manipulate exosomes for clinical advantage. Pancreatic ductal adenocarcinoma–derived exosomes induce caspase-mediated apoptosis in tumor cells that spares nonneoplastic cells.65,66 The lipid composition of PDAC-derived exosomes is distinct from that of microvesicles and the plasma membrane of parent cells, which facilitates exosome binding and downstream depression of Notch-1 signaling and apoptosis. Synthetic exosome-like nanoparticles (SELNs) with similar lipid composition to PDAC exosomes demonstrated similar effects on Notch-1 signaling and apoptosis in vitro.67 However, in vivo, SELNs are rapidly cleared from the circulation by blood monocytes, limiting their potential utility as therapeutics.68

More recently, exosomes are proving useful as a delivery vehicle for inhibitory siRNA against mutant KRAS.69 The ubiquity of KRAS mutation and its cancer growth–promoting activities in PDAC are well established, but the ability to target KRAS therapeutically remains elusive. Exosomes deliver anti-KRAS siRNA specifically to cancer cells and silence KRAS gene expression, leading to inhibition of tumor growth in vivo and improved survival of experimental mice. Importantly, exosomes persist in the circulation secondary to expression of CD47, which interacts with signal regulatory protein α on blood monocytes to confer a “do not eat me” signal.69 This proved to be a key finding explaining the failure of SELNs to inhibit tumor growth in vivo. The potential for exosomes and exosome-like manufactured nanoparticles as therapeutics is an exciting prospect that appears within reach.

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CONCLUSIONS

Continued developments in exosome research underscore a recurring conclusion: exosomes occupy a central role in the function of diverse biological systems (Fig. 3). The highly conserved, tightly regulated mechanisms by which exosomes are produced and elaborated and the ways exosomes exert their influences are a testament to their importance in both health and disease. Consequently, it is not surprising that for a disease such as PDAC exosomes appear to be involved in many seemingly divergent processes.

FIGURE 3

FIGURE 3

Ultimately, exosomes may represent an ideal system to manipulate in our continued effort to find more effective therapies for PDAC. There are numerous potential opportunities ranging from inhibiting exosome biosynthesis and release by PDAC cells, to alteration of exosome contents and blockade of uptake by target cells. Exosomes may also provide opportunities for advances in PDAC care, ranging from early detection and therapeutic monitoring to targeted drug delivery and biological therapies.

Continued work is needed to further refine and elucidate details of the largely theoretic constructs outlined above. As our understanding improves, we must then work to exploit the therapeutic opportunities made possible by these findings and extend them to clinically relevant treatments in PDAC patients. Although there is much work to be done to achieve these lofty goals, the ever-expanding body of exosome research has the potential to revolutionize the field.

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Keywords:

adenocarcinoma; diagnosis; exosome; metastasis; pancreas; treatment

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