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Liquid biopsy and tumor derived exosomes in clinical practice
+Model PATOL-375; No. of Pages 6
ARTICLE IN PRESS
Rev Esp Patol. 2016;xxx(xx):xxx---xxx
Patología R E V I S TA
E S PA Ñ O L A
D E
www.elsevier.es/patologia
REVIEW
Liquid biopsy and tumor derived exosomes in clinical practice Pablo Sánchez-Vela a , Nahuel Aquiles Garcia b , María Campos-Segura c , Jerónimo Forteza-Vila c,∗ a
Facultad de Medicina, Universidad Católica de Valencia, Valencia, Spain Mixed Unit for Cardiovascular Repair, Instituto de Investigación Sanitaria La Fe- Centro de Investigación Príncipe Felipe, Valencia, Spain c Instituto Valenciano de Patología, Centro de Investigación Príncipe Felipe/Universidad Católica de Valencia, Valencia, Spain b
Received 25 September 2015; accepted 12 November 2015
KEYWORDS Exosomes; Cancer; Biomarkers; Liquid-biopsy; microRNA
PALABRAS CLAVE Exosomas; Cáncer; Biomarcadores; Biopsia líquida; microARN
∗
Abstract Tumors are complex tissues that interact in many different ways. Tissue biopsies provide a great amount of information and remain the gold standard for tumor diagnosis. However, they cannot always be performed due to the invasive nature of the procedure and in such circumstances, a liquid biopsy could provide a solution. Liquid biopsy is defined as the search of biomarkers in peripheral blood. To date, there are three main research fields: (1) circulating tumor cells (CTCs); (2) circulating free tumor nucleic acids (cfNA) and (3) exosomes, small vesicles containing various types of signaling molecules capable of modulating a tumor-immune response. In recent years, exosomes have arisen as a powerful tool both to further our understanding of cancer biology and to improve clinical management. We review how the isolation and study of exosomes from liquid biopsies may affect clinical practice. © 2016 Sociedad Espa˜ nola de Anatomía Patológica. Published by Elsevier España, S.L.U. All rights reserved.
Biopsia líquida y exosomas derivados del tumor en la práctica clínica Resumen Las biopsias tisulares, aunque otorgan gran cantidad de información y son el gold standard para el diagnóstico tumoral, no pueden ser realizadas continuamente para monitorizar la evolución tumoral dada su inherente naturaleza invasiva. Por otro lado, la biopsia líquida surge como una herramienta capaz de compensar estas limitaciones. En este momento se han identificado 3 tipos principales de biomarcadores en la biopsia líquida: (1) células tumorales circulantes (CTC); (2) ácidos nucleicos tumorales circulantes libres (cfNA), y (3) exosomas. En los
Corresponding author. E-mail address: [email protected] (J. Forteza-Vila).
http://dx.doi.org/10.1016/j.patol.2015.11.004 1699-8855/© 2016 Sociedad Espa˜ nola de Anatomía Patológica. Published by Elsevier España, S.L.U. All rights reserved.
Please cite this article in press as: Sánchez-Vela P, et al. Liquid biopsy and tumor derived exosomes in clinical practice. Rev Esp Patol. 2016. http://dx.doi.org/10.1016/j.patol.2015.11.004
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P. Sánchez-Vela et al. últimos a˜ nos los exosomas (peque˜ nas vesículas que contienen moléculas capaces de modular la respuesta inmune-tumoral) han surgido como una potente herramienta para comprender mejor la biología del cáncer así como una manera para intentar mejorar el manejo de los pacientes oncológicos. El objetivo de este artículo es presentar una revisión sobre cómo la caracterización de los exosomas a través de la biopsia líquida podría influir sobre la práctica clínica. © 2016 Sociedad Espa˜ nola de Anatomía Patológica. Publicado por Elsevier España, S.L.U. Todos los derechos reservados.
Introduction to the liquid biopsy
Liquid biopsy possibilities
Tumors are complex tissues that interact in many different ways. Today we assume that these particular cells are defined by their ability to sustain proliferative signaling, evade growth suppressors, resist cell death, enable replicative immortality, induce angiogenesis, reprogram energy metabolism, evade immune destruction and activate invasion and metastasis.1 Genotyping tumor samples in search of genetic alterations with predictive and prognostic information has become a daily practice in clinical oncology. However, the method of their extraction still has some limitations. The decision to administer chemotherapy and/or other therapy subsequent to tumor resection usually depends on an oncologist’s assessment of the volume of the primary tumors (T), the lymph node extension (N) and the presence of metastatic disease (M). Now the expression analysis of certain proteins and the presence or absence of certain genetic mutations in the tissue biopsy is also considered and will be a biomarker for the application of targeted therapies. Although tumor biopsies provide a great amount of information and remain the gold standard for tumor diagnosis, they cannot always be performed due to the invasive nature of the procedure. The condition of the patient or the accessibility of the tumor may make a biopsy impossible and therefore impractical for monitoring the progression or recurrence of the disease.2 Molecular genetics can differ within the same tumor and thus the small area removed in a tissue biopsy may not reflect the overall picture. Similarly, a primary tumor biopsy does not reflect the ongoing clinical course during which there may be clonal evolution and tumor heterogeneity resulting in systemic drug resistance, transition of epithelium to mesenchyme, etc. In order to avoid such ‘‘snap-shot’’ results from tumor biopsies, rapid, cost-effective and non-invasive identification of biomarkers at various moments during the disease process are required.3 Liquid biopsy could provide such a method. Liquid biopsy is defined as the search of biomarkers in peripheral blood. At this moment three main research fields may be accepted as possible biomarkers4,5 : (1) circulating tumor cells (CTCs); (2) circulating free tumor nucleic acids (cfNA) including miRNAs, mRNA and long non-coding RNA, but mainly cfDNA; and (3) exosomes, small vesicles containing various types of signaling molecules capable of modulating tumor-immune response.
cfDNA Circulating free DNA is a normal finding in healthy individuals. However cfDNA levels are higher in cancer patients in comparison to non-cancer patients. Specific detection of tumor-derived cfDNA may be a dynamic way of monitoring tumor change in response to treatment and to indicate that certain subpopulations of resistant cells may continue to proliferate despite therapeutic measures. Tracking tumorassociated abnormalities in blood can be used to assess the presence of residual, recurrent, relapse and resistance diseases.6 For example, a colon cancer cohort study showed that cfDNA may be useful in evaluating if patients were tumor free after surgery. An evaluation of the presence or absence of residual disease could help patients to avoid needless, toxic adjuvant chemotherapy.7 Furthermore, cfDNA may be used as a biomarker of when a tumor resistance to targeted therapies: a patient with colon cancer with a wild-KRAS suitable for a treatment with a EGFR (Epidermal Growth Factor Receptor) blocker such as Cetuximab could be monitored for acquired resistance to this therapy by the mean of serial blood sampling and detection of de novo KRAS, NRAS or MET mutations.6 However, before implementing cfDNA testing, standardization and prospective clinical trials are required.2
CTCs CTCs are tumor cells which have become detached from the primary solid tumor mass. They have been described in breast, prostate, lung and colon tumors (Fig. 1) and are certainly involved in the process of metastasis. Clinical quantification of CTCs is highly valuable as they represent potential metastatic cells and facilitate real time monitoring during systemic and targeted therapies via blood sampling monitoring.8 Presently, research into the assessment of the clinical value of CTCs analysis is being carried out and has already proved them to be a significant prognostic factor in metastatic breast cancer9 and other solid tumors such as colorectal,10 prostate11 and lung.12 CTCs have even proven superior to conventional imaging for monitoring therapeutic response.13 While evidence of the tumor’s genomic evolution can be also obtained by isolating cfDNA, CTCs will lead
Please cite this article in press as: Sánchez-Vela P, et al. Liquid biopsy and tumor derived exosomes in clinical practice. Rev Esp Patol. 2016. http://dx.doi.org/10.1016/j.patol.2015.11.004
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Liquid biopsy and tumor derived exosomes in clinical practice
Figure 1 Circulating Tumor Cells (CTCs) obtained from a Breast Cancer patient blood before neoadjuvant chemotherapy and surgery. Courtesy of Dr. José Juan Gaforio Martínez and its team from the Immunology Department at the University of Jaén.
to a deeper understanding and could be useful in further in vivo studies.14 Consequently, in the future therapeutic decisions may be based on the characteristics of the circulating tumor cells. Their detection in peripheral blood as part of a liquid biopsy represents a minimally invasive and useful procedure, however, more sensitive methods to reinforce the isolation of CTCs must be found before it can be used on a daily basis.8
Exosomes Introduction Exosomes are a type of recently described vesicular structures excreted all types of cells which seem to be involved in the regulation of extracellular communication, cellular differentiation, migration and tissue homeostasis.15 These microvesicles whose size ranges from 30 to 100 nm are secreted via inward budding of the plasma membrane. This process generates multi-vesicular cell-derived bodies called exosomes (Figs. 2---4). Exosomes can be excreted by a variety of normal (epithelial, mesenchymal and immune) and cancerous cells in diverse conditions and environments like blood, urine, bronchoalveolar secretions and breast milk. Due to their endosomal origin; exosomes may contain nucleic acids (mRNA and miRNA) as well as proteins.16 The latter are related to microvesicular structure, antigen presentation, targeting and apoptosis. In some way they act as ‘‘hormones’’17 with the ability to trigger a receptormediated response in distant cells or in their own microenvironment. 4,18 Indeed, exosomes are capable of acting very efficiently toward the tumor’s escape from the host immune system.5,19 An important mechanism by which the exosomes interact with their microenvironment is by modifying the relation with the immune system. This can happen either
3
Figure 2 Different sized exosomes (40---100 nm) observed by means of electron microscopy (EM) through negative staining technique.
by deprivation or stimulation. The mechanism of action implicates a mix of bioactive proteins confined within the microvesicles that are related to antigen presentation (MHC, and heat shock proteins), exosome targeting and apoptosis.19 Anti-tumorigenic properties Exosomes regulate protein expression in cellular processes at transcriptional, posttranscriptional, and translational levels as part of the mechanism for intercellular communication.18 As an example of this, miR-124 was found
Figure 3 Exosomes secreted from a culture of neonatal rat cardiomyocytes observed via EM (Immunogold for CD63, specific exosomal tetraspanin). Scale 100 nm.
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P. Sánchez-Vela et al. there is more tissue invasion with increased potential for metastasis and in the end more exosomes are released by the expansion of these clonal cells.16 In some way it can be understood as the last expression of Virchow’s postulate: Omnis cellula e cellula (‘‘Every cell originates from another existing cell’’).31
Figure 4 Fluorescence immunocytochemistry for neonatal rat cardiomyocytes. Anti-desmin, specific cardiomyocyte protein (red); DAPI nuclei (blue) and anti-CD63, exosomes (green).
to be deeply downregulated in all types of gliomas. Furthermore, it has been shown to play an important role in the signal transducer and activator of transcription 3 (STAT3) pathways which mediates T-cell immunosuppression. The injection of miR-124 into murine models and the subsequent stimulation of T-cell proliferation resulted in an anti-glioma therapeutic effect.18,20 Protumorigenic properties The connection between the defects in genetic coding for proteins and noncoding microRNAs (miRNAs) have been one of the most important and unexpected findings in oncology in recent years. MiRNAs, produced from what was once considered the dark side of the genome (non-coding DNA) have proved to be crucial for cancer initiation, progression, and dissemination. Some authors consider that the inhibition of intercellular signaling may prevent the formation of metastasis since exosomes may be involved in the establishment of the so called ‘‘premetastatic niche’’.21---24 Indeed, exosome secretion is exacerbated in multiple cancerous cells as observed in plasma, ascites, and other body fluids of patients with cancer.25---27 For instance, it has been demonstrated that inflammatory cytokines such as inteleukin-1 beta and tumor necrosis factor-alpha (both upregulated in glioblastoma multiforme) stimulate exosome released CRYAB (HspB5). CRYAB plays an important role, conferring resistance to apoptosis by inhibiting the activity and maturation of caspase-3.28 That is to say that cytokine stimulation of glioblastoma multiforme (GM) is translated in changes of the secreted exosomal proteome, many of which are related to cancer progression.29 Furthermore, the development of the tumor microenvironment is driven by the genetic instability of neoplastic cells as they release exosomes carrying oncogenes and other bioactive molecules.30 Sometimes, tumor derived exosomes can modulate their host microenvironment via the creation of positive feed-back loops: as more exosomes are released,
Clinical applications There is plenty of evidence that involves exosomes in intracellular signaling processes, metastasis21---23,32 and chemoresistance.23,25,33 Recent publications describe the expression profile of miRNAs inside exosomes as ‘‘genetic signatures’’ of different tumors that may relate to the diagnostic and prognostic features of patients with cancer.25,34---38 Normal cell-derived exosomes and melanoma cellderived exosomes contain many mRNAs, most of them related to cellular growth, proliferation, movement and gene expression. This, coupled with the fact that exosome contained mRNA correlates with cellular mRNA, could mean that exosomal mRNA signatures may reveal important gene signatures from their parent cells.39 In fact, various studies have shown that tumor exosomes have the ability to transport RNA to promote tumor progression.40 In other words, this suggests that the potential role of exosome profiles as biomarkers is not only diagnostic but also prognostic and predictive of the therapeutic response.39 As an example of this, it is known that DNA methylation leads to the inadequate expression, or silencing, of genes involved in metastasis; this reaction is catalyzed by DNA methyltransferases (DNMT). Recently it has been demonstrated that decitabine (DAC), which is a cytidine analog that inhibits DNMT1, affects metastasis by disrupting cell migration and invasion abilities of the cancer cells.41 Many different mechanisms are believed to contribute to boost DNMT1 expression, but exosome function may play an important role in preservation of the genome DNA methylation level and therefore tumurigenesis.42 An increase in exosomal miRNA expression negatively correlated with invasion ability and therefore could be used as a biomarker for mesenchymal-epithelial transition of colorectal tumor cells.41 miRNA measuring may be used as a predictor of the metastatic potential of colorectal tumors. Other clinical uses of exosomes are currently being studied by different groups with varying results. Among them, we would highlight the different efforts being made to modulate immune response either by targeting exosomes themselves or by introducing immune modulators (such as miRNA) or chemotherapeutic agents (such as doxorubicin) into the exosomes so that they stimulate anti-cancerous activity around the tumor microenviroment.43,44 Quite recently Hoshino et al. reported that the integrins contained in exosomes may specifically direct and predict metastasis to certain organs. Using experimental murine models, they demonstrated that ␣V 5 directed exosomes to the liver, and ␣6 4 integrins promoted their seeding to the liver. Indeed, using integrin inhibitors might stop metastatic spreading to certain target organs.5,45
Conclusion As important as early diagnosis is the need of finding new targets for the development of new nanodrugs that could
Please cite this article in press as: Sánchez-Vela P, et al. Liquid biopsy and tumor derived exosomes in clinical practice. Rev Esp Patol. 2016. http://dx.doi.org/10.1016/j.patol.2015.11.004
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Liquid biopsy and tumor derived exosomes in clinical practice act upon exosomes. The inhibition of intercellular communication between neoplastic cells generated by the exosomes may significantly contribute to tumor death. Tumor-derived exosomes may prove a unique source of clinically relevant and non-invasive biomarkers.16,18,30,46---50 They can be isolated from minimally invasive procedures, such as blood samples,16 they are very stable under different storage conditions and their content may reflect the molecular signatures of the diseased cell from which they were secreted. Such characteristics qualify circulating serum exosomes as potential biomarkers to predict cancer burden at an early stage. We hope that exosome analysis will provide us with significant and unique information about the ‘‘personality’’ of cancer. Analysis of exosomes, cDNA, CTCs and other biomarkers in peripheral blood by means of liquid biopsies is becoming a powerful tool in furthering our understanding of cancer biology and improving the management and survival of cancer patients.
Ethical disclosures Protection of human and animal subjects. The authors declare that no experiments were performed on humans or animals for this study.
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Confidentiality of data. The authors declare that no patient data appear in this article.
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Right to privacy and informed consent. The authors declare that no patient data appear in this article.
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Conflict of interests
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The authors declare no conflict of interest. 22.
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ARTICLE IN PRESS
Rev Esp Patol. 2016;xxx(xx):xxx---xxx
Patología R E V I S TA
E S PA Ñ O L A
D E
www.elsevier.es/patologia
REVIEW
Liquid biopsy and tumor derived exosomes in clinical practice Pablo Sánchez-Vela a , Nahuel Aquiles Garcia b , María Campos-Segura c , Jerónimo Forteza-Vila c,∗ a
Facultad de Medicina, Universidad Católica de Valencia, Valencia, Spain Mixed Unit for Cardiovascular Repair, Instituto de Investigación Sanitaria La Fe- Centro de Investigación Príncipe Felipe, Valencia, Spain c Instituto Valenciano de Patología, Centro de Investigación Príncipe Felipe/Universidad Católica de Valencia, Valencia, Spain b
Received 25 September 2015; accepted 12 November 2015
KEYWORDS Exosomes; Cancer; Biomarkers; Liquid-biopsy; microRNA
PALABRAS CLAVE Exosomas; Cáncer; Biomarcadores; Biopsia líquida; microARN
∗
Abstract Tumors are complex tissues that interact in many different ways. Tissue biopsies provide a great amount of information and remain the gold standard for tumor diagnosis. However, they cannot always be performed due to the invasive nature of the procedure and in such circumstances, a liquid biopsy could provide a solution. Liquid biopsy is defined as the search of biomarkers in peripheral blood. To date, there are three main research fields: (1) circulating tumor cells (CTCs); (2) circulating free tumor nucleic acids (cfNA) and (3) exosomes, small vesicles containing various types of signaling molecules capable of modulating a tumor-immune response. In recent years, exosomes have arisen as a powerful tool both to further our understanding of cancer biology and to improve clinical management. We review how the isolation and study of exosomes from liquid biopsies may affect clinical practice. © 2016 Sociedad Espa˜ nola de Anatomía Patológica. Published by Elsevier España, S.L.U. All rights reserved.
Biopsia líquida y exosomas derivados del tumor en la práctica clínica Resumen Las biopsias tisulares, aunque otorgan gran cantidad de información y son el gold standard para el diagnóstico tumoral, no pueden ser realizadas continuamente para monitorizar la evolución tumoral dada su inherente naturaleza invasiva. Por otro lado, la biopsia líquida surge como una herramienta capaz de compensar estas limitaciones. En este momento se han identificado 3 tipos principales de biomarcadores en la biopsia líquida: (1) células tumorales circulantes (CTC); (2) ácidos nucleicos tumorales circulantes libres (cfNA), y (3) exosomas. En los
Corresponding author. E-mail address: [email protected] (J. Forteza-Vila).
http://dx.doi.org/10.1016/j.patol.2015.11.004 1699-8855/© 2016 Sociedad Espa˜ nola de Anatomía Patológica. Published by Elsevier España, S.L.U. All rights reserved.
Please cite this article in press as: Sánchez-Vela P, et al. Liquid biopsy and tumor derived exosomes in clinical practice. Rev Esp Patol. 2016. http://dx.doi.org/10.1016/j.patol.2015.11.004
+Model PATOL-375; No. of Pages 6
ARTICLE IN PRESS
2
P. Sánchez-Vela et al. últimos a˜ nos los exosomas (peque˜ nas vesículas que contienen moléculas capaces de modular la respuesta inmune-tumoral) han surgido como una potente herramienta para comprender mejor la biología del cáncer así como una manera para intentar mejorar el manejo de los pacientes oncológicos. El objetivo de este artículo es presentar una revisión sobre cómo la caracterización de los exosomas a través de la biopsia líquida podría influir sobre la práctica clínica. © 2016 Sociedad Espa˜ nola de Anatomía Patológica. Publicado por Elsevier España, S.L.U. Todos los derechos reservados.
Introduction to the liquid biopsy
Liquid biopsy possibilities
Tumors are complex tissues that interact in many different ways. Today we assume that these particular cells are defined by their ability to sustain proliferative signaling, evade growth suppressors, resist cell death, enable replicative immortality, induce angiogenesis, reprogram energy metabolism, evade immune destruction and activate invasion and metastasis.1 Genotyping tumor samples in search of genetic alterations with predictive and prognostic information has become a daily practice in clinical oncology. However, the method of their extraction still has some limitations. The decision to administer chemotherapy and/or other therapy subsequent to tumor resection usually depends on an oncologist’s assessment of the volume of the primary tumors (T), the lymph node extension (N) and the presence of metastatic disease (M). Now the expression analysis of certain proteins and the presence or absence of certain genetic mutations in the tissue biopsy is also considered and will be a biomarker for the application of targeted therapies. Although tumor biopsies provide a great amount of information and remain the gold standard for tumor diagnosis, they cannot always be performed due to the invasive nature of the procedure. The condition of the patient or the accessibility of the tumor may make a biopsy impossible and therefore impractical for monitoring the progression or recurrence of the disease.2 Molecular genetics can differ within the same tumor and thus the small area removed in a tissue biopsy may not reflect the overall picture. Similarly, a primary tumor biopsy does not reflect the ongoing clinical course during which there may be clonal evolution and tumor heterogeneity resulting in systemic drug resistance, transition of epithelium to mesenchyme, etc. In order to avoid such ‘‘snap-shot’’ results from tumor biopsies, rapid, cost-effective and non-invasive identification of biomarkers at various moments during the disease process are required.3 Liquid biopsy could provide such a method. Liquid biopsy is defined as the search of biomarkers in peripheral blood. At this moment three main research fields may be accepted as possible biomarkers4,5 : (1) circulating tumor cells (CTCs); (2) circulating free tumor nucleic acids (cfNA) including miRNAs, mRNA and long non-coding RNA, but mainly cfDNA; and (3) exosomes, small vesicles containing various types of signaling molecules capable of modulating tumor-immune response.
cfDNA Circulating free DNA is a normal finding in healthy individuals. However cfDNA levels are higher in cancer patients in comparison to non-cancer patients. Specific detection of tumor-derived cfDNA may be a dynamic way of monitoring tumor change in response to treatment and to indicate that certain subpopulations of resistant cells may continue to proliferate despite therapeutic measures. Tracking tumorassociated abnormalities in blood can be used to assess the presence of residual, recurrent, relapse and resistance diseases.6 For example, a colon cancer cohort study showed that cfDNA may be useful in evaluating if patients were tumor free after surgery. An evaluation of the presence or absence of residual disease could help patients to avoid needless, toxic adjuvant chemotherapy.7 Furthermore, cfDNA may be used as a biomarker of when a tumor resistance to targeted therapies: a patient with colon cancer with a wild-KRAS suitable for a treatment with a EGFR (Epidermal Growth Factor Receptor) blocker such as Cetuximab could be monitored for acquired resistance to this therapy by the mean of serial blood sampling and detection of de novo KRAS, NRAS or MET mutations.6 However, before implementing cfDNA testing, standardization and prospective clinical trials are required.2
CTCs CTCs are tumor cells which have become detached from the primary solid tumor mass. They have been described in breast, prostate, lung and colon tumors (Fig. 1) and are certainly involved in the process of metastasis. Clinical quantification of CTCs is highly valuable as they represent potential metastatic cells and facilitate real time monitoring during systemic and targeted therapies via blood sampling monitoring.8 Presently, research into the assessment of the clinical value of CTCs analysis is being carried out and has already proved them to be a significant prognostic factor in metastatic breast cancer9 and other solid tumors such as colorectal,10 prostate11 and lung.12 CTCs have even proven superior to conventional imaging for monitoring therapeutic response.13 While evidence of the tumor’s genomic evolution can be also obtained by isolating cfDNA, CTCs will lead
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Liquid biopsy and tumor derived exosomes in clinical practice
Figure 1 Circulating Tumor Cells (CTCs) obtained from a Breast Cancer patient blood before neoadjuvant chemotherapy and surgery. Courtesy of Dr. José Juan Gaforio Martínez and its team from the Immunology Department at the University of Jaén.
to a deeper understanding and could be useful in further in vivo studies.14 Consequently, in the future therapeutic decisions may be based on the characteristics of the circulating tumor cells. Their detection in peripheral blood as part of a liquid biopsy represents a minimally invasive and useful procedure, however, more sensitive methods to reinforce the isolation of CTCs must be found before it can be used on a daily basis.8
Exosomes Introduction Exosomes are a type of recently described vesicular structures excreted all types of cells which seem to be involved in the regulation of extracellular communication, cellular differentiation, migration and tissue homeostasis.15 These microvesicles whose size ranges from 30 to 100 nm are secreted via inward budding of the plasma membrane. This process generates multi-vesicular cell-derived bodies called exosomes (Figs. 2---4). Exosomes can be excreted by a variety of normal (epithelial, mesenchymal and immune) and cancerous cells in diverse conditions and environments like blood, urine, bronchoalveolar secretions and breast milk. Due to their endosomal origin; exosomes may contain nucleic acids (mRNA and miRNA) as well as proteins.16 The latter are related to microvesicular structure, antigen presentation, targeting and apoptosis. In some way they act as ‘‘hormones’’17 with the ability to trigger a receptormediated response in distant cells or in their own microenvironment. 4,18 Indeed, exosomes are capable of acting very efficiently toward the tumor’s escape from the host immune system.5,19 An important mechanism by which the exosomes interact with their microenvironment is by modifying the relation with the immune system. This can happen either
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Figure 2 Different sized exosomes (40---100 nm) observed by means of electron microscopy (EM) through negative staining technique.
by deprivation or stimulation. The mechanism of action implicates a mix of bioactive proteins confined within the microvesicles that are related to antigen presentation (MHC, and heat shock proteins), exosome targeting and apoptosis.19 Anti-tumorigenic properties Exosomes regulate protein expression in cellular processes at transcriptional, posttranscriptional, and translational levels as part of the mechanism for intercellular communication.18 As an example of this, miR-124 was found
Figure 3 Exosomes secreted from a culture of neonatal rat cardiomyocytes observed via EM (Immunogold for CD63, specific exosomal tetraspanin). Scale 100 nm.
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P. Sánchez-Vela et al. there is more tissue invasion with increased potential for metastasis and in the end more exosomes are released by the expansion of these clonal cells.16 In some way it can be understood as the last expression of Virchow’s postulate: Omnis cellula e cellula (‘‘Every cell originates from another existing cell’’).31
Figure 4 Fluorescence immunocytochemistry for neonatal rat cardiomyocytes. Anti-desmin, specific cardiomyocyte protein (red); DAPI nuclei (blue) and anti-CD63, exosomes (green).
to be deeply downregulated in all types of gliomas. Furthermore, it has been shown to play an important role in the signal transducer and activator of transcription 3 (STAT3) pathways which mediates T-cell immunosuppression. The injection of miR-124 into murine models and the subsequent stimulation of T-cell proliferation resulted in an anti-glioma therapeutic effect.18,20 Protumorigenic properties The connection between the defects in genetic coding for proteins and noncoding microRNAs (miRNAs) have been one of the most important and unexpected findings in oncology in recent years. MiRNAs, produced from what was once considered the dark side of the genome (non-coding DNA) have proved to be crucial for cancer initiation, progression, and dissemination. Some authors consider that the inhibition of intercellular signaling may prevent the formation of metastasis since exosomes may be involved in the establishment of the so called ‘‘premetastatic niche’’.21---24 Indeed, exosome secretion is exacerbated in multiple cancerous cells as observed in plasma, ascites, and other body fluids of patients with cancer.25---27 For instance, it has been demonstrated that inflammatory cytokines such as inteleukin-1 beta and tumor necrosis factor-alpha (both upregulated in glioblastoma multiforme) stimulate exosome released CRYAB (HspB5). CRYAB plays an important role, conferring resistance to apoptosis by inhibiting the activity and maturation of caspase-3.28 That is to say that cytokine stimulation of glioblastoma multiforme (GM) is translated in changes of the secreted exosomal proteome, many of which are related to cancer progression.29 Furthermore, the development of the tumor microenvironment is driven by the genetic instability of neoplastic cells as they release exosomes carrying oncogenes and other bioactive molecules.30 Sometimes, tumor derived exosomes can modulate their host microenvironment via the creation of positive feed-back loops: as more exosomes are released,
Clinical applications There is plenty of evidence that involves exosomes in intracellular signaling processes, metastasis21---23,32 and chemoresistance.23,25,33 Recent publications describe the expression profile of miRNAs inside exosomes as ‘‘genetic signatures’’ of different tumors that may relate to the diagnostic and prognostic features of patients with cancer.25,34---38 Normal cell-derived exosomes and melanoma cellderived exosomes contain many mRNAs, most of them related to cellular growth, proliferation, movement and gene expression. This, coupled with the fact that exosome contained mRNA correlates with cellular mRNA, could mean that exosomal mRNA signatures may reveal important gene signatures from their parent cells.39 In fact, various studies have shown that tumor exosomes have the ability to transport RNA to promote tumor progression.40 In other words, this suggests that the potential role of exosome profiles as biomarkers is not only diagnostic but also prognostic and predictive of the therapeutic response.39 As an example of this, it is known that DNA methylation leads to the inadequate expression, or silencing, of genes involved in metastasis; this reaction is catalyzed by DNA methyltransferases (DNMT). Recently it has been demonstrated that decitabine (DAC), which is a cytidine analog that inhibits DNMT1, affects metastasis by disrupting cell migration and invasion abilities of the cancer cells.41 Many different mechanisms are believed to contribute to boost DNMT1 expression, but exosome function may play an important role in preservation of the genome DNA methylation level and therefore tumurigenesis.42 An increase in exosomal miRNA expression negatively correlated with invasion ability and therefore could be used as a biomarker for mesenchymal-epithelial transition of colorectal tumor cells.41 miRNA measuring may be used as a predictor of the metastatic potential of colorectal tumors. Other clinical uses of exosomes are currently being studied by different groups with varying results. Among them, we would highlight the different efforts being made to modulate immune response either by targeting exosomes themselves or by introducing immune modulators (such as miRNA) or chemotherapeutic agents (such as doxorubicin) into the exosomes so that they stimulate anti-cancerous activity around the tumor microenviroment.43,44 Quite recently Hoshino et al. reported that the integrins contained in exosomes may specifically direct and predict metastasis to certain organs. Using experimental murine models, they demonstrated that ␣V 5 directed exosomes to the liver, and ␣6 4 integrins promoted their seeding to the liver. Indeed, using integrin inhibitors might stop metastatic spreading to certain target organs.5,45
Conclusion As important as early diagnosis is the need of finding new targets for the development of new nanodrugs that could
Please cite this article in press as: Sánchez-Vela P, et al. Liquid biopsy and tumor derived exosomes in clinical practice. Rev Esp Patol. 2016. http://dx.doi.org/10.1016/j.patol.2015.11.004
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Liquid biopsy and tumor derived exosomes in clinical practice act upon exosomes. The inhibition of intercellular communication between neoplastic cells generated by the exosomes may significantly contribute to tumor death. Tumor-derived exosomes may prove a unique source of clinically relevant and non-invasive biomarkers.16,18,30,46---50 They can be isolated from minimally invasive procedures, such as blood samples,16 they are very stable under different storage conditions and their content may reflect the molecular signatures of the diseased cell from which they were secreted. Such characteristics qualify circulating serum exosomes as potential biomarkers to predict cancer burden at an early stage. We hope that exosome analysis will provide us with significant and unique information about the ‘‘personality’’ of cancer. Analysis of exosomes, cDNA, CTCs and other biomarkers in peripheral blood by means of liquid biopsies is becoming a powerful tool in furthering our understanding of cancer biology and improving the management and survival of cancer patients.
Ethical disclosures Protection of human and animal subjects. The authors declare that no experiments were performed on humans or animals for this study.
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Confidentiality of data. The authors declare that no patient data appear in this article.
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Right to privacy and informed consent. The authors declare that no patient data appear in this article.
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Conflict of interests
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The authors declare no conflict of interest. 22.
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Please cite this article in press as: Sánchez-Vela P, et al. Liquid biopsy and tumor derived exosomes in clinical practice. Rev Esp Patol. 2016. http://dx.doi.org/10.1016/j.patol.2015.11.004