|Year : 2017 | Volume
| Issue : 2 | Page : 58-63
Exosomes biology: Function and clinical implications in lung cancer
Martin Frederik Dietrich1, Christian Rolfo2, Pablo Reclusa2, Marco Giallombardo2, Anna Valentino2, Luis E Raez1
1 Department of Thoracic Oncology, Memorial Cancer Institute, Florida International University, Pembroke Pines, FL, USA
2 Department of Oncology, Phase I-Early Clinical Trials Unit, Antwerp University Hospital, Center for Oncological Research, Antwerp University, Antwerp, Belgium
|Date of Submission||01-Aug-2016|
|Date of Acceptance||10-Jan-2017|
|Date of Web Publication||27-Apr-2017|
Luis E Raez
Department of Thoracic Oncology, Memorial Cancer Institute, Florida International University, 801 N. Flamingo Road, Suite 11, Pembroke Pines, FL 33028
Source of Support: None, Conflict of Interest: None
Lung cancer is the most common malignancy in the United States, totaling 225,000 cases per year. In recent years, several new treatment options have become available based on the molecular and cellular characterization of the disease. More recently, "liquid biopsies" have received attention to complement traditional tissue biopsies and to enhance the spectrum of analysis for tumor-derived factors. As one of these tumor characteristics, extracellular vesicles (EVs) are lipid bilayered EVs that can cargo a variety of factors, including growth factors and their receptors, RNA transcripts, microRNAs, and DNA, among others. Initial acceptance as mere physiological products has been attributed to the presence of exosomes in healthy individuals, and the large diversity of exosomes that have made the assignment of distinct pathophysiological roles difficult. While their role in clinical application has not yet been established, they have emerged from their once thought innocent role as a bystander to a critical mediator of intratumoral signaling, tumor progression, chemotherapy resistance, and metastasis. In this review, we have summarized the structure and biology of EVs, their role in lung cancer, and the potential diagnostic and therapeutic implications for the treatment of this complex disease.
Keywords: Exosomes, liquid biopsy, lung cancer, molecular targeting
|How to cite this article:|
Dietrich MF, Rolfo C, Reclusa P, Giallombardo M, Valentino A, Raez LE. Exosomes biology: Function and clinical implications in lung cancer. Cancer Transl Med 2017;3:58-63
|How to cite this URL:|
Dietrich MF, Rolfo C, Reclusa P, Giallombardo M, Valentino A, Raez LE. Exosomes biology: Function and clinical implications in lung cancer. Cancer Transl Med [serial online] 2017 [cited 2017 Jul 27];3:58-63. Available from: http://www.cancertm.com/text.asp?2017/3/2/58/202227
| Introduction|| |
Extracellular vesicles (EVs) encompass a broad family of bilayered lipid vesicles that have specific function in cell–cell communication. EVs are lipid-based, bilayered structures with a size range of 40–1000 nm that occur in a multitude, if not all, cell types. These exocytically formed EVs were originally described in reticulocytes in the early 1980s by several groups who followed the transferrin receptor as a marker of maturation from reticulocyte to erythrocyte and recognized initial endocytic uptake with subsequent extracellular secretion through the fusion of multivesicular bodies (MVBs) with the plasma membrane., Excretion of the transferrin receptor was thought to be a simple mechanism of discarding unnecessary cellular components following maturation into erythrocytes. Recognition of their importance as key mediators of intercellular communication in cancer was originally hindered by several factors, including a presence in healthy individuals, their large variety in size and cargo content, the unappreciated role of "liposomes" as vectors in physiological functions, and in part, through consideration as technical artifacts from membrane isolation procedures. In addition, the broad nomenclature and diversity of its usage in the literature contributed to this confusion. An overlap also seems to exist between endocellular and membrane-derived vesicles due to indiscriminate isolation techniques by centrifugation. Therefore, a clear distinction from the literature appears difficult, and clear differentiation by origin may remain technically challenging.
Based on the cellular origin within the cell, EVs are differentiated into ectosomes, also known as microparticles, that occur through fusion and budding with the plasma cell membrane,, whereas exosomes originate from MVBs inside the cell that is only later fused with the plasma cell membrane before their extracellular secretion. Common laboratory techniques using centrifugation and size exclusion filters are not able to reliably distinguish between these two based on cellular origin, and thus a large overlap between these two types of vesicles remains present in the literature. For purposes of this review, we will focus on the role of exosomes as part of the EV family and their implications in lung cancer. In particular, we will focus on disease initiation, conferred resistance through microenvironmental influences, and promotion of metastatic potential.
From the time that we used to treat with a "one size fits all" approach to "personalized medicine" in lung cancer, about 30% of lung cancer patients are now therapeutically benefitting from molecular analysis.,, Detection and serial monitoring of biomarkers have become increasingly more important to detect molecular vulnerabilities and follow their evolution., "Liquid biopsy" describes a more novel concept of tumor characterization through analysis of circulating components of tissue-resident cancer in various body fluids, predominantly obtained from blood or serum. Circulating tumor cells were first utilized in clinical application with some prognostic information in breast and other cancer types but with low utility in lung cancer. More recently, cell-free DNA (cfDNA) has been utilized to provide genomic information,, and epidermal growth factor receptor (EGFR) testing based on this methodology has become approved by the Food and Drug Administration for clinical companion testing in nonsmall cell lung cancer. There is always cfDNA shed from apoptotic or necrotic cells that can be isolated and utilized for genetic analysis through next-generation sequencing, with an increase in concentration from cancer cell populations. Another clinical application of "liquid biopsy" is the analysis of circulating protein composition from serum/plasma and is further utilized in the VeriStrat test, predicting a response to inhibitor erlotinib independent of mutation status in the EGFR tyrosine kinase domain. Other circulating factors, for example, circulating vascular endothelial growth factor (VEGF), have been investigated to predict therapy responses but have not yet reached a level of clinical applicability to guide therapy.
Although cancer-related exosomes, sometimes referred to as "oncosomes" in the literature, have been described over 30 years ago,, they have only more recently become a focus of clinical investigation in lung cancer. While the extent of involvement, physiological processes and clinical utility in disease of these particles remain to be fully understood, basic and translational science has made remarkable progress to elicit functions with a potential of clinical exploitation. The idea is to discover clinical utility, but most likely the benefit will be in assessing early detection, recurrence, prognosis or disease monitoring.,,
| Structural Properties of Exosomes|| |
Exosomes are a heterogeneous group of bilayered lipid vesicles that originate from cytosolic compartments of the cell [Figure 1]. All cell types are capable of secreting exosomes in both physiological and pathological conditions. Exosomes range in size from 40 to 150 nm., Technical preparation, cell of origin, and cargo may be some of the influences to contribute to this diversity. The inhibition of exosomes secretion could be an interesting treatment due to the many roles that exosomes play in tumor development; however, mechanisms of exosome secretion are still unclear. It has been demonstrated that exosomes can be released directly from the plasmatic membrane; however, the majority are released from the endosomal compartment. Several proteins have been described to be implied in the exosome secretion machinery with contradictory results., The endosomal sorting complex required for transport has been described as one of the most important members of the exosome formation since it seems to be the complex that organizes the cargo of the exosomes during its formation and may contribute to targeting the receptor to the intraluminal vesicles., In addition, some lipids seem to be required for exosome secretion as ceramide. In any case, the exosomes when released get coated by the plasmatic membrane, such that membrane composition mimics the content of any intracellular membrane, including cholesterol, sphingomyelin, etc. The content cargo of exosome includes DNA, virtually all types of RNA including mRNA, tRNA, microRNAs (miRNAs), mature proteins such as growth factors and growth factor receptors as well as lipids and metabolites. The exact composition of exosomes seems to be dependent on currently unknown factors; however, the presence of certain cancer-specific oncogenic drivers suggests a more directed role rather than a random process of formation.,
| Exosomes In Lung Cancer|| |
Exosomes are detected in healthy individuals as well as those affected by cancer. This omnipresence likely contributed to a delay in identifying the importance of exosomes in various processes that are important to cancer. The estimated number of exosomes in circulation is estimated around 2000 trillion in healthy individuals, with up to 4000 trillion reported in cancer patients., It is unclear whether this increase is related to increase exosome secretion through tumor cells or a more general phenomenon.
Exosomes have been described to play pleiotropic roles, such as tumor progression, metastasis, angiogenesis, immunomodulation, and horizontal transfer. Below, different experiments are described, which support these roles.
Al-Nedawi et al. demonstrated transfer of a truncated, oncogenic version of the EGFR, known as EGFRvIII, can be "shared" between glioma cells through exosomal transport. In these experiments, incubating wild-type EGFR carrying U373 with exosomes extracted from the supernatant of EGFRvIII carrying U373 demonstrated anchoring of the mutant receptor in the plasma membrane. Further, activation of downstream MEK/Erk1/2 signaling further suggested functional activation of the pathway by the mutant receptor, rather than inconsequential colocalization of the receptor with the plasma membrane. These findings support the idea that exosomes can promote the tumor growth through different mechanisms. The findings were complemented by a study that described EGFRvIII-containing exosomes in blood in 7 out of 25 glioblastoma patients., In the same study, mRNAs and miRNAs were detected in exosomes that are involved in a variety of cellular and metabolic processes relevant to glioblastoma. These were found in concentrations largely exceeding their normal cellular counterparts, thus suggesting a relevant physiological role. These findings supported the role of exosomes in tumor cell growth in a paracrine/intratumoral manner with an infection-like process of horizontal transfer of a malignant phenotype within the tumor microenvironment. The underlying signals that target specific cells remain unclear; however, the presence of mutant protein expression in only a subset of cells provides an intriguing contribution to tumor heterogeneity. Valadi et al. were the first to demonstrate exosomes carrying mRNA and miRNAs and their influence to modulate gene expression in recipient cells. In a translational study, Rolfo et al. were able to demonstrate a tumor histology-specific miRNA signature affecting the EGFR signaling pathway in nonsmall cell lung cancer (NSCLC), further supporting a clinical role for exosomal messenger function.
The tumor microenvironment consists not only of cancer cells but also of a variety of cell types necessary for tumor growth including nontransformed counterparts to cancer cells, fibroblasts, vascular endothelial cells, tissue-resident macrophages, and dendritic cells, among others. Exosomes have the ability to guide cell–cell communication between all of these types. For example, exosomes can transfer transformative molecules from a cancer cell to a nonaffected cell., Delivered cargo functional changes delivered by exosomal influence seem to evolve on induction of cellular transformation. In fact, this communication is bidirectional, and activation of cells in the microenvironment by exosomes can, in turn, deliver exosomes that can contribute to chemoresistance and immunomodulation of the primary breast cancer cells through activation of both STAT1/NOTCH3 and JAG1 cellular pathways. Further, exosomes carrying EGFR from A431, A549, and DLD-1 lung cancer cell lines were shown to enhance angiogenesis through upregulation of VEGF expression, an important pathway in local tumor growth. Fibroblast growth factor and platelet-derived growth factor were equally found in exosomes, both on the transcriptional and protein level. Exosomes containing matrix metalloproteinases can contribute to local tumor cell expansion and allow for malignant growth including tumor support structures of blood vessels and tumor-associated stroma.
Hoshino et al. were able to demonstrate the contribution of exosomes to the development of metastasis in an organ-specific manner through cellular alterations through upregulation of integrins to form the premetastatic niche in several xenograft models of NSCLC. The composition of exosomes may be underlying the preferential organotropism of certain cancer types. In an immunocompromised murine model, isolated exosomes from liver and/or lung metastatic tumor cell lines have demonstrated preferential localization of exosomes within their respective metastatic target environment, suggesting a role in the promotion of organ-specific metastasis before arrival of malignant cells from the primary tumor. The analysis demonstrated a correlation between the expression of integrins and the metastatic potential of the tumor cell. The uptake of malignant cells was then confirmed to be cell-type specific in their respective target organs. Lung-tropic exosomes from melanoma cells predominantly co-localized with S100A4-positive fibroblasts in the lung while exosomes derived from pancreatic cancer colocalized to Kupffer cells in the liver, the primary site of metastasis in this model. This study not only provided powerful evidence of exosome-directed organotropism through integrin-mediated signaling but also a proof-of-principle that blocking integrins through decoy peptides can successfully ablate exosome adhesion in an organic-specific manner. Similar results were obtained in a murine model of pancreatic cancer with predilection to hepatic metastasis through exosome-mediated initiation of a premetastatic niche. Increased exosome formation has been found in hypoxic tumor environments of breast cancer, glioma, leukemia and prostate cancer  models, suggesting that hypoxic environments within enlarging tumors could accelerate the development of distant metastasis through a forced secretion of exosomes.
One of the major developments in the treatment of lung cancer and melanomas was the recent advent of immunotherapy. These rely on blocking negative regulators, for example, PD-1 and PD-L1, of T-cell activation in the microenvironment as well as inflammatory signals., Modulation of immune signals into an immunosuppressive microenvironment has been described to be mediated by exosomes ,, through humoral and cellular mechanisms, therefore, allowing tumors to escape immune surveillance in the microenvironment and indicating a possible role in treatment resistance to the newly introduced checkpoint inhibitor therapies. In contrast, exosomes have demonstrated their versatility in (pre-) metastatic sites by upregulation of inflammatory processes and recruitment of bone marrow-derived immune cells, a critical step in preparing the "soil" for the premetastatic niche.
| Potential Clinical Applications Of Exosomes|| |
Most of the information implicating a contribution of exosomes to cancer development and progression is derived from in vitro or murine in vivo models. Human-derived data are still in its infancy. While the exact contribution of exosomes to cancer development and progression is still largely unclear, it is conceivable that targeting exosomes could attain a role in cancer diagnosis, prediction, and therapy. Given the versatile functions of exosomes, multiple diagnostic and therapeutic angles could prove beneficial.
Exosomes have been described as powerful tools for different cancer diagnoses. In lung cancer, this field is still under development due to the lack of information regarding NSCLC biomarkers. However, Jakobsen et al. have described a protein profile from exosomes derived from patients with advance NSCLC showing an improvement compared to the expression of CD91 described by Ueda et al. Our group is actively trying to use exosomes for the identification of actionable mutations and trying to find a role in the serial follow-up of specific mutations, such as T790M.
Many miRNAs and proteins have been described as prognostic factors for patients with NSCLC. For example, let-7f and miR-30-3p are related to poor outcome and miR-373 and miR-512 silenced miRNAs that suppress tumor growth, and when reactivation is produced, decrease the tumor growth and the invasiveness. However, prognostic factors inside the exosomes have yet to be implemented into clinical practice.
For conventional chemotherapy, exosomes have been described to shuttle agents such as cisplatin and doxorubicin out of the cell and thus could contribute to chemoresistance. Inhibition of this clearance mechanism would increase sensitivity to therapy and augment an anti-tumor effect. Further, targeted agents such as trastuzumab could lose their specificity, as Her2/ErbB2 receptors are negatively regulated through exosomal export, suggesting a lack of clinical efficacy of this agent. In addition, inhibition of the cellular cross-talk in the tumor environment could increase sensitivity to treatment by blocking microenvironmental-derived supportive signals, including growth factors, RNA transcripts, and miRNAs. In a study focusing on lung cancer-derived exosomes, epithelial-to-mesenchymal transition was demonstrated in human bronchial epithelial cells, suggesting a role of exosomes in malignant microenvironmental transformation. For most tumor types, early detection before development is crucial for curative intent approaches. Several studies now suggest that exosomes play a key role in the development of metastasis through activation of the target tissue environment through growth factors enhancing vascular permeability, increased tissue inflammation resulting in extracellular matrix remodeling, and enhanced angiogenesis as well as recruitment of bone marrow-derived immune cells. These immune cells have been previously implicated in the formation of the premetastatic niche, however, without a clear understanding of the activating signals. Blocking exosomal release from the cell may suppress the development of a favorable tumor microenvironment in the premetastatic niche, and thus prevent disease progression. Peptide-mediated blockades of integrins critical to this process have shown some preclinical promise although similar studies in humans, to the best our knowledge, have not yet been initiated. On the contrary, exosome-mediated suppression of anti-inflammatory signals within the tumor microenvironment could enhance the efficacy of recently introduced immunotherapy in lung cancer.
Liposomal formulation of chemotherapeutic agents, for example, doxorubicin or irinotecan, has been utilized clinically to enhance delivery to cancer cells. Another mechanism of cancer cell-specific delivery includes drug-antibody conjugate, for example, T-DM1 in breast cancer, utilizing a growth factor receptor as a homing beacon to a drug coupled antibody. It is plausible to assume that exosomes have the capability of combining properties of both, including a liposomal envelope with a yet-to-be-identified targeting structure for recipient cells. Given the results of several preclinical studies, it is less likely that exosomal delivery is merely a random process. The success of utilizing exosomes in clinical efforts will largely depend on an improved understanding of exosomal regulation, including the biogenic triggers of information, their specific cargo selection, and identification of "zip codes" for target cells.
| Conclusion|| |
Exosomes have emerged from innocent bystanders of physiological cellular exports to central regulators of signaling in cancer growth and development. The ability to freely access exosomes by simple phlebotomy further makes them an attractive object of investigation in cancer. Recognition of their reflection of intratumoral properties indeed widens the spectrum of diagnostic possibility. For example, the presence of EGFR-activating mutations through sequencing of exosomes enables clinicians to perform prognostic and predictive assessments as well as the possibility to guide therapeutic applications. The most important step in taking exosomes from a basic science phenomenon into clinical practice is gaining an improved understanding of exosome biology, in particular, the signals responsible for formation, cargo selection, and labeling for the respective target cell population. Modifying the signaling of exosomes into tumor suppressing properties could be a "trojan horse" approach to undermine tumor signaling although at this point in time this seems rather hypothetical. In clinical practice, delivery of messenger transcripts (siRNA, shRNA, etc.) for therapeutic purposes has largely failed due to low target concentration and lack of target cell specificity. The plethoric nature of exosome involvement in virtually all aspects of cellular function indicates that diagnostic and therapeutic approaches will have to focus on highly context-dependent applications. It remains intriguing that exosomes are freely accessible, and analysis of their content could be utilized to diagnose cancer early, guide therapy, and monitor disease recurrence in a noninvasive manner.
Financial support and sponsorship
Conflicts of interest
LR receives research support from Exosomes DX.
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