Current Search through Liquid Biopsy of Effective Biomarkers for Early Cancer Diagnosis into the Rich Cargoes of Extracellular Vesicles

There exist many different human cancers, but regardless of the cancer type, an early diagnosis is a necessary condition for further optimal outcomes from the disease. Therefore, efficient specific and sensitive cancer biomarkers are urgently needed. This is especially true for the cancers depicting a silent progression, and those only diagnosed in an already metastatic state with a poor survival prognostic. After a rapid overview of the previous methods for cancer diagnosis, the outstanding characteristics of extracellular vesicles (EVs) will be presented, as new interesting candidates for early cancer diagnosis in human biofluid non-invasive liquid biopsy. The present review aims to give the state-of-the-art of the numerous searches of efficient EV-mediated cancer diagnosis. The corresponding literature quest was performed by means of an original approach, using a powerful Expernova Questel big data platform, which was specifically adapted for a literature search on EVs. The chosen collected scientific papers are presented in two parts, the first one drawing up a picture of the current general status of EV-mediated cancer diagnosis and the second one showing recent applications of such EV-mediated diagnosis for six important human-specific cancers, i.e., lung, breast, prostate, colorectal, ovary and pancreatic cancers. However, the promising perspective of finally succeeding in the worldwide quest for the much-needed early cancer diagnosis has to be moderated by the many remaining challenges left to solve before achieving the efficient clinical translation of the constantly increasing scientific knowledge.     

Extracellular Vesicles: Messengers of p53 in Tumor–Stroma Communication and Cancer Metastasis

Tumor progression to a metastatic and ultimately lethal stage relies on a tumor-supporting microenvironment that is generated by reciprocal communication between tumor and stromal host cells. The tumor–stroma crosstalk is instructed by the genetic alterations of the tumor cells—the most frequent being mutations in the gene Tumor protein p53 (TP53) that are clinically correlated with metastasis, drug resistance and poor patient survival. The crucial mediators of tumor–stroma communication are tumor-derived extracellular vesicles (EVs), in particular exosomes, which operate both locally within the primary tumor and in distant organs, at pre-metastatic niches as the future sites of metastasis. Here, we review how wild-type and mutant p53 proteins control the secretion, size, and especially the RNA and protein cargo of tumor-derived EVs. We highlight how EVs extend the cell-autonomous tumor suppressive activity of wild-type p53 into the tumor microenvironment (TME), and how mutant p53 proteins switch EVs into oncogenic messengers that reprogram tumor–host communication within the entire organism so as to promote metastatic tumor cell dissemination.     

Extracellular Vesicles and Their Role in the Spatial and Temporal Expansion of Tumor–Immune Interactions

Extracellular vesicles (EVs) serve as trafficking vehicles and intercellular communication tools. Their cargo molecules directly reflect characteristics of their parental cell. This includes information on cell identity and specific cellular conditions, ranging from normal to pathological states. In cancer, the content of EVs derived from tumor cells is altered and can induce oncogenic reprogramming of target cells. As a result, tumor-derived EVs compromise antitumor immunity and promote cancer progression and spreading. However, this pro-oncogenic phenotype is constantly being challenged by EVs derived from the local tumor microenvironment and from remote sources. Here, we summarize the role of EVs in the tumor–immune cross-talk that includes, but is not limited to, immune cells in the tumor microenvironment. We discuss the potential of remotely released EVs from the microbiome and during physical activity to shape the tumor–immune cross-talk, directly or indirectly, and confer antitumor activity. We further discuss the role of proinflammatory EVs in the temporal development of the tumor–immune interactions and their potential use for cancer diagnostics.     

Molecular and Circulating Biomarkers of Brain Tumors

Brain tumors are the most common malignant primary intracranial tumors of the central nervous system. They are often recognized too late for successful therapy. Minimally invasive methods are needed to establish a diagnosis or monitor the response to treatment of CNS tumors. Brain tumors release molecular information into the circulation. Liquid biopsies collect and analyze tumor components in body fluids, and there is an increasing interest in the investigation of liquid biopsies as a substitute for tumor tissue. Tumor-derived biomarkers include nucleic acids, proteins, and tumor-derived extracellular vesicles that accumulate in blood or cerebrospinal fluid. In recent years, circulating tumor cells have also been identified in the blood of glioblastoma patients. In this review of the literature, the authors highlight the significance, regulation, and prevalence of molecular biomarkers such as O₆-methylguanine-DNA methyltransferase, epidermal growth factor receptor, and isocitrate dehydrogenase. Herein, we critically review the available literature on plasma circulating tumor cells (CTCs), cell-free tumors (ctDNAs), circulating cell-free microRNAs (cfmiRNAs), and circulating extracellular vesicles (EVs) for the diagnosis and monitoring of brain tumor. Currently available markers have significant limitations. While much research has been conductedon these markers, there is still a significant amount that we do not yet understand, which may account for some conflicting reports in the literature.     

Inactivation of EMILIN-1 by Proteolysis and Secretion in Small Extracellular Vesicles Favors Melanoma Progression and Metastasis

Several studies have demonstrated that melanoma-derived extracellular vesicles (EVs) are involved in lymph node metastasis; however, the molecular mechanisms involved are not completely defined. Here, we found that EMILIN-1 is proteolyzed and secreted in small EVs (sEVs) as a novel mechanism to reduce its intracellular levels favoring metastasis in mouse melanoma lymph node metastatic cells. Interestingly, we observed that EMILIN-1 has intrinsic tumor and metastasis suppressive-like properties reducing effective migration, cell viability, primary tumor growth, and metastasis. Overall, our analysis suggests that the inactivation of EMILIN-1 by proteolysis and secretion in sEVs reduce its intrinsic tumor suppressive activities in melanoma favoring tumor progression and metastasis.     

Extracellular Vesicles in Osteosarcoma: Antagonists or Therapeutic Agents?

Osteosarcoma (OS) is a skeletal tumor affecting mainly children and adolescents. The presence of distance metastasis is frequent and it is localized preferentially to the lung, representing the main reason for death among patients. The therapeutic approaches are based on surgery and chemotherapeutics. However, the drug resistance and the side effects associated with the chemotherapy require the identification of new therapeutic approaches. The understanding of the complex biological scenario of the osteosarcoma will open the way for the identification of new targets for its treatment. Recently, a great interest of scientific community is for extracellular vesicles (EVs), that are released in the tumor microenvironment and are important regulators of tumor proliferation and the metastatic process. At the same time, circulating extracellular vesicles can be exploited as diagnostic and prognostic biomarkers, and they can be loaded with drugs as a new therapeutic approach for osteosarcoma patients. Thus, the characterization of OS-related EVs could represent a way to convert these vesicles from antagonists for human health into therapeutic and/or diagnostic agents.     

MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.     

Extracellular Vesicles as Emerging Players in Intercellular Communication: Relevance in Mast Cell-Mediated Pathophysiology

Mast cells are major effector cells in eliciting allergic responses. They also play a significant role in establishing innate and adaptive immune responses, as well as in modulating tumor growth. Mast cells can be activated upon engagement of the high-affinity receptor FcεRI with specific IgE to multivalent antigens or in response to several FcεRI-independent mechanisms. Upon stimulation, mast cells secrete various preformed and newly synthesized mediators. Emerging evidence indicates their ability to be a rich source of secreted extracellular vesicles (EVs), including exosomes and microvesicles, which convey biological functions. Mast cell-derived EVs can interact with and affect other cells located nearby or at distant sites and modulate inflammation, allergic response, and tumor progression. Mast cells are also affected by EVs derived from other cells in the immune system or in the tumor microenvironment, which may activate mast cells to release different mediators. In this review, we summarize the latest data regarding the ability of mast cells to release or respond to EVs and their role in allergic responses, inflammation, and tumor progression. Understanding the release, composition, and uptake of EVs by cells located near to or at sites distant from mast cells in a variety of clinical conditions, such as allergic inflammation, mastocytosis, and lung cancer will contribute to developing novel therapeutic approaches.     

MicroRNAs in Metastasis and the Tumour Microenvironment

Metastasis is the process whereby cancer cells migrate from the primary tumour site to colonise the surrounding or distant tissue or organ. Metastasis is the primary cause of cancer-related mortality and approximately half of all cancer patients present at diagnosis with some form of metastasis. Consequently, there is a clear need to better understand metastasis in order to develop new tools to combat this process. MicroRNAs (miRNAs) regulate gene expression and play an important role in cancer development and progression including in the metastatic process. Particularly important are the roles that miRNAs play in the interaction between tumour cells and non-tumoral cells of the tumour microenvironment (TME), a process mediated largely by circulating miRNAs contained primarily in extracellular vesicles (EVs). In this review, we outline the accumulating evidence for the importance of miRNAs in the communication between tumour cells and the cells of the TME in the context of the pre-metastatic and metastatic niche.     

Movement of Mitochondria with Mutant DNA through Extracellular Vesicles Helps Cancer Cells Acquire Chemoresistance

Triple negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer with the worst prognosis after chemo- or radiation therapy. This is mainly due to the development of cancer chemoresistance accompanied by tumor recurrence. In this work, we investigated a new mechanism of acquired chemoresistance of TNBC cells. We showed that extracellular vehicles (EVs) of chemoresistant TNBC cells can transfer mitochondria to sensitive cancer cells, thus increasing their chemoresistance. Such transfer, but with less efficiency, can be carried out over short distances using tunneling nanotubes. In addition, we showed that exosome fractions carrying mitochondria from resistant TNBC cells contribute to acquired chemoresistance by increasing mtDNA levels with mutations in the mtND4 gene responsible for tumorigenesis. Blocking mitochondrial transport by exosome inhibitors, including GW4869, reduced acquired TNBC chemoresistance. These results could lead to the identification of new molecular targets necessary for more effective treatment of this type of cancer.     

Role of Energy Metabolism in the Progression of Neuroblastoma

Neuroblastoma is a common childhood cancer possessing a significant risk of death. This solid tumor manifests variable clinical behaviors ranging from spontaneous regression to widespread metastatic disease. The lack of promising treatments calls for new research approaches which can enhance the understanding of the molecular background of neuroblastoma. The high proliferation of malignant neuroblastoma cells requires efficient energy metabolism. Thus, we focus our attention on energy pathways and their role in neuroblastoma tumorigenesis. Recent studies suggest that neuroblastoma-driven extracellular vesicles stimulate tumorigenesis inside the recipient cells. Furthermore, proteomic studies have demonstrated extracellular vesicles (EVs) to cargo metabolic enzymes needed to build up a fully operative energy metabolism network. The majority of EV-derived enzymes comes from glycolysis, while other metabolic enzymes have a fatty acid β-oxidation and tricarboxylic acid cycle origin. The previously mentioned glycolysis has been shown to play a primary role in neuroblastoma energy metabolism. Therefore, another way to modify the energy metabolism in neuroblastoma is linked with genetic alterations resulting in the decreased activity of some tricarboxylic acid cycle enzymes and enhanced glycolysis. This metabolic shift enables malignant cells to cope with increasing metabolic stress, nutrition breakdown and an upregulated proliferation ratio.     

Extracellular Vesicles: Potential Mediators of Psychosocial Stress Contribution to Osteoporosis?

Osteoporosis is characterized by low bone mass and damage to the bone tissue’s microarchitecture, leading to increased fracture risk. Several studies have provided evidence for associations between psychosocial stress and osteoporosis through various pathways, including the hypothalamic-pituitary-adrenocortical axis, the sympathetic nervous system, and other endocrine factors. As psychosocial stress provokes oxidative cellular stress with consequences for mitochondrial function and cell signaling (e.g., gene expression, inflammation), it is of interest whether extracellular vesicles (EVs) may be a relevant biomarker in this context or act by transporting substances. EVs are intercellular communicators, transfer substances encapsulated in them, modify the phenotype and function of target cells, mediate cell-cell communication, and, therefore, have critical applications in disease progression and clinical diagnosis and therapy. This review summarizes the characteristics of EVs, their role in stress and osteoporosis, and their benefit as biological markers. We demonstrate that EVs are potential mediators of psychosocial stress and osteoporosis and may be beneficial in innovative research settings.     

Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.     

Proteomic Landscape of Extracellular Vesicles for Diffuse Large B-Cell Lymphoma Subtyping

The role of extracellular vesicles (EVs) proteome in diffuse large B-cell lymphoma (DLBCL) pathology, subclassification, and patient screening is unexplored. We analyzed by state-of-the-art mass spectrometry the whole cell and secreted extracellular vesicles (EVs) proteomes of different molecular subtypes of DLBCL, germinal center B cell (GCB subtype), and activated B cell (ABC subtype). After quality control assessment, we compared whole-cell and secreted EVs proteomes of the two cell-of-origin (COO) categories, GCB and ABC subtypes, resulting in 288/1115 significantly differential expressed proteins from the whole-cell proteome and 228/608 proteins from EVs (adjust p-value < 0.05/p-value < 0.05). In our preclinical model system, we demonstrated that the EV proteome and the whole-cell proteome possess the capacity to separate cell lines into ABC and GCB subtypes. KEGG functional analysis and GO enrichment analysis for cellular component, molecular function, and biological process of differential expressed proteins (DEP) between ABC and GCB EVs showed a significant enrichment of pathways involved in immune response function. Other enriched functional categories for DEPs constitute cellular signaling and intracellular trafficking such as B-cell receptor (BCR), Fc_gamma R-mediated phagocytosis, ErbB signaling, and endocytosis. Our results suggest EVs can be explored as a tool for patient diagnosis, follow-up, and disease monitoring. Finally, this study proposes novel drug targets based on highly expressed proteins, for which antitumor drugs are available suggesting potential combinatorial therapies for aggressive forms of DLBCL. Data are available via ProteomeXchange with identifier PXD028267.     

Extracellular Vesicles in Acute Kidney Injury and Clinical Applications

Acute kidney injury (AKI)––the sudden loss of kidney function due to tissue damage and subsequent progression to chronic kidney disease––has high morbidity and mortality rates and is a serious worldwide clinical problem. Current AKI diagnosis, which relies on measuring serum creatinine levels and urine output, cannot sensitively and promptly report on the state of damage. To address the shortcomings of these traditional diagnosis tools, several molecular biomarkers have been developed to facilitate the identification and ensuing monitoring of AKI. Nanosized membrane-bound extracellular vesicles (EVs) in body fluids have emerged as excellent sources for discovering such biomarkers. Besides this diagnostic purpose, EVs are also being extensively exploited to deliver therapeutic macromolecules to damaged kidney cells to ameliorate AKI. Consequently, many successful AKI biomarker findings and therapeutic applications based on EVs have been made. Here, we review our understanding of how EVs can help with the early identification and accurate monitoring of AKI and be used therapeutically. We will further discuss where current EV-based AKI diagnosis and therapeutic applications fall short and where future innovations could lead us.     

Organ-on-a-Chip for Studying Gut-Brain Interaction Mediated by Extracellular Vesicles in the Gut Microenvironment

Extracellular vesicles (EVs) are a group of membrane vesicles that play important roles in cell-to-cell and interspecies/interkingdom communications by modulating the pathophysiological conditions of recipient cells. Recent evidence has implied their potential roles in the gut–brain axis (GBA), which is a complex bidirectional communication system between the gut environment and brain pathophysiology. Despite the evidence, the roles of EVs in the gut microenvironment in the GBA are less highlighted. Moreover, there are critical challenges in the current GBA models and analyzing techniques for EVs, which may hinder the research. Currently, advances in organ-on-a-chip (OOC) technologies have provided a promising solution. Here, we review the potential effects of EVs occurring in the gut environment on brain physiology and behavior and discuss how to apply OOCs to research the GBA mediated by EVs in the gut microenvironment.     

Characterization of Urine Stem Cell-Derived Extracellular Vesicles Reveals B Cell Stimulating Cargo

Elucidation of the biological functions of extracellular vesicles (EVs) and their potential roles in physiological and pathological processes is an expanding field of research. In this study, we characterized USC–derived EVs and studied their capacity to modulate the human immune response in vitro. We found that the USC–derived EVs are a heterogeneous population, ranging in size from that of micro–vesicles (150 nm–1 μm) down to that of exosomes (60–150 nm). Regarding their immunomodulatory functions, we found that upon isolation, the EVs (60–150 nm) induced B cell proliferation and IgM antibody secretion. Analysis of the EV contents unexpectedly revealed the presence of BAFF, APRIL, IL–6, and CD40L, all known to play a central role in B cell stimulation, differentiation, and humoral immunity. In regard to their effect on T cell functions, they resembled the function of mesenchymal stem cell (MSC)–derived EVs previously described, suppressing T cell response to activation. The finding that USC–derived EVs transport a potent bioactive cargo opens the door to a novel therapeutic avenue for boosting B cell responses in immunodeficiency or cancer.     

Biophysical and Biochemical Comparison of Extracellular Vesicles Produced by Infective and Non-Infective Stages of Trypanosoma cruzi

Extracellular vesicles (EVs) are small lipid vesicles released by either any prokaryotic or eukaryotic cell, or both, with a biological role in cell-to-cell communication. In this work, we characterize the proteomes and nanomechanical properties of EVs released by tissue-culture cell-derived trypomastigotes (mammalian infective stage; (TCT)) and epimastigotes (insect stage; (E)) of Trypanosoma cruzi, the etiologic agent of Chagas disease. EVs of each stage were isolated by differential centrifugation and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy and atomic force microscopy (AFM). Measurements of zeta-potential were also included. Results show marked differences in the surface molecular cargos of EVs between both stages, with a noteworthy expansion of all groups of trans-sialidase proteins in trypomastigote’s EVs. In contrast, chromosomal locations of trans-sialidases of EVs of epimastigotes were dramatically reduced and restricted to subtelomeric regions, indicating a possible regulatable expression of these proteins between both stages of the parasite. Regarding mechanical properties, EVs of trypomastigotes showed higher adhesion compared to the EVs of epimastigotes. These findings demonstrate the remarkable surface remodeling throughout the life cycle of T. cruzi, which shapes the physicochemical composition of the extracellular vesicles and could have an impact in the ability of these vesicles to participate in cell communication in completely different niches of infection.     

Exosomes as A Next-Generation Diagnostic and Therapeutic Tool in Prostate Cancer

Extracellular vesicles (EVs) have brought great momentum to the non-invasive liquid biopsy procedure for the detection, characterization, and monitoring of cancer. Despite the common use of PSA (prostate-specific antigen) as a biomarker for prostate cancer, there is an unmet need for a more specific diagnostic tool to detect tumor progression and recurrence. Exosomes, which are EVs that are released from all cells, play a large role in physiology and pathology, including cancer. They are involved in intercellular communication, immune function, and they are present in every bodily fluid studied—making them an excellent window into how cells are operating. With liquid biopsy, EVs can be isolated and analyzed, enabling an insight into a potential therapeutic value, serving as a vehicle for drugs or nucleic acids that have anti-neoplastic effects. The current application of advanced technology also points to higher-sensitivity detection methods that are minimally invasive. In this review, we discuss the current understanding of the significance of exosomes in prostate cancer and the potential diagnostic value of these EVs in disease progression.     

Biologically Active Tissue Factor-Bearing Larger Ectosome-Like Extracellular Vesicles in Malignant Effusions from Ovarian Cancer Patients: Correlation with Incidence of Thrombosis

The development of malignant effusions such as ascites reflects a massive progression of a malignant disease. In patients with ovarian carcinoma, a high amount of ascites (>500 mL) is an independent negative prognostic marker. The composition and constituents of ascites reflect the inflammatory environment of the underlying tumor. Increased cellular resistance of ascites-derived tumor cells and the development of venous thromboembolic events (VTE) are major risks for these patients, especially in patients with advanced ovarian carcinoma. In this study, we discuss the release of tissue factor-bearing extracellular vesicles (TF⁺ EVs) from tumor cells into the environment (ascites fluid) and their systemic spreading as a possible causal explanation of the pathologic coagulation status in these patients. We obtained ascites from patients with advanced ovarian carcinoma, collected during surgery or therapeutic paracentesis (n = 20). Larger ectosome-like EVs were isolated using sequential centrifugation, quantified by high-resolution flow cytometry and analyzed using nanoparticle tracking analysis. Furthermore, the pro-coagulant properties (TF activity) of EVs were determined. Compared to published TF activities of EVs from healthy persons, TF activities of EVs derived from ascites of patients with ovarian cancer were very high, with a median of 80 pg/mL. The rate of VTE, as reported in the patient files, was high as well (35%, 7 out of 20). Furthermore, all but one patient with VTE had EV concentrations above the median within their ascetic fluid (p < 0.02). Since VTE continues to be a frequent cause of death in cancer patients, prophylactic antithrombotic treatment might be worth considering in these patients. However, given the risk of bleeding, more clinical data are warranted. Although the study is too small to enable reaching a conclusion on direct clinical implementation, it can well serve as a proof of principle and a rationale to initiate a prospective clinical study with different patient subgroups. We also show ex vivo that these larger ectosome-like EVs induce intracellular ERK phosphorylation and tumor cell migration, which is not directly related to their pro-coagulative potency, but might help to understand why cancer patients with thromboembolic events have a poorer prognosis.