Introduction
Despite persistent and great efforts thrown into basic and clinical cancer research, pancreatic cancer remains one of the most lethal diseases. Compared to the obvious progress in many other cancer types, the current 5-year survival rate of pancreatic cancer is still <10%.[1] At present, surgery remains the only way to eradicate pancreatic cancer. However, due to the mild or asymptomatic early stage, about 80% of patients are diagnosed to be advanced stage with obvious arterial tumor contact, thus losing opportunities to receive operation.[2] Therefore, systematic therapy including chemotherapy remains the main treatment to manage pancreatic cancer patients. After decades of research, several drugs are currently recommended to be the first-line chemotherapy regimens, such as gemcitabine and FOLFIRINOX (folinic acid, 5-fluorouracil, irinotecan, and oxaliplatin).[3–5] However, they still failed to dramatically improve the prognosis of pancreatic cancer patients due to chemoresistance.[6] In addition to chemotherapy, radiotherapy, another approved method to treat a variety of solid tumors, currently serves as a palliative manner to relieve cancerous pain, but with undesirable side effects.[7] Therefore, effective therapy against pancreatic cancer is urgently needed to solve the current dilemma.
As the prerequisite, understanding pancreatic cancer clearly and thoroughly is the basis for developing novel therapy. Compared to massive previous studies on malignant cells themselves, the tumor microenvironment is recently gaining more attention,[8] which is a complex structure that predominantly consists of not only pancreatic cancer cells (PCCs), but abundant extracellular matrix (ECM), cancer-associated fibroblasts (CAFs), cancer stem cells (CSCs), immune cells, blood vessels, and a variety of soluble proteins.[9] All components elaborately coordinate with each other, contributing to pancreatic cancer progression.[10] Therefore, communication among them is vital for pancreatic cancer progression.
Exosomes are preferred to be researched and referred to as small extracellular vesicles (about 40–160 nm in diameter), containing proteins, nucleic acids, lipids, and metabolites, which could be generated by essentially all cell types and found in multiple body fluids, such as blood plasma, serum, and urine. Protected by the lipid bilayer membrane with identifiable proteins (such as CD9, CD63, and CD81), exosomes can mediate cell–cell communication in both health and disease.[11] With the accumulated evidence, exosomes are suggested to importantly mediate the communication among different populations of cells within the tumor microenvironment, which could be divided into PCCs–PCCs, PCCs–CAFs, PCCs–immune cells, and others.[12] As a result, the proliferation, invasion, chemoresistance, immune response, and metabolism of pancreatic cancer are largely affected.
Database search strategy
A computer-based online search of the PubMed database was performed to retrieve articles published until May 31, 2022. A combination of the following text words (MeSH terms) was used to maximize search specificity and sensitivity: “Pancreatic cancer”; “tumor microenvironment”; “exosome”; “extracellular vesicles”; “cancer cells”; “endothelial cells”; “immune cells”; “cancer-associated fibroblasts”; and “cancer stem cells”. The results were further screened by title and abstract, and only those studies exploring the relationship between exosomes and pancreatic cancer were included. No language or study type restrictions were applied.
Pancreatic cancer cells–pancreatic cancer cells
Heterogenous subpopulations of PCCs within the tumor microenvironment could coordinate with each other through exosomes (Fig. 1).
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Figure 1.: The communication among different pancreatic cancer cells via exosomes. Pancreatic cancer cells are heterogeneous within pancreatic tumor. Highly malignant cancer cells and cancer stem cells could load a variety of molecules, such as protein and RNA, into multiple vesicle body, and secret them via exosomes. Upon uptake, downstream pathways and biological behaviors of recipient pancreatic cancer cells are dramatically changed. Arrows represent positive regulation. Blunt ends represent inhibition.
Exosome RNAs
In addition to mature proteins, exosomes also contained massive RNAs. mRNAs of superoxide dismutase 2 and catalase were packed into exosomes in gemcitabine-treated PCCs, conferring chemoresistance to the recipient cancer cells by suppressing gemcitabine-induced ROS overproduction. In addition, those exosomes also had miR-155, downregulating deoxycytidine kinase, a gemcitabine-activating enzyme, and finally contributing to chemoresistance.[13] Moreover, Exosomes could also carry lncRNA Sox2ot from highly invasive PCCs to others, which downregulated miR-200 and subsequently increased Sox2 and Snail in the recipient PCCs. As a result, EMT and invasive ability of recipient PCCs were largely enhanced.[14] Upon stimulation by TGF-β, lncRNA HULC was upregulated in PCCs, which could transmit proliferation and EMT abilities among different PCCs via exosomes.[15] However, miR-622 overexpressing PCCs could reverse the effects of lncRNA HULC by secreting exosomal miR-622, which targeted lncRNA HULC.[16] Periostin could promote the expression of LncRNA LINC01133 in PCCs, activating the Wnt/β-catenin pathway and inducing EMT among PCCs through Exosomes.[17] Additionally, highly invasive PCCs also conferred exosomal circRNA PDE8A and miR-125b-5p to other PCCs, contributing to their invasive ability,[18,19] and even liver metastasis.[18] Due to the dense and hypovascular stroma, oxygen levels are significantly low in pancreatic tumors. PCCs under hypoxia conditions preferred to release exosomal circZNF91, which could deprive the suppression of miR-23b-3p on Sirtuin1. Then, the hypoxia‐inducible factor‐1α stabilization and glycolysis were promoted, leading to the chemoresistance and hypoxic survival of recipient PCCs.[20,21]
Interestingly, in addition to the promoting effects, some other PCCs-derived exosomes with abundant lipid rafts could effectively inhibit the growth of relatively differentiated PCCs, which was mainly dependent on the mitochondria apoptotic pathway and could compromise the Notch-1 survival pathway of recipient cells.[22–24] Additionally, upon melittin treatment, lncRNA NONHSAT105177 was upregulated in PCCs, inhibiting their EMT and proliferation by suppressing the cholesterol biosynthesis pathway in vitro and in vivo. Besides the role in individual cells, melittin-induced lncRNA NONHSAT105177 could also be transmitted to other PCCs via Exosomes.[25]
Exosome proteins
PCCs with mutant KRAS, an extremely common and hyperactive small-GTPase in pancreatic cancer, could express more Survivin and transmit it to other PCCs via exosomes, thus promoting the integral survival and therapy resistance of pancreatic tumors.[26] Highly malignant PCCs also could confer high proliferation and invasion abilities to moderately malignant PCCs through exosomal zinc transporter protein (ZIP4). Moreover, ZIP4 levels in serous exosomes potentially served as a novel diagnostic biomarker which was confirmed by clinical blood samples.[27] In addition to ZIP4, Tenascin-c, an ECM protein, could be transmitted from highly invasive PCCs to others with less-invasive ability, contributing to the increased proliferation and epithelial-mesenchymal transition (EMT) through respectively activating nuclear factor-κB pathway and Wnt/β-catenin pathway in vitro.[28] Aspartate β-hydroxylase, highly expressed in PCCs on the cell surface, could enhance the invasive ability of PCCs by activating the Notch cascade. Furthermore, it also potentiated PCCs to release exosomes carrying pro-invasive cargoes, such as matrix metalloproteinases, resulting in the metastasis of pancreatic cancer.[29] Besides that, ephrin type-A receptor 2 on the exosomes membrane conferred chemoresistance and metastatic potential between distinct PCCs.[30,31] Additionally, cytoskeleton-associated protein 4 (CKAP4), another transmembrane protein on exosomes, could also be transmitted among different PCCs after binding with a secretory protein named Dickkopf 1 (DKK1). After uptake, the Phosphatidylinositol 3-kinase (PI3K)-AKT pathway was activated. Therefore, proliferation, migration, and chemoresistance were then enhanced in pancreatic cancer. Anti-CKAP4 antibody effectively blocked DKK1-CKAP4 interaction and followed pancreatic cancer development in mouse models.[32] Similarly, another study suggested that asparaginyl endopeptidase in the exosomes derived from PCCs could also stimulate the PI3K-AKT pathway in the recipient cells.[33]
Pancreatic cancer cells–cancer stem cells
Among heterogenous PCCs, CSCs have unique malignant properties, exhibiting extraordinary abilities in continuous self-renewal and chemoresistance.[34] CSCs secreted exosomes carrying CD44 variant isoform 6 and Tspan8, 2 membrane proteins, which could transfer “stem”-like phenotypes to other non-stem cancer cells. CD44 variant isoform 6primarily induced extensively altered mRNA and miRNA profiles in recipient PCCs, which could widely change their signaling, transport, and transcription. Tspan8 mainly facilitated the binding and uptake. Therefore, combining the anti-Tspan8 antibody (CO029) with gemcitabine exhibited promising therapeutic effects in mouse models.[35] Additionally, again was also enriched in CSC-derived exosomes, activating YAP in recipient PCCs and contributing to cancer progression.[36] Besides proteins, CSCs also secreted exosomes with miR-210 and subsequently activated the mammalian target of rapamycin signaling pathway in recipient PCCs, which provided chemoresistance to mouse models with subcutaneous grafts.[37] For the radiotherapy of pancreatic cancer, tumor-repopulating cells sharing some CSCs properties are the main cause of treatment failure. During radiation, irradiated dying cancer cells secreted exosomal miR-194-5p. Upon uptake, miR-194-5p downregulates E2F3, facilitates DNA damage, pair, and finally potentiates survival and followed the proliferation of tumor-repopulating cells.[38] Therefore, the functions of minority subpopulations of PCCs could be amplified through exosomes (Fig. 1).
Pancreatic cancer cells–cancer-associated fibroblasts
Pancreatic tumor is characterized by the massive and dense ECM, which is synthesized by CAFs, the primary cellular components of the stroma.[39,40] There are several sources of CAFs, such as resident fibroblasts, bone marrow-derived mesenchymal stem cells, epithelial cells, and pancreatic stellate cells (PSCs). Among them, PSCs are activated during carcinogenesis, serving as the most prominent origin of CAFs in pancreatic cancer.[41] There are amounts of crosstalk mediated by exosomes between PCCs and CAFs/activated PSCs (Fig. 2).
Figure 2.: The communication between pancreatic cancer cells and cancer-associated fibroblasts via exosomes. Pancreatic cancer cells and cancer-associated fibroblasts have extensive interactions mediated by exosomes. Pancreatic cancer cell could transfer miR-1246 and miR-1290 to cancer-associated fibroblasts and activate them. In turn, cancer-associated fibroblast could transmit miR-21, miR-4465, miR-146a, miR-5703, miR-106b, miR-616-3p, Snail (mRNA), lncRNA UCA1, and metabolites to pancreatic cancer cell, finally promote cancer progression.
PCCs could turn adjacent quiescent PSCs to be activated by exosomes containing miR-1246 and miR-1290.[42] Interestingly, PCCs could attract more activated PSCs around them via exosomes as well. Lin28B, an RNA-binding protein, was released from PCCs to other malignant cells via exosomes. Upon uptake, platelet-derived growth factor B secretion from recipient PCCs would be upregulated, facilitating the recruitment of activated PSCs.[43]
In return, activated PSCs widely improved chemokine ligands expression, proliferation, and migration of PCCs through Exosomes.[44] Those CAFs/activated PSCs-derived Exosomes were rich in miR-21,[45] which indirectly activated ERK and AKT pathways and subsequently promoted EMT and migration of recipient PCCs.[46] Activated PSCs under hypoxia preferred to synthesize exosomal miR-4465 and miR-616-3p, which targeted PTEN and subsequently activated AKT signaling in PCCs as well.[47] Besides, the activated PSCs also released Exosomes miR-5703, which downregulated CMTM4 and then stimulated the PI3K/AKT pathway, finally promoting the proliferation of recipient PCCs.[48] Additionally, CAFs also play a role in inducing chemoresistance of pancreatic cancer. CAFs have an innate ability in chemoresistance and could release more exosomes upon gemcitabine treatment. Those Exosomes improved chemoresistance and proliferation of PCCs, which might be mediated by Snail (mRNA), miR-146a, miR-106b, and LncRNA UCA1.[49–51] Among them, miR-106b might contribute to chemoresistance by inhibiting TP53INP1 in the recipient PCCs,[50] LncRNA UCA1 could epigenetically inhibit the transcription of suppressor of cytokine signaling 3 and promote chemoresistance.[51] Moreover, GW4869, an exosome secretion inhibitor, effectively reversed the promotive role of CAFs-derived Exosomes in vitro,[44,49] exhibiting obvious anticancer effects in mouse models as well.[49]
Besides that, CAFs also assisted the survival of PCCs within a harsh tumor microenvironment. Under the nutrient-stressed condition, CAFs released exosomes containing massive intact metabolites, such as TCA-cycle intermediates, amino acids, and lipids, which could be used by adjacent PCCs for growth.[52] Therefore, given the rising concern on CAFs in pancreatic cancer,[40,52,53] the role of exosomes in connecting PCCs and CAFs deserves further research.
Pancreatic cancer cells–immune cells
In addition to heterogenous PCCs and CAFs, there are massive immune cells within the tumor microenvironment, including CD8+ cytotoxic T lymphocytes, CD4+ helper T cells (TH1, TH2, Th17, and regulatory T cells), natural killers (NKs), tumor-associated macrophages (TAMs), myeloid-derived suppress cells, and dendritic cells (DCs). Those immune cells engage the extensive crosstalk with PCCs via exosomes, which subsequently affects the progression of pancreatic cancer (Fig. 3).[54,55]
Figure 3.: The communication between pancreatic cancer cells and immune cells within tumor microenvironment via exosomes. Tumor microenvironment contains many types of immune cells. Immune cells and pancreatic cancer cells could communicate with each other by exosomes, regulating local immunity and cancer progression. Pancreatic cancer cells transmit KRAS, Ezrin, miR-301-3p, miR-155, and miR-125b2 to tumor-associated macrophages and regulate their M2 polarization. Tumor-associated macrophages deliver miR-501-3p, miR-365, miR-21-5p, lncRNA SBF2-AS1, fibronectin, and Chitinase-3-like protein 1 to accelerate cancer growth. While natural killers-exosomes could inhibit pancreatic cancer cells proliferation and metastasis by miR-3607-3p. Besides tumor-associated macrophage, pancreatic cancer cells-exosomes also could carry miR-203 and miR-212-3p to dendritic cells, transforming growth factor-β, and heat shock protein70 to nature killers, changing their anticancer abilities. Additionally, T lymphocytes are also turned to be T regulatory phenotype upon pancreatic cancer cells-exosomes treatment.
Integrated analysis showed that there were extensive alterations of lncRNAs and mRNAs in the DCs cocultured with PCCs-derived exosomes.[56] Specifically, exosomal miR-203 could be transmitted from PCCs to DCs, which inhibited the expression of toll-like receptor 4 and its downstream cytokines, such as interleukin-12 and tumor necrosis factor-α, finally contributing to the immunosuppression.[57] Moreover, PCC-derived exosomes were also rich in miR-212-3p, which could inhibit major histocompatibility complex II expression in recipient DCs and result in immune tolerance.[58] Although the DCs cocultured with PCCs-derived Exosomes exhibited an immunosuppressive role in vitro, injecting those DCs could also effectively delay pancreatic tumor growth in mouse models as vaccination, especially combining with cytotoxic drugs, including gemcitabine, ATRA, and sunitinib.[59]
The tumor microenvironment is characterized by hypoxia and oxidative stress.[60] Under oxidative stress, PCCs secreted exosomes containing mutant KRAS to TAMs. Upon uptake, the increased STAT3-dependent fatty acid oxidation switched TAMs to M2 polarization (immunosuppressive and pro-tumor phenotype), which could promote tumor progression.[61] In addition to KRAS, PCCs also transmitted exosomal Ezrin to TAMs and accelerated their M2 polarization.[62] Under hypoxia, PCCs secreted exosomal miR-301a-3p and promoted M2 polarization of TAMs as well through activating PTEN/PI3K signaling pathway.[63] However, PCCs transfected with miR-155 and miR-125b2 could release exosomes rich in these 2 microRNAs, transforming immunosuppressive M2 TAMs to M1 phenotype with anticancer properties.[64] Besides that, TAMs also could release exosomes to affect PCCs. M2 TAMs released Exosomes containing miR-501-3p, which could inhibit TGF-β receptor III expressions and then activate the TGF-β signaling pathway. As a result, proliferative, migrative, and invasive abilities were improved in recipient PCCs.[65] LncRNA SBF2-AS1 in M2 TAMs-derived Exosomes repressed miR-122-5p in PCCs upon uptake, upregulating X‐linked inhibitor of apoptosis protein level and accelerating pancreatic cancer progression.[66] TAMs played a role in the chemoresistance of pancreatic cancer as well. Those M2 TAMs-derived Exosomes also carried miR-365 which could increase the triphosphate-nucleotide pool and upregulate cytidine deaminase (a gemcitabine-inactivating enzyme) in PCCs, promoting the chemoresistance of pancreatic cancer. Therefore, transferring artificial antagomiR-365 to murine peritoneal macrophages and subsequently re-injecting them into mice bearing pancreatic cancer showed obvious anticancer effects.[67] Macrophages, independent of M1 or M2 types, also secreted Exosomes chitinase 3-like-1 and fibronectin 1 and promoted chemoresistance upon PCCs uptake.[68] Furthermore, another recent report also found that M2 TAM could secrete exosomal miR-21-5p, which targeted Krüppel-like factor 3 in PCC and promoted the stemness of pancreatic cancer.[69] Interestingly, some exosome-mediated communication between PCCs and macrophages could be inhibited by REG3β, a lectin released by healthy acinar cells surrounding the tumor.[70]
Besides DCs and TAMs, PCCs also communicated with other immune cells by exosomes within the tumor microenvironment. Cytotoxic activity of peripheral T lymphocytes could be impaired by PCCs-derived exosomes, which increased FOXP3 expression in recipient T lymphocytes and induced Treg phenotype.[71] Additionally, PCCs-derived exosomes also contained TGF-β, phosphorylating Smad2/3 in NKs and contributing to their dysfunction.[72] However, some other exosome-mediated communication showed anticancer effects. PCCs could release heat shock protein 70 surface-positive exosomes, which enhanced the migratory and cytolytic abilities of NKs.[73] As feedback, NKs-derived exosomes transmitted miR-3607-3p to inhibit IL-26 expression in PCCs, suppressing their proliferation and metastasis.[74] Furthermore, after miRNA depletion, exosome proteins from PCCs could more effectively activate DCs/cytokine-induced killer cells than regular immune activators (lipopolysaccharide), exhibiting promising anticancer effects.[75] Besides that, recent studies suggested that exosomes released by PCC contained programmed death-ligand 1, which could elicit the immune checkpoint response. Therefore, inhibiting exosome release might provide novel insights into promoting the efficacy of immune therapy.[76,77]
Angiogenesis and lymphomagenesis
Besides PCCs, CAFs, and immune cells, there are many other cell types within the tumor microenvironment.
Angiogenesis and lymphomagenesis are vital for tumor growth and metastasis, which also could be stimulated by PCCs via exosomes.[78] Exosomal miR-27a released by PCCs directly targeted B‐cell translocation gene 2 in endothelial cells and promoted angiogenesis.[79] Exosomal LncRNA CCAT1 derived from PCCs could induce angiogenesis as well, which was mainly mediated by miR-138-5p and high mobility group A1.[80] Moreover, rat PCCs also secreted exosomes carrying tetraspanin, D6.1A/CO-029, which could increase endothelial cell branching and angiogenesis.[81] Hypoxia has an intricate relationship with angiogenesis, especially in solid tumors.[82] Hypoxia PCCs could secrete exosomal lncRNA UCA1, which served as a sponge of miR-96-5p and indirectly upregulated AMOTL2 expression and ERK pathway in adjacent endothelial cells. As a result, angiogenesis was promoted.[83]
In addition to the abovementioned interactions, exosome signaling also could coordinate PCCs and endothelial cells synergistically. PCCs with low levels of dual-specificity phosphatase-2 had activated ERK pathway and proprotein convertase, which increased vascular endothelial growth factor-C expression. Subsequently, amounts of vascular endothelial growth factor-C in PCCs were packaged into exosomes and transmitted to other PCCs and lymphatic/vascular endothelial cells, finally mediating PCCs migration and lymphangiogenesis.[84] Annexin A1, a transmembrane protein located in PCCs-derived exosomes, interacted with formyl peptide receptors on PCCs, fibroblasts, and endothelial cells, finally inducing metastasis and angiogenesis.[85] Therefore, the role of exosomes in the tumor microenvironment is significant and remains further studied.
Limitations
There may be some possible limitations in this study. The included original research may have reporting bias, which deserves further study to confirm. Additionally, we only retrieved the articles published until May 31, 2022. The latest studies in recent months still need further review.
Discussion
The tumor microenvironment is a highly heterozygous organization. Multiple cells and ECM coordinate with each other, synergistically promoting pancreatic cancer progression. Exosomes are vital mediators to realize rapid and abundant communication among different PCCs, CAFs, and immune cells within the tumor microenvironment.
Recently, a novel method showed that pure exosomes could be effectively isolated from tissues directly.[86,87] Therefore, the role of exosomes in the tumor microenvironment is ready to be clear in future studies. Besides that, although the RNA or protein contents within exosomes were widely researched, the biogenesis process should also be paid attention to, which could provide more insights into liquid biopsy and engineered exosomes. Engineered exosome paved a novel way for targeted therapy. For example, at present, a clinical trial led by MD Anderson Cancer Center is recruiting, which attempts to treat metastatic pancreatic cancer by engineered exosome loading with small interfering RNA which targeted mutant KRAS (NCT03608631).[88] However, there are still some barriers restraining exosome research. For example, exosomes could be extracted from cell medium or body fluid through a variety of methods, such as ultracentrifugation and size exclusion chromatography, which may contribute to unrepeatable results in different studies. Therefore, the standard in isolating exosomes from various fluids remains to be worked out. Exosomes could carry a variety of cargo including proteins, RNAs, and metabolites, mediating the communication among heterogeneous cellular components within the tumor microenvironment. In conclusion, a better understanding of pancreatic cancer Exosomes will benefit liquid biopsy and novel treatments, finally improving the prognosis of patients.
Conclusion
In this review, we summarize current knowledge about exosomes in connecting PCCs and the tumor microenvironment. First, exosomes could coordinate different PCCs subpopulations and promote integral proliferation, migration, and chemoresistance. Second, exosomes also play a key role in the communication between cancer cells and stroma or immune cells. Third, research in exosomes not only improves the understanding of the carcinogenesis and progression of pancreatic cancer but also provides insights into liquid biopsy and targeted therapy.
Acknowledgments
None.
Author contributions
WW designed this manuscript. CQ, BZ, and YW collected related literature and drafted this manuscript. CQ, BZ, YW, TL, ZL, TL, YZ, and WW made critical revisions to this manuscript. All authors read and approved the final manuscript.
Financial support
This work was supported by the National Natural Science Foundation of China Grants (No. 81773215, 82173074), and CAMS Innovation Fund for Medical Sciences (CIFMS) (No. 2021-I2M-1-002). The funders had no role in the manuscript design, data collection, analysis, preparation of the manuscript, or decision to publish.
Conflicts of interest
The authors declare no conflicts of interest.
Ethics approval
Not applicable.
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