The potential roles of exosomes in pancreatic cancer initiation and metastasis

PaCa-derived exosomes (PaCaExos) contain various protein molecules that can activate surrounding stromal cells and induce extracellular matrix (ECM) remodeling and neovascularization, thus establishing a TME to facilitate metastasis. In vivo studies using PaCa animal models have demonstrated that PaCaExos are rich in Tspan8, CD106, and CD49d [118]. Upon uptake by rat aortic epithelial cells (ECs), those PaCaExos activate the intracellular expression of VWF (von Willebrand factor), TSPAN8, CXCL5, MIF (migration inhibitory factor), CCR1, VEGF and VEGFR2, which lead to neovascularization by inducing EC proliferation, migration, sprouting, and progenitor maturation. Notably, Tspan8-enriched exosomes produced by PaCa cells can induce VEGF-independent angiogenesis around tumor tissues [118]. Costa Silva and colleagues have pointed out that PaCa can utilize exosomes to establish a pre-metastatic niche in distal organs, such as liver or lungs [130]. In this case, after exosomes derived from mouse PaCa cells were injected into healthy mice, they could be found in the liver [130]. Mechanistic analysis have shown that MIF-positive exosomes derived from PaCa cells can promote liver metastasis by increasing TGF-β expression in Kupffer cells (KCs) and also activating hepatic stellate cells (HSCs) to secret fibronectin [130]. Compared with healthy subjects or individuals with 5-year progression-free PaCa, PaCa patients with liver metastases usually exhibit elevated exosomal MIF levels in the serum [116]. Therefore, exosomal MIF may prominently function in the formation of the liver pre-metastatic niche. Additional evidence has demonstrated that PaCaExos that are positive for integrin αvβ5 usually reach the liver, whereas integrin α6β4- and α6β1-containing exosomes are transported to the lungs [116]. A recent study has demonstrated that protein kinase D1 (PRKD-1) expression is significantly downregulated in PaCa tissues when compared to non-tumor tissues [131]. Particularly, PRKD-1 knockout can induce PaCa cells (Panc-1) to produce more exosomes. Moreover, PaCa xenograft mouse experiments have confirmed that PRKD-1 knockout can increase the content of exosomes in the serum, thus promoting PaCa invasion. Mechanistic analysis has showed that alteration on PRKD-1 may stimulate PaCa cells to produce more integrin α6β4 positive exosomes to promote PaCa lung metastasis [131]. Furthermore, Li and colleagues have figured out that the formation of a pre-metastatic niche also requires the generation of new blood vessels [128]. Upon uptake by human umbilical vein endothelial cells (HUVECs), PaCaExos can activate Akt and ERK1/2 signaling pathways. This pathway activation promotes tube formation, by increasing Ras homolog gene family member A (RhoA) activity, as well as cytoskeleton remodeling, which drive a cell shrinkage due to the decreased expression of tight junction ligand protein Zonula occludens-1 (ZO-1), and also induce endothelial barrier dysfunction by enhancing local hyperpermeability [128]. In another study, Satake and colleagues have injected double fluorescence-labeled Mia-PaCa-2 cells into the spleen of nude mice and then demonstrated that PaCaExos reach the liver where they are uptaken by KCs, but also appear in the bone marrow and lung [132].

It has been shown that the knockout of CD151 or TSPAN8 expression (CD151^−/− or TSPAN8^−/−, respectively) results in impaired metastasis of PaCa cells [111]. Remarkably, the re-introduction of regular PaCaExos into CD151^−/− or TSPAN8^−/− cells can restore metastasis. Functional analysis has shown that CD151- and Tspan8-postive exosomes are able to (i) activate the expression of EMT-related genes in PaCa cells, (ii) induce ECM remodeling by activating stromal cells, and (iii) up-regulate the expression of pro-inflammatory factors in hematopoietic cells [111]. Furthermore, lymphangiogenesis is impaired and the hypersensitivity reaction is delayed in TSPAN8^−/− mice, while angiogenesis is severely impaired in both CD151^−/− or TSPAN8^−/− mice. Still, metastasis of PaCa cells transplanted into either TSPAN8^−/− or TSPAN8^−/−/CD151^−/− mice is effectively inhibited, suggesting that host Tspan8 or CD151 can significantly affect tumor progression [112]. Totally, PaCa exosomal CD151 and Tspan8 may promote matrix degradation and reprogramming of the stroma and hematopoietic cells, which are essential steps for PaCa metastasis. CD44 variant isoform 6 (CD44v6) is highly expressed in PaCa cells and can be integrated into exosomes [133]. Upon uptake of PaCa-derived CD44v6-positive exosomes by other PaCa cells, they activate Wnt/β-Catenin signaling and up-regulate the expression of plasminogen activator inhibitor 1 (PAI-1), MMP, and tissue inhibitor of metalloproteases 1 (TIM-1), thus enhancing PaCa cell migration and metastatsis [110]. Since CD44v6 can promote TSPAN8 expression at the transcriptional level, CD44v6 gene silencing effectively attenuates Tspan8-induced PaCa cell metastasis [134, 135]. Similarly, Jung and colleagues have observed that CD44v6 gene knockout (CD44v6^−/−) severely impairs PaCa cell metastasis. Co-treatment of CD44v6^−/− cells with soluble matrix (SM), produced by regular PaCa cells and PaCa-derived CD44v6-positive exosomes, can effectively restore the metastatic pattern of these cells, suggesting that PaCa may form a (pre-)metastatic niche microenvironment in distal metastasized organs by synergized effects derived of produced exosomes and other factors [136].

Myoferlin (MYOF) plays a crucial role in cell migration and invasion, as well as cell membrane endocytosis and vesicle transportation [137, 138]. It has been reported that MYOF can promote the migration and invasion of PaCa cells by regulating the mitochondrial structure and energy production [139, 140]. In PaCa cells, MYOF mediates the inclusion of VEGF into exosomes to promote tumor growth and angiogenesis. Accordingly, knockdown of MYOF expression can largely inhibit the growth and proliferation of PaCa cells [119]. Inhibition of MYOF function is also capable of reducing the volume of exosomes produced by PaCa cells as well as decreasing the levels of exosomal Rab7a and CD63. Although these exosomes with smaller volume are uptaken by human ECs, they fail to promote EC proliferation and migration, which eventually leads to inhibition of angiogenesis [118]. LIN28 is a 25-kDa RNA-binding protein that has been shown to promote PaCa growth and metastasis by inhibiting the biogenesis of a group of microRNAs, including let-7. The NAD(+)-dependent histone deacetylase sirtuin 6 (SIRT6) is able to induce PaCa growth inhibition by reducing LIN28 in PaCa cells [141]. Liver metastasis studies using PaCa tumor-bearing mice have demonstrated that LIN28B-positive exosomes produced by PaCa cells may reach target cells and activate the LIN28B/let-7/HMGA2/PDGFB signaling axis to further promote PaCa metastasis after injection via caudal vein [115].

Claudin 7 (Cld7) is a key structural protein present in tight junctions that interconnect cells [142]. It has also been shown that Cld7 can be distributed beyond TJ sites. For instance, palmitoylated Cld7 (Palm-Cld7) is localized in glycolipid-enriched membrane microdomains (GEMM) [113]. Cld7 in tight junction (TJ-Cld7) is shown to regulate the entry of related proteins into PaCaExos and affect the function of exosomes derived from CICs (CIC-Exos) by modulating the composition of exosomal transporters and lipid metabolites, while Palm-Cld7-positive exosomes have the capability of regulating cell migration [113]. Importantly, Kyuno and colleagues have found that murine pancreatic cancer initiating cells (PaCICs) can produce Cld7-positive exosomes which are capable of inducing re-programming of non-metastatic cancer cells to further increase their invasiveness [113]. Another PaCa-derived Wnt5β-positive exosomes have been reported to enter and activate the Wnt5β signaling in other cancer cells lines such as PaCa, A549 and Caco-2, where they stimulate migration and proliferation. Wnt5β knockout and TSG101 silencing can both abrogate the exosomal Wnt5β-dependent PaCa cell proliferation and migration [143]. Under normal physiological conditions, plectin is usually localized in the cytoplasm where it functions as a scaffolding protein. Plectin is expressed in PaCa, but usually undetectable in non-PaCa tissues [144]. In PaCa cells, integrin β4 mediates the transfer of overexpressed plectin into exosomes, eventually leading to the proliferation, migration, and invasion of these cells [117].

Zinc transporter ZIP4-positive exosomes, produced by highly metastatic PaCa cells, can stimulate the proliferation, migration, and invasion of non-metastatic PaCa cells [145]. Accordingly, exosomal ZIP4 from the serum of PaCa patients can be used as a diagnostic marker for cancer progression [145]. Compared with exosomes derived from human pancreatic ductal epithelial cells (HPDE), exposure of non-tumorigenic cells to PaCaExos potentially induces transformation as well as tumorigenesis in vivo of non-malignant cells [120]. Functional analysis have indicated that PaCaExos are capable of inducing random gene mutations in recipient cells, while only certain cell populations with PaCaExo-induced mutations can undergo transformation and, eventually, become tumors. Considering the stochastic nature of mutations, the mechanism of PaCaExo-induced tumorigenesis in transformed cells may differ from each other [120]. Specifically, it has been reported that mutated DNA segments from KRAS, CDKN2A, P53, and SMAD4 can be internalized into PaCaExos. Thus, these exosomes may effectively promote the transformation of normal cells as well as subsequent tumor formation [146].

It has been shown that miR-27a is overexpressed in cancer tissues from PaCa patients as well as PaCa cell lines [147]. PaCa-derived exosomes containing miR-27a can induce proliferation, invasion and angiogenesis in human microvascular endothelial cells (HMVECs) by suppressing B-cell translocation gene 2 (BTG2), which promotes PaCa cell survival and growth [121]. In contrast, in vivo studies using PaCa animal models have demonstrated miR-339-5p can inhibit cell invasion and migration by down-regulating the expression the zinc finger protein ZNF689. MiR-339-5p levels are significantly reduced in exosomes from highly metastatic PaCa cells. Accordingly, the exogenous introduction of miR-339-5p can effectively inhibit PaCa migration and invasion [124]. MiR-222 is overexpressed in highly invasive PaCa cells, where it is assimilated into exosomes. Upon uptake by poorly invasive PaCa cells, exosomal miR-222 is then released to further decrease the expression, phosphorylation, and nuclear exit of p27 via the PPP2R2A/Akt axis, which ultimately promotes the proliferation and invasion of respective cancer cells [122]. Moreover, abnormal ECM accumulation and blood vessel depletion in the TME can cause high desmoplasia and extreme hypoxia in PaCa tissues, which in turn stimulates cancer cells to ensure their survival by offsetting the hypoxic/ischemic environment via compensatory metabolic mechanisms that promote PaCa progression and apoptosis resistance [148]. The hypoxic environment inside the tumor, which is caused by rapid cell growth, can stimulate the production exosomal miR-301a-3p in PaCa cells [123]. After being acquired by other PaCa cells, miR-301a-3p-positive PaCaExos can promote the metastatic ability and invasiveness of these cancer cells. Upon uptake by macrophages, miR-301a-3p can also induce HIF1α/2α-dependent M2 phenotype transformation due to the activation of PTEN/PI3K signaling cascade [123]. Hypoxia has been shown to stimulate PaCa cells to generate more of small-volume exosomes via HIF1α, which increases the survival, proliferation, and metastasis of PaCa cells [149]. Additionally, exosomal miR-1246 has been found in the serum from patients with breast and prostate cancers [150, 151]. High levels of miR-1246 have been associated with GEM-resistance in PaCa cells, which can promote PaCa metastasis, invasion, cancer stemness, and angiogenesis due to the inhibition of CCNG2 expression [152]. However, it still remains unclear whether miR-1246 can enter exosomes to affect the chemo-resistance in pancreatic cancer.

Besides the above distinct miRNAs, cancer tissues originated from PaCa patients have presented high levels of circular RNA IARS (circ-IARS) [128]. Exosomal circ-IARS produced by PaCa cells can promote cancer metastasis by increasing endothelial monolayer permeability and activating HUVECs to enhance angiogenesis. Mechanistic analyses have revealed that circ-IARS-positive exosomes may contribute to tumor invasion by (i) down-regulating miR-122 and ZO-1, (ii) up-regulating RhoA, RhoA-GTP, and F-actin and (iii) promoting focal adhesion. The high expression of circ-IARS has been positively correlated with liver metastasis, vascular invasion, and tumor-node-metastasis (TNM) of PaCa [128]. Li and colleagues have verified that metastatic PaCa cells in the liver present high levels of circular RNA PDE8A (circ-PDE8A). Serum circ-PDE8A-positive exosomes can induce invasive growth of PaCa cells by counteracting with miR-338 to activate the MACC/MET/ERK/Akt signaling axis [129]. Therefore, exosomal circ-PDE8A may be considered as a putative marker to predict PaCa metastatic progression. Additionally, exosomes from the serum of PaCa patients may also contain human telomerase reverse transcriptase (hTERT) mRNA [127]. PaCa-derived exosomes that are hTERT mRNA-positive can induce the transformation of non-malignant pancreatic fibroblasts (PF) into cells with high telomerase activity, thus stimulating cell proliferation and delaying aging [127].

Cancer-associated fibroblasts (CAFs) that comprise main constituent cells of PaCa are essential for establishing the TME [153]. In TME, CAFs are able to regulate various behaviors and characteristics of PaCa cells, including epithelial-mesenchymal transition (EMT), proliferation, migration, invasion, metabolic transformation, and chemotherapy resistance [154, 155]. It has been shown that CAF-derived exosomes can reprogram the energy metabolism and up-regulate mitochondrial oxidation in PaCa cells, which induces glycolysis and glutamine-dependent reductive carboxylation to provides amino acids, fatty acids, as well as tricarboxylic acid cycle (TAC) intermediates for PaCa cells that are nutritionally deficient, thereby promoting the survival and growth of these cancer cells [156]. CAF-derived exosomes induced by gemcitabine (GEM) can activate miR-146a and the Snail signaling cascade in PaCa cells, thus promoting survival, proliferation, and drug resistance [157]. Interestingly, extracellular vesicles (EVs) containing annexin A6/LDL receptor-related protein 1/thrombospondin 1 (ANXA6/LRP1/TSP1) are solely present in the serum from PaCa patients. ANXA6/LRP1/TSP1-positive EVs are only produced by CAFs from PaCa patients, which are essential for liver metastasis [158]. Nevertheless, further studies are required to validate whether the ANXA6/LRP1/TSP1 complex may enter exosomes to support the aggressiveness of PaCa.

Tumor-associated macrophages (TAMs), a class of TME-infiltrating macrophages, are capable of promoting the radio/chemotherapy resistance, angiogenesis, migration, invasion and metastasis of tumor cells [159]. It has been demonstrated that TAM-producing exosomes can promote PaCa metastasis and progression. For instance, exosomal miR-501-3p derived from M2 macrophages is able to inhibit TGFBR3 expression and then activate TGF-β signaling, therefore inducing the formation and metastasis of PaCa xenografts in nude mice [125]. Analysis of clinical PaCa tissue specimens has indicated that miR-501-3p is also highly expressed in PaCa tissues. Exosomes produced by different types of PaCa cells (AsPC-1, BxPC-3, Panc-1, and MiaPaCa-2) contain distinct membrane proteins and lipid components that may affect the communication between PaCa cells and TAMs. For instance, exosomes produced by the ascites-derived human PDAC cell line AsPC-1 (AsPC-1-Exos) contain a large amount of ICAM-1 and arachidonic acid (AA). Since ICAM-1 can recognize and bind to CD11c on the surface of THP-1-derived macrophages (TDMs), AsPC-1-Exos can be uptaken rapidly by TDMs. The hydrolysis of AA in AsPC-1-Exos, catalyzed by phospholipase A2, has been shown to effectively reduce the fusion of AsPC-1-Exos with TDMs [114]. Treating non-polarized M0 macrophages with AsPC-1-Exos can induce M0 macrophages to transition into immunosuppressive M2 macrophages. Moreover, AsPC-1-Exos can simulate TDMs to secrete a subset of cytokines, such as VEGF, MCP-1, IL-6, IL-1β, MMP-9, and TNF-α, thereby promoting the growth and progression of PaCa [114].

Cancer initiating cells (CICs) play a major role in the initiation of cancer cell migration and metastasis [160]. PaCa-related CICs (PaCICs) can induce tumor stroma reorganization, stimulate angiogenesis, and promote hematopoietic cells to generate immunosuppressive cells via the production of exosomes (PaCIC-Exos), which eventually create a (pre-)metastatic niche in the distal metastatic organ [161, 162]. PaCIC-Exos can transfer certain characteristics of CICs to non-CICs, therefore inducing their reprogramming and promoting transformation characteristics such as anchorage-independent growth, apoptosis resistance, migration, and invasion, until they are phenotypically modified into CICs [163, 164]. In addition, PaCIC-Exos can be uptaken by non-CICs and then increase the levels of p-Akt, Bcl-2, Bcl-X_L, and MDR1, which potentially leads into the induction of metastatic growth, cisplatin resistance, EMT, migration, and invasion. Intravenous injection of Tspan8 antibody (CO029) has been shown to properly inhibit the drug resistance of non-CICs induced by CICs via exosomes [165], suggesting its use as a potential therapeutic target for PaCa.

In PaCa cells, the expression of Tspan8 and other CIC marker proteins, such as integrin α6β1, CD104, EpCAM, CXCR4, and CD44v6, are mutually regulated. It has been demonstrated that knockdown of CD44v6 expression is capable of decreasing the invasiveness of PaCICs [165]. In contrast, CD151-positive exosomes produced by CICs can induce EMT and migration of PaCa cells [111]. CIC-produced Cld7-positive exosomes exhibit some activity to promote non-metastatic PaCa dissemination and metastatic growth, by increasing cell migration, invasion, and angiogenesis. Still, Cld7-positive exosomes do not have an apparent impact in apoptotic resistance, proliferation and EMT of tumor cells [166]. Functional studies have demonstrated that CIC-Exos can rescue the defects caused by Cld7 loss in CLD7^−/− PaCa cells, by activating integrin signaling pathway, proteases (such as uPA) and lymphangiogenic receptor (for instance, VEGFR3). Interestingly, the ability of CIC-Exos to promote tumor progression by activating receptor tyrosine kinase (RTK) can be blocked by the RTK inhibitor Sunitinib, indicating that RTK inhibition could be serves as a therapeutic approach in PaCa [166]. Furthermore, incubation of rat PaCa cells with Tspan8-positive exosomes carrying Cld7-specific miRNA may cause CLD7 gene silencing in vitro. Tspan8 can significantly enhance the targeting of exosomal Cld7-related miRNAs to PaCa sites, leading to decreased Cld7 levels and further reduction on the expression of other CIC markers and Notch1, which suppress tumor cell growth, motility, and invasion [167]. Therefore, these modified exosomes may effectively load and carry nucleic acid fragments to tumor sites and help inhibit tumor progression.

Pancreatic stellate cells (PSCs) play crucial roles in chronic pancreatitis and pancreatic fibrosis [72]. These cells are capable of interacting with tumor and surrounding stromal cells (such as immune and endothelial cells) to respectively promote cell growth and distant metastasis [168]. Exosomes produced by PSCs can stimulate PaCa cells to express a number of chemokines, including CCL20, CXCL1/2, PDZK1IP1, SAA1/2, SMCR7L, and ZNF619, which in turn promote the proliferation and migration of PaCa cells [169]. PaCa cell lines including Panc-1 and SUIT-2 can produce miR-1246 and miR-1290-positive exosomes to induce cell proliferation and migration of PSCs by up-regulating the expression of α-smooth muscle actin (α-SMA/ACTA2), increasing procollagen type I C-peptide production, and activating ERK/Akt signaling cascades [126]. Activated PSCs subsequently produce exosomes containing CD9, CCN2 and miR-21, which exacerbate tumor tissue fibrosis by stimulating other PSCs to secret and deposit more collagen [39]. Thus, PSCs communicate with PaCa cells via exosomes in the TME, which is essential for PaCa progression.