Clinical Therapeutics/Volume ], Number ], 2014
Potential of Cancer Cell–Derived Exosomes in Clinical Application: A Review of Recent Research Advances Yu Sun, PhD1; and Jing Liu, PhD1,2 1
Regenerative Medicine Centre, First Affiliated Hospital, Dalian Medical University, Dalian, P.R. China; and 2Institute of Integrative Medicine, Dalian Medical University, Dalian, P.R. China
ABSTRACT Background: Exosomes are 30- to 100-nm, membrane-bound vesicles that are released by most types of cells, including tumor cells. Exosomes contain a great variety of bioactive molecules, including signal peptides, microRNA, lipids, and DNA. In cancer, tumor cells aberrantly secrete large quantities of exosomes to transport paracrine signals or to contribute to tumor–environment interaction at a distance. Objective: The goal of this review was to discuss the recent advances on the mechanism of cancerderived exosomes in tumor regulation. Methods: Pertinent articles and abstracts were identified through searches of PubMed for literature published from 1983 to December 2013. Search terms included exosome, tumor, cancer, diagnosis, and therapy. Results: All of the exposed evidence points to communication between cancer cells and their surroundings, either mediated by cancer cell–derived exosomes or by stromal cell–derived exosomes. This communication probably supports tumor proliferation, motility, invasion, angiogenesis, and premetastatic niche preparation. In addition, recent research implies that cancer cell–derived exosomes play a suppressive role in cancer-directed immune response. Conclusions: The biomarkers detected in bodily fluid– derived exosomes imply a potential for exosomes in cancer diagnosis. Also, exosomes could be used as a vehicle to selectively deliver therapeutic nucleic-acid drugs or conventional drugs for tumor therapy. The tolerability and feasibility of cancer exosomes in diagnosis and therapy need to be further evaluated. (Clin Ther. 2014;]:]]]–]]]) & 2014 Elsevier HS Journals, Inc. All rights reserved. Key words: cancer, diagnosis, exosomes, therapy.
vesicles containing definite receptors to ensure traffic specificity. The research accumulated over the past 10 years has demonstrated that a heterogeneous group of vesicles is also released from the cell surface and is used as intercellular signalosomes in information exchange, even over a long distance.1 These membranous vesicles, released by a variety of cells and generally termed extracellular vesicles, can be divided into 3 main classes: exosomes, microvesicles (100– 1000 nm), and apoptotic bodies (1–5 μm). Exosomes are 30 to 100 nm in diameter and shed from many different types of cells under both normal and pathologic conditions.2 These exosomes can be formed through inward budding of endosomal membranes, giving rise to intracellular multivesicular bodies that later fuse with the plasma membrane, releasing the exosomes to the exterior.3 They are released from many different cell types in the body, such as red blood cells, platelets, lymphocytes, dendritric cells (DCs), and tumor cells.4 Depending on the cellular origin, exosomes contain various cellular proteins. These proteins may be different from proteins that are normally located in the plasma membrane, including major histocompatibility complex (MHC) molecules, tetraspanins, adhesion molecules, and metalloproteinases.5,6 Exosomes also contain signal proteins and/or peptides, microRNAs (miRs), mRNAs, and lipids that can be transferred to a recipient cell by fusion of the exosome with the target cell membrane.7,8 It has been proposed that this transfer of protein and RNAs from one cell to another might represent a novel mechanism of intercellular communication.9,10 Initially, exosomes were described as vesicles released by reticulocytes. They were thought to function as a way to remove unnecessary proteins, such as the transferrin
INTRODUCTION
Accepted for publication April 24, 2014. http://dx.doi.org/10.1016/j.clinthera.2014.04.018 0149-2918/$ - see front matter
Cells are known to deliver proteins and molecules between the intracellular organelles by membrane
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& 2014 Elsevier HS Journals, Inc. All rights reserved.
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Clinical Therapeutics receptor, during the process of maturation into erythrocytes.3 Emerging evidence indicates exosomes as important mediators of cellular communication. Exosomes are involved not only in normal physiologic processes, such as lactation, immune response, and neuronal function, but also in the development and progression of diseases, such as liver disease, neurodegenerative diseases, and cancer.11 However, the detailed mechanisms of exosomes in physiologic and pathologic regulation are still being researched. Exosomes can also be isolated from various bodily fluids, such as breast milk, serum, plasma, malignant ascites, and urine.4 Therefore, ex vivo analysis of exosomes may provide biomarker-discovery platforms and facilitate disease diagnosis and monitoring. The goal of this review was to discuss the recent advances on the mechanism of cancer-derived exosomes in tumor regulation.
MATERIALS AND METHODS Pertinent articles and abstracts were identified through searches of PubMed for literature published from 1983 to December 2013. Search terms included exosome, tumor, cancer, diagnosis, and therapy.
RESULTS Exosomes and Cancer Exosomes are secreted by 2 different mechanisms: (1) constitutive release by the trans-Golgi network and (2) inducible release.12 In the past decade, studies have found that tumor cells constitutively release exosomes. Like most nontransformed cells, tumor cells release exosomes whose compositions vary depending on the nature and conditions of the cells. Tumor cell–secreted exosomes include many of the common exosomal proteins; they also contain tumor antigens that are reflective of the tumors from which they are derived,2
Cancer cells Wnt-PCP signaling
Matrix
Target cell
Horizontal transfer
FasL
Cytokine, ECM production
CAF
mRNA protein
TGF-
microRNA exosome
Stromal reorganization
Angiogenic proteins or mRNAs
MHC-II MDSCs
exosome Endothelium pancrea
Immune suppression
Lymph node
pre-metastatic niche formation
Blood vessel
Diagnosis
Cancer cell exosomes CAF-derived exosomes
Angiogenesis
Cancer cells Cancer-associated fibroblasts(CAF)
Cancer-derived exosomes mediated the crosstalk
Immune cells Myeloid-derived suppressor cells (MDSCs)
Figure. Cancer-derived exosomes are implicated in transferring miRNAs, mRNA and proteins to target cells. They are able to reorganize extracellularmatrix, alter the premetastatic niche and promote angiogenesis. They also create an immunosuppressive microenvironment by impairing the immune cells, by inducing the apoptosis of T cells through FAS-FASL ligation.
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Y. Sun and J. Liu such as the tumor antigens melanoma antigen recognized by T cells 1 (MelanA/Mart-1) and glycoprotein 100 (GP100), carcinoembryogenic antigen (CEA), and human epidermal growth factor receptor 2 (Her2).13,14 Cancer cell–derived exosomes are emerging as local and systemic intercellular mediators of oncogenic information through the horizontal transfer of mRNAs, miRs, and proteins during tumorigenesis.15,16 For example, gliomas often express a truncated and oncogenic form of the epidermal growth factor receptor (EGFR) vIII. Within each tumor, only a small percentage of gliomal cells may actually express EGFRvIII; however, most of the cells exhibit a transformed phenotype. It has been revealed that gliomal cell–derived exosomes support the propagation of malignancy through the transfer of EGFRvIII.17 McCready et al18 found that one of the multiple isoforms of heat shock protein (Hsp) 90 is secreted by exosomes from invasive cancer cells and contributes to their motility and invasiveness. Cancer-derived exosomes are of great importance in the communication between cancer cells and the tumor-associated microenvironment or the immune system. This subject has been heavily investigated, but we are still awaiting a clear answer. Additionally, tumor-derived exosomes can be isolated from the bodily fluids of patients with tumors. Both cancer cell–derived exosomes and bodily fluid– derived exosomes contain proteins and RNAs originating from neoplastic cells.19 Therefore, these characteristics reveal the potential for cancer-derived exosomes as biomarkers in cancer diagnosis.
Internalization of Cancer Cell–Derived Exosomes The exosomal protein profile is rich in molecules located in membrane domains and is prone to internalization such as rafts and tetraspanin-enriched microdomains, as well as molecules engaged in fission, scission, and vesicular transport; adhesion molecules; and proteases.20 Studies aimed at revealing the mechanism of cancer cell–derived exosome uptake by target cells have reported that cancer cell–derived exosomes can also use heparin sulfate proteoglycans (HSPGs) for their internalization. HSPGs are a family of proteins substituted with glycosaminoglycan polysaccharides, which are extensively modified by sulfation to determine their functional interactions. Several viruses have previously been reported to enter cells
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through HSPGs. Christianson et al21 provided evidence that HSPGs function as receptors of cancer cell–derived exosomes, which was dependent on intact HS, specifically on the 2-O and N-sulfation groups. Importantly, their data indicated that the HSPGdependent uptake route is highly relevant to the biological activity of exosomes.21 In addition, like all exosomes, tumor exosomes can transfer their contents to alter the physiologic states of recipient cells. O’Brien et al22 found that exosomes from triple-negative breast cancer sera significantly increased recipient cells’ invasion, conferring phenotypic traits to secondary cells. Given all of this evidence, it appears that HSPG may be a potential target for inhibition of exosomemediated content transfer and/or subsequent exosome-associated tumor development.
Cancer Cell–Derived Exosomes and Mediation of Communication Between Tumor Cells and the Tumor Microenvironment One important factor in tumor biology is that it relies on the capacity of tumor cells to create a microenvironment by recruiting and modulating nontransformed cells that support tumor cell survival and spread/propagation.23 The tumor-associated microenvironment consists of an extracellular matrix (ECM), cancer-associated fibroblasts, inflammatory immune cells, and tumor-associated vasculature.17 Tumorderived exosomes are of great importance in the communication between cancer cells and the tumorassociated environment, a subject being heavily discussed but still awaiting a clear answer. In this section, we provide the recent achievements on the mechanism of the communication between cancer cell–derived exosomes and the tumor microenvironment (Figure).
Cancer Cell–Derived Exosomes and the Modulation of Stromal Cells The ECM is an essential constituent of nontransformed tissues and tumors. It is now well-established that primary tumors are composed of a multitude of stromal cell types in addition to cancerous cells.24 By using green fluorescent protein–tagged CD63, a general marker of exosomes, Suetsugu et al25 imaged exosomes transferred from breast cancer cells to host cells, including red fluorescent protein (RFP) cancer– associated fibroblasts and RFP–lung tissue stromal cells in orthotopic nude mouse models. There is also evidence that cancer cell–derived exosomes stimulate
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Clinical Therapeutics secretion of the oncogenic receptor EGFRvIII20 and several proangiopoietic factors, such as matrix metalloproteinase 9, vascular endothelial growth factor, interleukin 8, and hepatocyte growth factor by stromal cells; induce proliferation of endothelial cells; and promote angiogenesis in metastatic organs.26 These results suggest that cancer cell–derived exosomes are incorporated into stromal cells, which may have consequences on the secretion of some biological factors by stromal cells to promote tumor metastasis.
Cancer Cell–Derived Exosomes and Reorganization of the Extracellular Matrix In addition to the effect of cancer cell–derived exosomes on stromal cells, recent data have also demonstrated a pivotal role for cancer cell–derived exosomes in the organization of the ECM. It has been reported that cancer cell–derived exosomes, being rich in proteases, modulate the ECM for degradation of collagens, laminin, and fibronectin, and that matrix degradation by cancer cell–derived exosomes has severe consequences on tumor and host cell adhesion, motility, and invasiveness.20 In that study, the investigators also provided evidence that tumor cell– derived exosomes bind to individual components of the ECM, the preferential partner depending on the exosomes’ adhesion-molecule profile, such that high CD44 expression is accompanied by hyaluronic acid binding and high α6β4 expression by laminin-332 binding. All of these findings imply that cancer cell–derived exosomes are able to alter the stroma, either by modulating the stromal cells or by degradating stroma.
Stromal Cell Exosomes and Regulation of Cancer Cells Stroma in the tumor microenvironment plays a crucial role in cancer progression. In addition to the effect of cancer cell–derived exosomes on stroma, the latest evidence has reported that stroma can also influence the biological behavior of cancer cells, which is mediated by stromal cell exosomes. Luga et al27 reported that exosomes secreted by stromal cells, such as cancer-associated fibroblasts, are taken up by breast cancer cells and promote the protrusive activity and motility of breast cancer cells by Wnt/planar cell polarity signaling. This work highlights the role of
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stromal cell–derived exosomes in modulating tumor cell metastasis. So far, all of these results suggest that exosomes are agents of communication between cancer cells and stroma to stimulate metastasis.
Cancer Cell–Derived Exosomes and Promotion of Angiogenesis Cancer cell–derived exosomes are also important for inducing angiogenesis. Most of the experimental evidence points to a potential involvement of exosomes in angiogenesis. Angiogenic proteins or mRNAs that promote proliferation of endothelial cells have been observed in cancer cell–secreted exosomes. For example, glioblastoma tumor cells release microvesicles (exosomes) containing mRNA, miRs, and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells.28 Hong et al29 demonstrated that colorectal cancer cell–derived exosomes are enriched in cell cycle–related mRNAs that promote proliferation of endothelial cells, suggesting that the microvesicles of cancer cells may be involved in tumor growth and metastasis by facilitating angiogenesisrelated processes. Recent research has also demonstrated that in a model of highly malignant brain tumor glioblastoma multiforme, exosomes derived from glioblastoma multiforme cells grown in hypoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells.30 So far, all of the exposed evidence points to communication between cancer cells their surroundings, either mediated by cancer cell–derived exosomes or by stromal cell–derived exosomes. This probably supports tumor proliferation, motility, invasion, angiogenesis, and premetastatic niche preparation.
Cancer Cell–Derived Exosomes and Modulation of Metastatic Niche Preparation A premetastatic niche is a distant target site for metastasis adapted to cancer cell seeding before the arrival of the first cancer cells. Jung et al31 found that cancer cell–derived exosomes modulated premetastatic organs for cancer cell settlement and proliferation in a rat model of pancreatic adenocarcinoma. An in vivo study showed melanoma exosomes homed to sentinel lymph nodes and imposed synchronized molecular signals that effected melanoma cell
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Y. Sun and J. Liu recruitment, ECM deposition, and vascular proliferation in the lymph nodes.32 These findings imply that cancer cell–derived exosomes are able to mediate the process of the microanatomic niche preparation that facilitates cancer cell metastasis.
Cancer Cell–Derived Exosomes and Suppression of Immune Response Exosomes play an important role in modulating immune responses. When derived from DCs, they bear MHC proteins and co-stimulatory molecules.33 These vesicles are effective in stimulating antigen-specific Tcell responses in vitro and in vivo. However, cancer cell–derived exosomes can suppress cancer-directed immune response.34 The interactions between cancer cell–derived exosomes and immune cells are mediated through direct signaling interactions by surfaceexpressed molecules or by the transfer of exosomes and/or their contents to immune cells. For example, tumor necrosis superfamily, member 6 ligand protein (FasL) on the surfaces of tumor cell–derived exosomes mediates cleavage of the T-cell surface glycoprotein CD3 (TCR)-ζ chain, a crucial T-cell–signaling molecule that is required for activation.35 Tumor-derived exosomes also display transforming growth factor β on their surfaces, which maintains the numbers and immunosuppressive effects of regulatory T cells in vitro.36 Additionally, a recent study has reported that cancer cell–derived exosomes can modulate T cells by hydrolyzing ATP into adenosine in the tumor microenvironment. Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune responses. A study by Clayton et al37 demonstrated that cancer cell–derived exosomes possess enzymatic activity that causes hydrolysis of ATP into adenosine in the tumor microenvironment, which negatively regulates T-cell activation. Myeloidderived suppressor cells have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T-cell activation. Chalmin et al38 showed that in both mice and humans, Hsp72-expressing exosomes restrain tumor immunosurveillance by promoting the suppressive functions of myeloid-derived suppressor cells, indicating that cancer cell–derived exosomes can modulate other crucial components of the immune response, not only T cells. Moreover, it was demonstrated that plasmaderived exosomes from tumor-bearing mice suppress
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tumor antigen–specific immune responses. Also noteworthy, depletion of MHC Class IIþ vesicles from plasma-derived exosomes or using plasma-derived exosomes isolated from MHC Class II–deficient mice resulted in significant abrogation of the suppressive effect.39 These results highlight the role of MHC Class IIþ exosomes in suppressing the immune response specific to tumor antigens. Exosomes derived from tumor cells have drawn intense attention because of the immunogenic activities of these exosomes in the induction of antitumor immune response. Exosomes derived from a rat metastatic pancreas adenocarcinoma BSp73ASML cell line support leukocyte-effector functions and have only a minor impact on leukocyte activation.40 This observation suggests that ASML exosomes might well serve as an adjuvant to immunotherapy. Chemokinecontaining exosomes derived from heat-stressed tumor cells are functionally competent in chemoattracting and activating CD11cþDC and CD4þ/CD8þ T cells both in vitro and in vivo, thus inhibiting tumor growth and prolonging survival of tumor-bearing mice.41 These immunogenic activities of exosomes confer the potential for being an efficient tumor vaccine to cancer-derived exosomes.
Potential of Cancer Cell–Derived Exosomes in Diagnosis Tumor-derived exosomes can be isolated from tumors and bodily fluids in patients with tumors. Exosomes contain many proteins and RNAs from neoplastic cells of origin.19 Therefore, some clinicians and researchers have turned to the study of exploring the potential of exosomes as biomarkers. Ex vivo analysis of exosomes may provide biomarkerdiscovery platforms and facilitate disease diagnosis and monitoring.
Potential of Cancer Cell–Derived Exosomes as Cancer Biomarkers A large amount of proteomic/miR/mRNA data regarding cancer cell–derived exosomes has been obtained from studies of human colorectal cancer breast cancer, bladder cancer, mesothelioma, melanoma, and medulloblastoma.7,42 These exosomes carry the genomic and proteomic signatures characteristic of the tumor cells from which they were derived. For example, as an oncogene, full-length EGFR (170 kDa) has been identified in exosomes isolated from
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Clinical Therapeutics pancreatic cell lines.43 Let-7 miR, correlated with poor survival in patients with lung cancer, has been identified in exosomes isolated from a metastatic gastric cancer cell line.44 Hong et al29 profiled the mRNA of exosomes derived from the colorectal cancer cell line SW480 and identified 411,000 distinct mRNA molecules. Of these, 2% were overexpressed at least 2-fold in exosomes (compared with levels in the parental cell line). These unique signatures make exosomes a potential tool for cancer detection.
Potential for Circulating Exosomes and Urinary Exosomes as Cancer Biomarkers Exosomes in the bodily fluids of patients with tumors are the same exosomes found in healthy individuals plus cancer-derived exosomes. Biological molecules characteristic of cancers can be detected in exosomes derived from various bodily fluids, such as serum, plasma, malignant ascites, and urine.4 Among them, cancer-derived circulating exosomes and urinary exosomes attract more attention because they are easy to obtain without invasive sampling procedures. Several studies have investigated the different possibilities of circulating exosomes as cancer biomarkers. Recent evidence has suggested that cancer cell–derived exosomes in the circulation are a clinical marker of poor prognosis.45 For example, the concentration of exosomes reported in the circulation of patients with ovarian cancer was 3- to 4-fold higher compared with that in healthy individuals.46 However, marker proteins that allow enrichment of tumor-derived exosomes over normal exosomes are less well-defined. Rupp et al47 observed that CD24 cells were selectively present on ascites exosomes of patients with tumor and were present on serum exosomes in patients with breast cancer, which suggests that CD24 could be an additional marker for the enrichment of tumor-derived exosomes from blood. Expression patterns of a recently identified biomarker family, miR, appear to be characteristic of tumor type and developmental origin. Therefore, an miR signature of circulating cancer cell–derived exosomes could be a candidate diagnostic biomarker of cancer. For example, Rabinowits et al45 evaluated the circulating levels of tumor exosomes, exosomal small RNA, and specific exosomal miRs in patients with and without lung adenocarcinoma, correlating the levels with the American Joint Committee on Cancer disease stage to validate it as an acceptable marker for
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diagnosis and prognosis in patients with adenocarcinoma of the lung. Taylor and Gercel-Taylor48 reported that the levels of the 8 specific miRs previously demonstrated as diagnostic were similar to those from ovarian tumor cells and from sera specimens. Additionally, sera exosomal miR from patients with ovarian cancer was significantly distinct from profiles observed in benign disease. Similarly, circulating miR signatures of tumor-derived exosomes can be used for early diagnosis of non–small-cell lung cancer.49 These results suggest that miR profiling of circulating tumor exosomes could potentially be used as a surrogate diagnostic marker for biopsy profiling. Urine-derived exosomes could be candidate diagnostic biomarkers or treatment-response markers of urologic cancer. The 2 known prostate cancer biomarkers, prostate cancer antigen 3 (PCA-3) and v-ets erythroblastosis virus E26 oncogene homolog (TMPRSS2:ERG), have been detected in the urinary exosomes of patients with prostate cancer.50 Similarly, 2 prostate markers, prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA), were present in 20 of 24 of urinary exosome specimens derived from prostate cancers and were not detected in healthy donor specimens. There was a clear treatment-related decrease in exosomal prostate markers in 1 of 8 patients.51 Although in their study the quantity and quality of exosomes present in urine was highly variable, this approach holds promise as a noninvasive source of multiple markers of malignancy that could provide clinically useful information. All this evidence implies that bodily fluid–derived exosomes could be a noninvasive tool for the early diagnosis and monitoring of cancer. More studies need to be conducted to evaluate the feasibility of bodily fluid–derived exosomes for diagnosis.
Potential of Exosomes in Therapy Immune Therapy Because cancer cell–derived exosomes contain many molecules characteristic of their matched tumors, exosomes have been proposed as a vehicle by which cytotoxic T-lymphocyte responses could be increased by the interaction of tumor exosomes and DCs. In murine models, mature DC-derived exosomes elicit immune activation, resulting in tumor eradication and bacterial or virus elimination.52 Given the fact that exosomes are stable during purification and will not go through phenotypic changes in vivo,53 patients with
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Y. Sun and J. Liu malignant diseases were vaccinated with autologous DC-derived exosomes in Phase I and II clinical trials; this treatment was reported to provide a curative effect with few adverse events.54,55 However, immature or suppressive DC-derived exosomes harbor anti-inflammatory properties distinct from mature DC-derived exosomes. In murine models of autoimmune disease and transplantation, immature DC-derived exosomes reduced T-cell–dependent immunoactivation, relieved clinical manifestation of autoimmune disease, and prolonged survival time after transplantation.56
cartridges that integrate into standard dialysis units or continuous renal-replacement therapy machines. As a patient’s blood passes through the device, plasma components (o200 nm) travel through the porous fibers and interact with the immobilized affinity agent (s) to which target molecules are selectively adsorbed while blood cells and unbound serum components pass through the device.59 This strategy offers an approach for targeting exosomes that should be examined for its utility as an therapeutic candidate adjunct to standard-of-care cancer treatments.
Drug Delivery
DISCUSSION
Because exosomes have a specific cell tropism according to their characteristics, they can be used to target specific tissues and/or organs. For example, modified exosomes GE11 peptide (amino acid sequence YHWYGYTPQNVI) or EGF on their surfaces delivered miR to EGFR-expressing cancer tissues; intravenously injected exosomes targeting EGFR delivered let-7a specifically to xenograft breast cancer cells in recombination activating gene 2 (Rag2)–/– mice. These data indicate that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.57 Some studies have provided evidence of using exosomes as a delivery vehicle loaded with conventional drugs. For example, curcumin, a natural polyphenol found in the rhizomes of Curcuma longa (turmeric), exhibits anti-inflammatory, antineoplastic, antioxidant, and chemopreventive activity. However, the poor solubility of curcumin, due to its hydrophobic property and preferential interaction with lipid membranes, remains a major barrier in its bioavailability and clinical efficacy. Sun et al58 reported that exosomes can deliver curcumin, increasing its solubility and bioavailability to activate myeloid cells in vivo, without unwanted off-target effects that limit their utility. This result suggests an opportunity for using exosomes as a carrier of treatments of many inflammation-related diseases.
All of the exposed evidence points to communication between cancer cells and their surroundings, either mediated by cancer cell–derived exosomes or by stromal cell–derived exosomes, which probably supports tumor proliferation, motility, invasion, angiogenesis, and premetastatic niche preparation. In addition, recent research implies that cancer cell– derived exosomes play a suppressive role in cancerdirected immune response. The biomarkers detected in bodily fluid–derived exosomes imply a potential for exosomes in cancer diagnosis. Also, exosomes could be used as a vehicle to selectively deliver therapeutic nucleic-acid drugs or conventional drugs for tumor therapy. The tolerability and feasibility of cancer exosomes in diagnosis and therapy need to be further evaluated.
Removal of Cancer Exosomes Given the recent appreciation for the roles of cancer exosomes as malignancy-associated factors, an extracorporeal strategy for specifically targeting exosomes is an attractive therapeutic option for cancer. A therapeutic hemofiltration approach* has been derived, which consists of immobilized affinity agents in the outer capillary space of hollow-fiber plasma-separator
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CONCLUSIONS The biomarkers detected in bodily fluid–derived exosomes implicate a potential for exosomes in cancer diagnosis. Also, exosomes could be used as a vehicle to selectively deliver therapeutic nucleic-acid drugs or conventional drugs for tumor therapy. The tolerability and feasibility of cancer exosomes in diagnosis and therapy need to be further evaluated.
ACKNOWLEDGMENTS This work was funded by the Chinese National Natural Science Foundation (grants 81071009 and 81271412); International S&T Cooperation Project of the Ministry of S&T of China (grant 2010D FR30850); The People’s Livelihood S&T Project, Bureau of S&T of Dalian (grants 2010E11SF008 *Trademark: ADAPT™ (Adaptive Dialysis-Like Affinity Platform Technology; Aethlon Medical Inc, San Diego, California).
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Clinical Therapeutics and 2011E12SF030); and the Scientific Research Foundation for Returned Overseas Chinese Scholars, Ministry of Human Resources and Social. All authors contributed equally to the literature search, data interpretation, and writing of the manuscript.
CONFLICTS OF INTEREST The authors have indicated that they have no conflicts of interest regarding the content of this article.
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tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008;10:1470–1476. Hong BS, Cho JH, Kim H, et al. Colorectal cancer cell–derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells. BMC Genomics. 2009;10:556. Kucharzewska P, Christianson HC, Welch JE, et al. Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. Proc Natl Acad Sci U S A. 2013;110:7312–7317. Jung T, Castellana D, Klingbeil P, et al. CD44v6 dependence of premetastatic niche preparation by exosomes. Neoplasia. 2009;11: 1093–1105. Hood JL, San RS, Wickline SA. Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis. Cancer Res. 2011;71:3792–3801. Viaud S, Théry C, Ploix S, et al. Dendritic cell-derived exosomes for cancer immunotherapy: what’s next? Cancer Res. 2010;70:1281–1285. Filipazzi P, Burdek M, Villa A, et al. Recent advances on the role of tumor exosomes in immunosuppression and disease progression. Semin Cancer Biol. 2012;22:342–349. Taylor DD, Gercel-Taylor C, Lyons KS, et al. T-cell apoptosis and suppression of T-cell receptor/CD3-zeta by Fas ligand containing membrane vesicles shed from ovarian tumors. Clin Cancer Res. 2003;9:5113–5119. Wada J, Onishi H, Suzuki H, et al. Surface-bound TGF-beta1 on effusion-derived exosomes participates in maintenance of number and suppressive function of regulatory T-cells in malignant effusions. Anticancer Res. 2010;30: 3747–3457. Clayton A, Al-Taei S, Webber J, et al. Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production. J Immunol. 2011;187:676–683.
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38. Chalmin F, Ladoire S, Mignot G, et al. Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J Clin Invest. 2010;120:457– 471. 39. Yang C, Ruffner MA, Kim SH, Robbins PD, Plasma-derived MHC. class IIþ exosomes from tumorbearing mice suppress tumor antigen-specific immune responses. Eur J Immunol. 2012;42:1778– 1784. 40. Zech D, Rana S, Buchler MW, Zoller M. Tumor-exosomes and leukocyte activation: an ambivalent crosstalk. Cell Commun Signal. 2012;10:37. 41. Chen T, Guo J, Yang M, et al. Chemokine-containing exosomes are released from heat-stressed tumor cells by lipid raft-dependent pathway and act as efficient tumor vaccine. J Immunol. 2011; 186:2219–2228. 42. Henderson MC, Azorsa DO. The genomic and proteomic content of cancer cell–derived exosomes. Front Oncol. 2012;2:38. 43. Adamczyk KA, Klein-Scory S, Tehrani MM, et al. Characterization of soluble and exosomal forms of the EGFR released from pancreatic cancer cells. Life Sci. 2011;89:304–312. 44. Ohshima K, Inoue K, Fujiwara A, et al. Let-7 microRNA family is selectively secreted into the extracellular environment by exosomes in a metastatic gastric cancer cell line. PLoS One. 2010;5:e13247. 45. Rabinowits G, Gercel-Taylor C, Day JM, et al. Exosomal microRNA: a diagnostic marker for lung cancer. Clin Lung Cancer. 2009;10: 42–46. 46. Went PT, Lugli A, Meier S, et al. Frequent EpCam protein expression in human carcinomas. Hum Pathol. 2004;35:122–128. 47. Rupp AK, Rupp C, Keller S, et al. Loss of EpCAM expression in breast
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cancer derived serum exosomes: role of proteolytic cleavage. Gynecol Oncol. 2011;122:437–446. Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol. 2008;110:13–21. Rosell R, Wei J, Taron M. Circulating microRNA signatures of tumorderived exosomes for early diagnosis of non-small-cell lung cancer. Clin Lung Cancer. 2009;10:8–9. Nilsson J, Skog J, Nordstrand A, et al. Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer. Br J Cancer. 2009;100:1603–1607. Mitchell PJ, Welton J, Staffurth J, et al. Can urinary exosomes act as treatment response markers in prostate cancer? J Transl Med. 2009;7:4. del Cacho E, Gallego M, Lee SH, et al. Induction of protective immunity against Eimeria tenella. Eimeria maxima, and Eimeria acervulina infections using dendritic cell-derived exosomes. Infect Immun. 2012;80:1909–1916. Luketic L, Delanghe J, Sobol PT, et al. Antigen presentation by exosomes released from peptide-pulsed dendritic cells is not suppressed by the presence of active CTL. J Immunol. 2007;179:5024–5032. Escudier B, Dorval T, Chaput N, et al. Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical trial. J Transl Med. 2005;3:10. Morse MA, Garst J, Osada T, et al. A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer. J Transl Med. 2005;3:9. Yin W, Ouyang S, Li Y, et al. Immature dendritic cell-derived exosomes: a promise subcellular vaccine for autoimmunity. Inflammation. 2013;36:232–240. Ohno S, Takanashi M, Sudo K, et al. Systemically injected exosomes
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Clinical Therapeutics targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol Ther. 2013;21:185–191. 58. Sun D, Zhuang X, Xiang X, et al. A novel nanoparticle drug delivery system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes. Mol Ther. 2010;18:1606–1614. 59. Marleau AM, Chen CS, Joyce JA, Tullis RH. Exosome removal as a therapeutic adjuvant in cancer. J Transl Med. 2012;10:134.
Address correspondence to: Jing Liu, Regenerative Medicine Center, First Affiliated Hospital of Dalian Medical University, No. 222 Zhongshan Road, Dalian 116011, P.R. China. E-mail: [email protected]
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Potential of Cancer Cell–Derived Exosomes in Clinical Application: A Review of Recent Research Advances Yu Sun, PhD1; and Jing Liu, PhD1,2 1
Regenerative Medicine Centre, First Affiliated Hospital, Dalian Medical University, Dalian, P.R. China; and 2Institute of Integrative Medicine, Dalian Medical University, Dalian, P.R. China
ABSTRACT Background: Exosomes are 30- to 100-nm, membrane-bound vesicles that are released by most types of cells, including tumor cells. Exosomes contain a great variety of bioactive molecules, including signal peptides, microRNA, lipids, and DNA. In cancer, tumor cells aberrantly secrete large quantities of exosomes to transport paracrine signals or to contribute to tumor–environment interaction at a distance. Objective: The goal of this review was to discuss the recent advances on the mechanism of cancerderived exosomes in tumor regulation. Methods: Pertinent articles and abstracts were identified through searches of PubMed for literature published from 1983 to December 2013. Search terms included exosome, tumor, cancer, diagnosis, and therapy. Results: All of the exposed evidence points to communication between cancer cells and their surroundings, either mediated by cancer cell–derived exosomes or by stromal cell–derived exosomes. This communication probably supports tumor proliferation, motility, invasion, angiogenesis, and premetastatic niche preparation. In addition, recent research implies that cancer cell–derived exosomes play a suppressive role in cancer-directed immune response. Conclusions: The biomarkers detected in bodily fluid– derived exosomes imply a potential for exosomes in cancer diagnosis. Also, exosomes could be used as a vehicle to selectively deliver therapeutic nucleic-acid drugs or conventional drugs for tumor therapy. The tolerability and feasibility of cancer exosomes in diagnosis and therapy need to be further evaluated. (Clin Ther. 2014;]:]]]–]]]) & 2014 Elsevier HS Journals, Inc. All rights reserved. Key words: cancer, diagnosis, exosomes, therapy.
vesicles containing definite receptors to ensure traffic specificity. The research accumulated over the past 10 years has demonstrated that a heterogeneous group of vesicles is also released from the cell surface and is used as intercellular signalosomes in information exchange, even over a long distance.1 These membranous vesicles, released by a variety of cells and generally termed extracellular vesicles, can be divided into 3 main classes: exosomes, microvesicles (100– 1000 nm), and apoptotic bodies (1–5 μm). Exosomes are 30 to 100 nm in diameter and shed from many different types of cells under both normal and pathologic conditions.2 These exosomes can be formed through inward budding of endosomal membranes, giving rise to intracellular multivesicular bodies that later fuse with the plasma membrane, releasing the exosomes to the exterior.3 They are released from many different cell types in the body, such as red blood cells, platelets, lymphocytes, dendritric cells (DCs), and tumor cells.4 Depending on the cellular origin, exosomes contain various cellular proteins. These proteins may be different from proteins that are normally located in the plasma membrane, including major histocompatibility complex (MHC) molecules, tetraspanins, adhesion molecules, and metalloproteinases.5,6 Exosomes also contain signal proteins and/or peptides, microRNAs (miRs), mRNAs, and lipids that can be transferred to a recipient cell by fusion of the exosome with the target cell membrane.7,8 It has been proposed that this transfer of protein and RNAs from one cell to another might represent a novel mechanism of intercellular communication.9,10 Initially, exosomes were described as vesicles released by reticulocytes. They were thought to function as a way to remove unnecessary proteins, such as the transferrin
INTRODUCTION
Accepted for publication April 24, 2014. http://dx.doi.org/10.1016/j.clinthera.2014.04.018 0149-2918/$ - see front matter
Cells are known to deliver proteins and molecules between the intracellular organelles by membrane
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& 2014 Elsevier HS Journals, Inc. All rights reserved.
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Clinical Therapeutics receptor, during the process of maturation into erythrocytes.3 Emerging evidence indicates exosomes as important mediators of cellular communication. Exosomes are involved not only in normal physiologic processes, such as lactation, immune response, and neuronal function, but also in the development and progression of diseases, such as liver disease, neurodegenerative diseases, and cancer.11 However, the detailed mechanisms of exosomes in physiologic and pathologic regulation are still being researched. Exosomes can also be isolated from various bodily fluids, such as breast milk, serum, plasma, malignant ascites, and urine.4 Therefore, ex vivo analysis of exosomes may provide biomarker-discovery platforms and facilitate disease diagnosis and monitoring. The goal of this review was to discuss the recent advances on the mechanism of cancer-derived exosomes in tumor regulation.
MATERIALS AND METHODS Pertinent articles and abstracts were identified through searches of PubMed for literature published from 1983 to December 2013. Search terms included exosome, tumor, cancer, diagnosis, and therapy.
RESULTS Exosomes and Cancer Exosomes are secreted by 2 different mechanisms: (1) constitutive release by the trans-Golgi network and (2) inducible release.12 In the past decade, studies have found that tumor cells constitutively release exosomes. Like most nontransformed cells, tumor cells release exosomes whose compositions vary depending on the nature and conditions of the cells. Tumor cell–secreted exosomes include many of the common exosomal proteins; they also contain tumor antigens that are reflective of the tumors from which they are derived,2
Cancer cells Wnt-PCP signaling
Matrix
Target cell
Horizontal transfer
FasL
Cytokine, ECM production
CAF
mRNA protein
TGF-
microRNA exosome
Stromal reorganization
Angiogenic proteins or mRNAs
MHC-II MDSCs
exosome Endothelium pancrea
Immune suppression
Lymph node
pre-metastatic niche formation
Blood vessel
Diagnosis
Cancer cell exosomes CAF-derived exosomes
Angiogenesis
Cancer cells Cancer-associated fibroblasts(CAF)
Cancer-derived exosomes mediated the crosstalk
Immune cells Myeloid-derived suppressor cells (MDSCs)
Figure. Cancer-derived exosomes are implicated in transferring miRNAs, mRNA and proteins to target cells. They are able to reorganize extracellularmatrix, alter the premetastatic niche and promote angiogenesis. They also create an immunosuppressive microenvironment by impairing the immune cells, by inducing the apoptosis of T cells through FAS-FASL ligation.
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Y. Sun and J. Liu such as the tumor antigens melanoma antigen recognized by T cells 1 (MelanA/Mart-1) and glycoprotein 100 (GP100), carcinoembryogenic antigen (CEA), and human epidermal growth factor receptor 2 (Her2).13,14 Cancer cell–derived exosomes are emerging as local and systemic intercellular mediators of oncogenic information through the horizontal transfer of mRNAs, miRs, and proteins during tumorigenesis.15,16 For example, gliomas often express a truncated and oncogenic form of the epidermal growth factor receptor (EGFR) vIII. Within each tumor, only a small percentage of gliomal cells may actually express EGFRvIII; however, most of the cells exhibit a transformed phenotype. It has been revealed that gliomal cell–derived exosomes support the propagation of malignancy through the transfer of EGFRvIII.17 McCready et al18 found that one of the multiple isoforms of heat shock protein (Hsp) 90 is secreted by exosomes from invasive cancer cells and contributes to their motility and invasiveness. Cancer-derived exosomes are of great importance in the communication between cancer cells and the tumor-associated microenvironment or the immune system. This subject has been heavily investigated, but we are still awaiting a clear answer. Additionally, tumor-derived exosomes can be isolated from the bodily fluids of patients with tumors. Both cancer cell–derived exosomes and bodily fluid– derived exosomes contain proteins and RNAs originating from neoplastic cells.19 Therefore, these characteristics reveal the potential for cancer-derived exosomes as biomarkers in cancer diagnosis.
Internalization of Cancer Cell–Derived Exosomes The exosomal protein profile is rich in molecules located in membrane domains and is prone to internalization such as rafts and tetraspanin-enriched microdomains, as well as molecules engaged in fission, scission, and vesicular transport; adhesion molecules; and proteases.20 Studies aimed at revealing the mechanism of cancer cell–derived exosome uptake by target cells have reported that cancer cell–derived exosomes can also use heparin sulfate proteoglycans (HSPGs) for their internalization. HSPGs are a family of proteins substituted with glycosaminoglycan polysaccharides, which are extensively modified by sulfation to determine their functional interactions. Several viruses have previously been reported to enter cells
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through HSPGs. Christianson et al21 provided evidence that HSPGs function as receptors of cancer cell–derived exosomes, which was dependent on intact HS, specifically on the 2-O and N-sulfation groups. Importantly, their data indicated that the HSPGdependent uptake route is highly relevant to the biological activity of exosomes.21 In addition, like all exosomes, tumor exosomes can transfer their contents to alter the physiologic states of recipient cells. O’Brien et al22 found that exosomes from triple-negative breast cancer sera significantly increased recipient cells’ invasion, conferring phenotypic traits to secondary cells. Given all of this evidence, it appears that HSPG may be a potential target for inhibition of exosomemediated content transfer and/or subsequent exosome-associated tumor development.
Cancer Cell–Derived Exosomes and Mediation of Communication Between Tumor Cells and the Tumor Microenvironment One important factor in tumor biology is that it relies on the capacity of tumor cells to create a microenvironment by recruiting and modulating nontransformed cells that support tumor cell survival and spread/propagation.23 The tumor-associated microenvironment consists of an extracellular matrix (ECM), cancer-associated fibroblasts, inflammatory immune cells, and tumor-associated vasculature.17 Tumorderived exosomes are of great importance in the communication between cancer cells and the tumorassociated environment, a subject being heavily discussed but still awaiting a clear answer. In this section, we provide the recent achievements on the mechanism of the communication between cancer cell–derived exosomes and the tumor microenvironment (Figure).
Cancer Cell–Derived Exosomes and the Modulation of Stromal Cells The ECM is an essential constituent of nontransformed tissues and tumors. It is now well-established that primary tumors are composed of a multitude of stromal cell types in addition to cancerous cells.24 By using green fluorescent protein–tagged CD63, a general marker of exosomes, Suetsugu et al25 imaged exosomes transferred from breast cancer cells to host cells, including red fluorescent protein (RFP) cancer– associated fibroblasts and RFP–lung tissue stromal cells in orthotopic nude mouse models. There is also evidence that cancer cell–derived exosomes stimulate
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Clinical Therapeutics secretion of the oncogenic receptor EGFRvIII20 and several proangiopoietic factors, such as matrix metalloproteinase 9, vascular endothelial growth factor, interleukin 8, and hepatocyte growth factor by stromal cells; induce proliferation of endothelial cells; and promote angiogenesis in metastatic organs.26 These results suggest that cancer cell–derived exosomes are incorporated into stromal cells, which may have consequences on the secretion of some biological factors by stromal cells to promote tumor metastasis.
Cancer Cell–Derived Exosomes and Reorganization of the Extracellular Matrix In addition to the effect of cancer cell–derived exosomes on stromal cells, recent data have also demonstrated a pivotal role for cancer cell–derived exosomes in the organization of the ECM. It has been reported that cancer cell–derived exosomes, being rich in proteases, modulate the ECM for degradation of collagens, laminin, and fibronectin, and that matrix degradation by cancer cell–derived exosomes has severe consequences on tumor and host cell adhesion, motility, and invasiveness.20 In that study, the investigators also provided evidence that tumor cell– derived exosomes bind to individual components of the ECM, the preferential partner depending on the exosomes’ adhesion-molecule profile, such that high CD44 expression is accompanied by hyaluronic acid binding and high α6β4 expression by laminin-332 binding. All of these findings imply that cancer cell–derived exosomes are able to alter the stroma, either by modulating the stromal cells or by degradating stroma.
Stromal Cell Exosomes and Regulation of Cancer Cells Stroma in the tumor microenvironment plays a crucial role in cancer progression. In addition to the effect of cancer cell–derived exosomes on stroma, the latest evidence has reported that stroma can also influence the biological behavior of cancer cells, which is mediated by stromal cell exosomes. Luga et al27 reported that exosomes secreted by stromal cells, such as cancer-associated fibroblasts, are taken up by breast cancer cells and promote the protrusive activity and motility of breast cancer cells by Wnt/planar cell polarity signaling. This work highlights the role of
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stromal cell–derived exosomes in modulating tumor cell metastasis. So far, all of these results suggest that exosomes are agents of communication between cancer cells and stroma to stimulate metastasis.
Cancer Cell–Derived Exosomes and Promotion of Angiogenesis Cancer cell–derived exosomes are also important for inducing angiogenesis. Most of the experimental evidence points to a potential involvement of exosomes in angiogenesis. Angiogenic proteins or mRNAs that promote proliferation of endothelial cells have been observed in cancer cell–secreted exosomes. For example, glioblastoma tumor cells release microvesicles (exosomes) containing mRNA, miRs, and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells.28 Hong et al29 demonstrated that colorectal cancer cell–derived exosomes are enriched in cell cycle–related mRNAs that promote proliferation of endothelial cells, suggesting that the microvesicles of cancer cells may be involved in tumor growth and metastasis by facilitating angiogenesisrelated processes. Recent research has also demonstrated that in a model of highly malignant brain tumor glioblastoma multiforme, exosomes derived from glioblastoma multiforme cells grown in hypoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells.30 So far, all of the exposed evidence points to communication between cancer cells their surroundings, either mediated by cancer cell–derived exosomes or by stromal cell–derived exosomes. This probably supports tumor proliferation, motility, invasion, angiogenesis, and premetastatic niche preparation.
Cancer Cell–Derived Exosomes and Modulation of Metastatic Niche Preparation A premetastatic niche is a distant target site for metastasis adapted to cancer cell seeding before the arrival of the first cancer cells. Jung et al31 found that cancer cell–derived exosomes modulated premetastatic organs for cancer cell settlement and proliferation in a rat model of pancreatic adenocarcinoma. An in vivo study showed melanoma exosomes homed to sentinel lymph nodes and imposed synchronized molecular signals that effected melanoma cell
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Y. Sun and J. Liu recruitment, ECM deposition, and vascular proliferation in the lymph nodes.32 These findings imply that cancer cell–derived exosomes are able to mediate the process of the microanatomic niche preparation that facilitates cancer cell metastasis.
Cancer Cell–Derived Exosomes and Suppression of Immune Response Exosomes play an important role in modulating immune responses. When derived from DCs, they bear MHC proteins and co-stimulatory molecules.33 These vesicles are effective in stimulating antigen-specific Tcell responses in vitro and in vivo. However, cancer cell–derived exosomes can suppress cancer-directed immune response.34 The interactions between cancer cell–derived exosomes and immune cells are mediated through direct signaling interactions by surfaceexpressed molecules or by the transfer of exosomes and/or their contents to immune cells. For example, tumor necrosis superfamily, member 6 ligand protein (FasL) on the surfaces of tumor cell–derived exosomes mediates cleavage of the T-cell surface glycoprotein CD3 (TCR)-ζ chain, a crucial T-cell–signaling molecule that is required for activation.35 Tumor-derived exosomes also display transforming growth factor β on their surfaces, which maintains the numbers and immunosuppressive effects of regulatory T cells in vitro.36 Additionally, a recent study has reported that cancer cell–derived exosomes can modulate T cells by hydrolyzing ATP into adenosine in the tumor microenvironment. Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune responses. A study by Clayton et al37 demonstrated that cancer cell–derived exosomes possess enzymatic activity that causes hydrolysis of ATP into adenosine in the tumor microenvironment, which negatively regulates T-cell activation. Myeloidderived suppressor cells have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T-cell activation. Chalmin et al38 showed that in both mice and humans, Hsp72-expressing exosomes restrain tumor immunosurveillance by promoting the suppressive functions of myeloid-derived suppressor cells, indicating that cancer cell–derived exosomes can modulate other crucial components of the immune response, not only T cells. Moreover, it was demonstrated that plasmaderived exosomes from tumor-bearing mice suppress
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tumor antigen–specific immune responses. Also noteworthy, depletion of MHC Class IIþ vesicles from plasma-derived exosomes or using plasma-derived exosomes isolated from MHC Class II–deficient mice resulted in significant abrogation of the suppressive effect.39 These results highlight the role of MHC Class IIþ exosomes in suppressing the immune response specific to tumor antigens. Exosomes derived from tumor cells have drawn intense attention because of the immunogenic activities of these exosomes in the induction of antitumor immune response. Exosomes derived from a rat metastatic pancreas adenocarcinoma BSp73ASML cell line support leukocyte-effector functions and have only a minor impact on leukocyte activation.40 This observation suggests that ASML exosomes might well serve as an adjuvant to immunotherapy. Chemokinecontaining exosomes derived from heat-stressed tumor cells are functionally competent in chemoattracting and activating CD11cþDC and CD4þ/CD8þ T cells both in vitro and in vivo, thus inhibiting tumor growth and prolonging survival of tumor-bearing mice.41 These immunogenic activities of exosomes confer the potential for being an efficient tumor vaccine to cancer-derived exosomes.
Potential of Cancer Cell–Derived Exosomes in Diagnosis Tumor-derived exosomes can be isolated from tumors and bodily fluids in patients with tumors. Exosomes contain many proteins and RNAs from neoplastic cells of origin.19 Therefore, some clinicians and researchers have turned to the study of exploring the potential of exosomes as biomarkers. Ex vivo analysis of exosomes may provide biomarkerdiscovery platforms and facilitate disease diagnosis and monitoring.
Potential of Cancer Cell–Derived Exosomes as Cancer Biomarkers A large amount of proteomic/miR/mRNA data regarding cancer cell–derived exosomes has been obtained from studies of human colorectal cancer breast cancer, bladder cancer, mesothelioma, melanoma, and medulloblastoma.7,42 These exosomes carry the genomic and proteomic signatures characteristic of the tumor cells from which they were derived. For example, as an oncogene, full-length EGFR (170 kDa) has been identified in exosomes isolated from
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Clinical Therapeutics pancreatic cell lines.43 Let-7 miR, correlated with poor survival in patients with lung cancer, has been identified in exosomes isolated from a metastatic gastric cancer cell line.44 Hong et al29 profiled the mRNA of exosomes derived from the colorectal cancer cell line SW480 and identified 411,000 distinct mRNA molecules. Of these, 2% were overexpressed at least 2-fold in exosomes (compared with levels in the parental cell line). These unique signatures make exosomes a potential tool for cancer detection.
Potential for Circulating Exosomes and Urinary Exosomes as Cancer Biomarkers Exosomes in the bodily fluids of patients with tumors are the same exosomes found in healthy individuals plus cancer-derived exosomes. Biological molecules characteristic of cancers can be detected in exosomes derived from various bodily fluids, such as serum, plasma, malignant ascites, and urine.4 Among them, cancer-derived circulating exosomes and urinary exosomes attract more attention because they are easy to obtain without invasive sampling procedures. Several studies have investigated the different possibilities of circulating exosomes as cancer biomarkers. Recent evidence has suggested that cancer cell–derived exosomes in the circulation are a clinical marker of poor prognosis.45 For example, the concentration of exosomes reported in the circulation of patients with ovarian cancer was 3- to 4-fold higher compared with that in healthy individuals.46 However, marker proteins that allow enrichment of tumor-derived exosomes over normal exosomes are less well-defined. Rupp et al47 observed that CD24 cells were selectively present on ascites exosomes of patients with tumor and were present on serum exosomes in patients with breast cancer, which suggests that CD24 could be an additional marker for the enrichment of tumor-derived exosomes from blood. Expression patterns of a recently identified biomarker family, miR, appear to be characteristic of tumor type and developmental origin. Therefore, an miR signature of circulating cancer cell–derived exosomes could be a candidate diagnostic biomarker of cancer. For example, Rabinowits et al45 evaluated the circulating levels of tumor exosomes, exosomal small RNA, and specific exosomal miRs in patients with and without lung adenocarcinoma, correlating the levels with the American Joint Committee on Cancer disease stage to validate it as an acceptable marker for
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diagnosis and prognosis in patients with adenocarcinoma of the lung. Taylor and Gercel-Taylor48 reported that the levels of the 8 specific miRs previously demonstrated as diagnostic were similar to those from ovarian tumor cells and from sera specimens. Additionally, sera exosomal miR from patients with ovarian cancer was significantly distinct from profiles observed in benign disease. Similarly, circulating miR signatures of tumor-derived exosomes can be used for early diagnosis of non–small-cell lung cancer.49 These results suggest that miR profiling of circulating tumor exosomes could potentially be used as a surrogate diagnostic marker for biopsy profiling. Urine-derived exosomes could be candidate diagnostic biomarkers or treatment-response markers of urologic cancer. The 2 known prostate cancer biomarkers, prostate cancer antigen 3 (PCA-3) and v-ets erythroblastosis virus E26 oncogene homolog (TMPRSS2:ERG), have been detected in the urinary exosomes of patients with prostate cancer.50 Similarly, 2 prostate markers, prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA), were present in 20 of 24 of urinary exosome specimens derived from prostate cancers and were not detected in healthy donor specimens. There was a clear treatment-related decrease in exosomal prostate markers in 1 of 8 patients.51 Although in their study the quantity and quality of exosomes present in urine was highly variable, this approach holds promise as a noninvasive source of multiple markers of malignancy that could provide clinically useful information. All this evidence implies that bodily fluid–derived exosomes could be a noninvasive tool for the early diagnosis and monitoring of cancer. More studies need to be conducted to evaluate the feasibility of bodily fluid–derived exosomes for diagnosis.
Potential of Exosomes in Therapy Immune Therapy Because cancer cell–derived exosomes contain many molecules characteristic of their matched tumors, exosomes have been proposed as a vehicle by which cytotoxic T-lymphocyte responses could be increased by the interaction of tumor exosomes and DCs. In murine models, mature DC-derived exosomes elicit immune activation, resulting in tumor eradication and bacterial or virus elimination.52 Given the fact that exosomes are stable during purification and will not go through phenotypic changes in vivo,53 patients with
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Y. Sun and J. Liu malignant diseases were vaccinated with autologous DC-derived exosomes in Phase I and II clinical trials; this treatment was reported to provide a curative effect with few adverse events.54,55 However, immature or suppressive DC-derived exosomes harbor anti-inflammatory properties distinct from mature DC-derived exosomes. In murine models of autoimmune disease and transplantation, immature DC-derived exosomes reduced T-cell–dependent immunoactivation, relieved clinical manifestation of autoimmune disease, and prolonged survival time after transplantation.56
cartridges that integrate into standard dialysis units or continuous renal-replacement therapy machines. As a patient’s blood passes through the device, plasma components (o200 nm) travel through the porous fibers and interact with the immobilized affinity agent (s) to which target molecules are selectively adsorbed while blood cells and unbound serum components pass through the device.59 This strategy offers an approach for targeting exosomes that should be examined for its utility as an therapeutic candidate adjunct to standard-of-care cancer treatments.
Drug Delivery
DISCUSSION
Because exosomes have a specific cell tropism according to their characteristics, they can be used to target specific tissues and/or organs. For example, modified exosomes GE11 peptide (amino acid sequence YHWYGYTPQNVI) or EGF on their surfaces delivered miR to EGFR-expressing cancer tissues; intravenously injected exosomes targeting EGFR delivered let-7a specifically to xenograft breast cancer cells in recombination activating gene 2 (Rag2)–/– mice. These data indicate that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.57 Some studies have provided evidence of using exosomes as a delivery vehicle loaded with conventional drugs. For example, curcumin, a natural polyphenol found in the rhizomes of Curcuma longa (turmeric), exhibits anti-inflammatory, antineoplastic, antioxidant, and chemopreventive activity. However, the poor solubility of curcumin, due to its hydrophobic property and preferential interaction with lipid membranes, remains a major barrier in its bioavailability and clinical efficacy. Sun et al58 reported that exosomes can deliver curcumin, increasing its solubility and bioavailability to activate myeloid cells in vivo, without unwanted off-target effects that limit their utility. This result suggests an opportunity for using exosomes as a carrier of treatments of many inflammation-related diseases.
All of the exposed evidence points to communication between cancer cells and their surroundings, either mediated by cancer cell–derived exosomes or by stromal cell–derived exosomes, which probably supports tumor proliferation, motility, invasion, angiogenesis, and premetastatic niche preparation. In addition, recent research implies that cancer cell– derived exosomes play a suppressive role in cancerdirected immune response. The biomarkers detected in bodily fluid–derived exosomes imply a potential for exosomes in cancer diagnosis. Also, exosomes could be used as a vehicle to selectively deliver therapeutic nucleic-acid drugs or conventional drugs for tumor therapy. The tolerability and feasibility of cancer exosomes in diagnosis and therapy need to be further evaluated.
Removal of Cancer Exosomes Given the recent appreciation for the roles of cancer exosomes as malignancy-associated factors, an extracorporeal strategy for specifically targeting exosomes is an attractive therapeutic option for cancer. A therapeutic hemofiltration approach* has been derived, which consists of immobilized affinity agents in the outer capillary space of hollow-fiber plasma-separator
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CONCLUSIONS The biomarkers detected in bodily fluid–derived exosomes implicate a potential for exosomes in cancer diagnosis. Also, exosomes could be used as a vehicle to selectively deliver therapeutic nucleic-acid drugs or conventional drugs for tumor therapy. The tolerability and feasibility of cancer exosomes in diagnosis and therapy need to be further evaluated.
ACKNOWLEDGMENTS This work was funded by the Chinese National Natural Science Foundation (grants 81071009 and 81271412); International S&T Cooperation Project of the Ministry of S&T of China (grant 2010D FR30850); The People’s Livelihood S&T Project, Bureau of S&T of Dalian (grants 2010E11SF008 *Trademark: ADAPT™ (Adaptive Dialysis-Like Affinity Platform Technology; Aethlon Medical Inc, San Diego, California).
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Clinical Therapeutics and 2011E12SF030); and the Scientific Research Foundation for Returned Overseas Chinese Scholars, Ministry of Human Resources and Social. All authors contributed equally to the literature search, data interpretation, and writing of the manuscript.
CONFLICTS OF INTEREST The authors have indicated that they have no conflicts of interest regarding the content of this article.
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Address correspondence to: Jing Liu, Regenerative Medicine Center, First Affiliated Hospital of Dalian Medical University, No. 222 Zhongshan Road, Dalian 116011, P.R. China. E-mail: [email protected]
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