Extracellular vesicles as mediators of the progression and chemoresistance of pancreatic cancer and their potential clinical applications

Pancreatic ductal adenocarcinoma (PDAC, hereafter referred to as pancreatic cancer [PC]) is the seventh most common malignancy, ranking as the fourth and sixth leading causes of cancer-related death in the USA and China, respectively. Patients with PC have a 5-year survival rate of approximately 6% and a median survival rate of 6 months [1‐3]. PC is expected to become the second leading cause of cancer-related death in the USA by 2030 [4]. The dismal prognosis of PC is mainly attributed to poor detection rates at early stages, rapid progression and disappointing surgical resection outcomes. Most patients with PC lack diagnostic symptoms during early stages, and the existing screening biomarkers, such as CA19–9, are not sufficient for a highly sensitive and specific diagnosis of PC [5]. When a patient is diagnosed with localized PC, surgical resection is regarded as the only potential curative treatment [6]. However, patients should expect only a 5-year survival rate of 23–26% before recurrence [7]. Unfortunately, approximately 80% of patients have reached an unresectable stage at the time of diagnosis. Chemotherapy with gemcitabine is an indispensable treatment for these patients. However, the effectiveness of chemotherapeutic drugs is often plagued by chemoresistance, worsening the outcomes of patients with metastatic disease [8, 9]. Thus, a better understanding of the underlying cellular and molecular mechanisms of PC progression and chemoresistance is urgently needed.

Extracellular vesicles (EVs), which were considered containers of cellular debris, have recently been highlighted as intercellular communication tools and mechanisms of molecular transfer [10‐12]. EVs include exosomes, ectosomes, microvesicles, apoptotic bodies and oncosomes, according to their sizes and biogenesis mechanisms [13]. Many types of cells have the capacity to secrete EVs, including dendritic cells (DCs) [14], B and T cells [15], neurons [16], fibroblasts [17], stem cells [18], and cancer cells [19]. EVs are also detected in and isolated from multiple biological fluids, such as blood, urine, ascitic fluid, saliva and supernatants [20, 21]. EV-mediated intercellular communication is achieved by biologically active substances, such as proteins and nucleic acids, carried by EVs. Similarly, the interactions between cells and the tumor microenvironment are also partially mediated by EVs [22]. EVs have been shown to play an important role in the tumorigenesis, progression, metastasis and chemoresistance of various malignancies, such as breast cancer [23], hepatocellular carcinoma [24] and prostate cancer [25]. The functions of EVs in PC have also been investigated [26]. In this review, we will discuss the roles of EVs in the progression, metastasis and chemoresistance of PC as well as their promising applications as biomarkers and therapeutic targets in PC.

The histology of PC is characterized by a complex microenvironment consisting of PC cells and other components including vascular endothelial cells, immune cells, fibroblasts, myofibroblasts, stellate cells and extracellular matrix(ECM). Cancer-associated fibroblasts (CAFs) promote remodeling of ECM and tumor growth. Immune cells in the microenvironment of PC have a highly immunosuppressive composition and further contribute to immune evasion [45]. The interactions between cancer cells and the tumor microenvironment are crucial steps in tumor progression [46‐48], contributing to the altered metabolism and hyper-proliferation of cancer cells as well as tumor metastasis and abnormal tumor-associated immunity [46, 49, 50]. The role of EVs in PC is illustrated by these interactions across this part because these interactions are partially modulated by EVs. For example, EVs from fibroblasts promote invasive behavior and upregulate drug resistance pathways in cancer cells, whereas EVs from tumor cells reprogram normal fibroblasts into CAFs [51‐53]. Here, we describe the following functions of EVs in PC: (1) regulating the proliferation of PC cells, (2) promoting PC invasion and metastasis, and (3) modulating tumor-associated immunity. Figure 1 summarizes the functions of EVs in PC.

Currently, chemotherapy is an indispensable treatment option for patients with advanced pancreatic cancer. Several clinical trials have shown that adjuvant chemotherapy based on fluorouracil and gemcitabine (GEM) improved survival in patients with resected PC [74, 75]. A randomized phase III trial has proven that the non-inferiority of oral S-1, a fluoropyrimidine derivative had a higher 2-year overall survival than GEM in adjuvant chemotherapy [76]. For metastatic PC, chemotherapy includes GEM monotherapy or GEM plus novel regimens, such as erlotinib [77] and nanoparticle albumin-bound paclitaxel [78]. Besides, the FOLFIRINOX regimen which consists of oxaliplatin, folinic acid (leucovorin), irinotecan, bolus fluorouracil, and infusional fluorouracil has better chemotherapy response than GEM monotherapy in metastatic PC [79]. Unfortunately, however, chemoresistance occurs in most cases after long-term exposure to chemotherapeutics, particularly GEM, the standard chemotherapeutic agent for unresectable pancreatic cancer. Based on accumulating evidence, EVs may play a role as intercellular communicators in promoting chemoresistance in multiple cancers, including leukemia [80], glioblastoma [81], lung cancer [82], gastric cancer [83], breast cancer [84], prostate cancer [25], ovarian cancer [85] and PC [9]. The mechanisms underlying the EV-mediated chemoresistance in cancers include transferring the drug-resistance-related gene multidrug resistant-1 (MDR-1), P glycoprotein [86], survivin [87] and ubiquitin carboxyl terminal hydrolase-L1 [84] to recipient cells.

The effects of EVs on PC chemoresistance partially rely on the RNA it transfers. When incubated with GEM, PC cells upregulated the expression of miR-155, which was transferred to other PC cells via exosomes. MiR-155 contributed to resistance among PC cells via anti-apoptosis pathways and suppression of deoxycytidine kinase (dCK), a key gemcitabine-metabolizing enzyme [9, 88]. Furthermore, miR-155 overexpression upregulated the synthesis and secretion of exosomes and miR-155 contents in exosomes [9], which formed a positive loop in regulating GEM resistance. The positive role of EVs in transporting RNA among cancer cells to induce resistance has also been observed in breast and lung cancers [82, 89].

In addition, EVs conferred chemoresistance to PC cells by promoting ROS detoxification through increases in the expression of the ROS detoxifying genes superoxide dismutase 2 (SOD2) and catalase (CAT) [88]. Moreover, CAFs, which were intrinsically resistant, play an active role in GEM resistance by increasing EV release upon exposure to GEM, leading to the upregulation of chemoresistance-inducing factor Snail in recipient PC cells [54]. The expression of Snail and its target, miR-146a, were also upregulated in GEM-treated CAF-derived EVs [54], which may partially explained the increase in Snail expression in recipient cells. GEM increases Snail expression in PC cells [90]; therefore, CAF-derived EVs may increase Snail expression in recipient cells by transferring GEM or its metabolite to PC cells at a dose that is not sufficient to induce cytotoxicity but induces resistance. In the tumor microenvironment, miR-21 derived from macrophages and CAFs are also transferred to cancer cells via EVs, inducing chemoresistance by activating the phosphoinositol 3-kinase (PI3K)/AKT signaling pathway or binding apoptotic peptidase activating factor 1 (APAF1) [83, 85]. Taken together, EVs from PC cells or other cell types in the tumor microenvironment facilitate chemoresistance by regulating RNAs, proteins, relevant genes and signaling pathways, and extensive investigations are required to further explain EV-related chemoresistance in PC.

Due to the non-specific symptoms in early stages, most patients (approximately 80–85%) are diagnosed with metastatic or locally advanced PC at the initial examination [91, 92]. Patients who are diagnosed incidentally during medical examinations have a better prognosis than patients with evident symptoms, and patients with PC diameters smaller than 10 mm can expect a 5-year survival rate of 80.4% [93, 94]. Therefore, methods for the early detection of PC are urgently needed to improve the overall survival of patients with PC. Currently, the most widely used and the only FDA-approved biomarker for PC is CA19–9. However, CA19–9 is not optimal for PC screening because of its relatively low sensitivity and specificity (70–90% and 68–91%, respectively) [95]; it is therefore primarily used to monitor progression and the therapeutic response [94]. More importantly, 5%–10% of the population are Lewis antigen negative and unable to produce CA19–9 [94]. Hence, it is urgent to find new biomarkers. The ideal biomarkers for PC should detect the initial lesions at early stage with high sensitivity and specificity, differentiating PC with healthy ones and benign pancreatic diseases. Besides, they should be able to predict progression and prognosis, contributing to more effective therapy managements. Up to now, extensive investigations have been conducted to identify EVs as novel biomarkers for PC. Because EVs contain specific molecules of original cells, and display stability and abundance in various biological fluids [96], which may lead to a higher sensitivity and specificity in PC diagnosis. Compositions of EVs cover most cancer cell-associated biomarkers, including proteins, mRNAs, miRNAs, and DNA [97]. Proteins and miRNAs are the focus of EV biomarker research.

EVs used for biomarker detection are usually isolated from blood. Researchers applied RT-PCR to analyze the exosomal miRNAs extracted from the blood of 22 individuals with PC (9 at early stage and 13 at advanced stage) and 27 individuals without PC (6 with ampullary carcinoma, 7 with benign pancreatic tumors, 6 with chronic pancreatitis and 8 healthy participants) and assess the roles of four miRNAs (miR-17-5p, miR-21, miR-155 and miR-196a) as biomarkers of PC [98]. Levels of miR-17-5p and miR-21 were elevated in patients with PC, with sensitivity and specificity values of 72.7% and 92.6% and 95.5% and 81.5%, respectively. MiR-17-5p expression was elevated in metastasis and advanced PC, indicating that it was a potential biomarker for unresectable PC [98]. Similar results were reported in the investigations of miR-550 [99] and miR-10b [100], which displayed increased levels in exosomes isolated from the plasma of patients with PC and conditioned media from PC cell lines, suggesting that they may serve as early biomarkers in PC diagnosis.

In addition to miRNAs, EV proteins play a significant role in PC diagnosis. MIF was expressed at high level in plasma exosomes isolated from PC mouse models with liver metastasis compared with healthy ones. Besides, the increased MIF level was also present in mice with PanIN lesions, suggesting that it might serve as a biomarker for early diagnosis and predicting liver metastasis [3]. In 2015, Nature published an article presenting a near-perfect diagnostic biomarker for PC – glypican-1 (GPC1) – a cell surface proteoglycan that is specifically enriched on cancer cell-derived exosomes [101]. GPC1⁺ circulating exosomes were significantly elevated in patients with PC (including carcinoma-in-situ, stage I–IV) compared to healthy individuals, indicating that it may act as an biomarker for all stages of PC and aid in distinguishing PC from benign pancreatic disease and healthy individuals, while CA 19–9 levels in the serum fail to distinguish patients with PC from those with benign pancreatic disease. The sensitivity and specificity of GPC1+ circulating exosomes in diagnosing PC were both 100% [101]. Furthermore, the GPC1⁺ exosome level reflected the tumor burden and distant metastasis, and a reduction in the number of GPC1⁺ exosomes was related to increased survival [101]. Interestingly, however, another study indicated that high levels of exosomal miR-10b, miR-21, miR-30c, and miR-181a and low levels of miR-let7a differentiated PC from normal control and chronic pancreatitis samples, while GPC1 level was not significantly different between normal, PC and chronic pancreatitis samples [102]. The difference may be explained by different sample volumes, or different antibodies for GPC1. Except for single EV biomarker, investigators utilized a combination of exosomal proteins and serum/exosomal miRNAs to diagnose PC [103]. In this study, the exosomal protein markers included CD44v6, TSPAN8, epithelial cell adhesion molecule (EpCAM), MET, and CD104, whereas miR-1246, miR-4644, miR-3976, and miR-4306 were selected as exosomal miRNA markers. Concomitant evaluation of these markers exhibited a sensitivity of 1.00 (confidence interval (CI) 0.95–1) and specificity of 0.80 (CI 0.67–0.90) for PC compared with all other groups and a sensitivity of 0.93 (CI 0.81–0.98) when non-PC-malignancies were excluded [103].

In addition to plasma/serum biomarkers, salivary biomarkers have shown potential in diagnosing PC, possibly because miRNA expression profiles in saliva are similar to those in serum [104]. In the study including 12 pancreatobiliary tract cancer patients and 13 healthy donors, salivary exosomal miR-1246 and miR-4644 exhibit great potential as pancreatobiliary tract cancer early biomarkers [105]. Besides, Salivary exosomal proteins are also utilized for the detection of PC.[107]Thus, tests of salivary EV-derived miRNAs and proteins may be a novel method for diagnosing PC.

The extraction of EV biomarkers from body fluids typically requires multiple ultracentrifugation steps, which are time consuming. Several investigators have proposed new technologies to simplify this process. A microfluidic-based platform to isolate circulating exosomes comprised an ExoChip with an antibody against CD63 to capture exosomes and a standard plate-reader for subsequent exosome quantification [107]. An ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed to quantify microRNA-10b levels, which are elevated in exosomes from the plasma of patients with PC [100]. In addition, another novel microfluidics-based approach was presented by researchers from the University of Notre Dame. This platform consisted of surface acoustic wave (SAW) exosome lysis and ion-exchange nanomembrane RNA sensing performed concurrently on two separate chips; this platform has the advantages of a shorter analysis time (1.5 h for the total analysis), smaller sample volumes (100 μL), and reduced sample loss [99]. Table 1 summarizes the current diagnostic biomarkers for PC.

Majority of these studies included both patients with benign pancreatic diseases and healthy individuals as controls to assess the role of EV biomarkers for PC diagnosis, and various EV biomarkers proved to indicate disease progression and predict prognosis. However, further studies on EVs biomarkers need to be conducted to assess response to therapy, such as chemotherapy and radiotherapy. Besides, several obstacles remain to be overcome before the clinical application of EVs as biomarkers: prolonged EV isolation procedures, unavailability of specific markers to separate tumor cell-derived EVs from normal cell-derived EVs and lack of sensitive system for large cohorts of clinical samples.

An increasing number of studies has aimed to apply EVs to PC therapy. The roles of EVs in PC treatment in these studies are divided into three categories: (1) tumor-associated immunity stimulators, (2) drug carriers and (3) therapeutic targets.

EVs have various roles in modulating PC progression, chemoresistance, diagnosis and treatment. The underlying mechanisms remain to be further explored, particularly the seemingly contradictory effects of EVs on tumor cell proliferation and tumor-associated immunity. Biomarkers related to exosomes prove to be promising in diagnosing PC and determining patient prognosis; further investigations are required to explore the potential clinical applications of EVs, which may usher in a new era for PC treatment.