Crosstalk between ferroptosis and extracellular vesicles in cancer: from interaction to clinical application

AimNowadays, extracellular vesicles are of great interest in prostate cancer (PCa) research. Asparagine (N)-linked glycosylation could play a significant role in the pathological mechanism of these vesicles. We investigated if...

Extracellular vesicles transport variable content and have crucial functions in cell–cell communication. The role of extracellular vesicles in cancer is a current hot topic, and no bibliometric study has ever analyzed research production regarding their role in breast cancer and indicated the trends in the field. In this way, we aimed to investigate the trends in breast cancer management involved with extracellular vesicle research. Articles were retrieved from Scopus, including all the documents published concerning breast cancer and extracellular vesicles. We analyzed authors, journals, citations, affiliations, and keywords, besides other bibliometric analyses, using R Studio version 3.6.2. and VOSviewer version 1.6.0. A total of 1151 articles were retrieved, and as the main result, our analysis revealed trending topics on biomarkers of liquid biopsy, drug delivery, chemotherapy, autophagy, and microRNA. Additionally, research related to extracellular vesicles in breast cancer has been focused on diagnosis, treatment, and mechanisms of action of breast tumor-derived vesicles. Future studies are expected to explore the role of extracellular vesicles on autophagy and microRNA, besides investigating the application of extracellular vesicles from liquid biopsies for biomarkers and drug delivery, enabling the development and validation of therapeutic strategies for specific cancers.

Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype.

<p dir="ltr">Extracellular vesicles (EVs) are small mediators of cellular interactions, secreted by all cell types, and abundantly found in body fluids. They specifically package a wide variety of bioactive molecules, such as proteins, nucleic acids, or metabolites, which are often altered in the context of diseases.</p><p dir="ltr">In lung diseases and particularly sarcoidosis, a granulomatous disease with unknown etiology, EVs have been shown to have an altered protein signature. In Study I, EVs originating from sarcoidosis patients were used to stimulate monocytic cells, which secreted more pro-inflammatory cytokines than healthy controls, suggesting a role of EVs in sustaining a pro-inflammatory environment. Study II focused on the characterization of the metabolomic content of EVs, comparing sarcoidosis to two other lung diseases (chronic obstructive pulmonary disease, COPD, and a variant of Myositis, anti-synthetase syndrome, ASyS). In line with clinical manifestations, patients with sarcoidosis and ASyS showed similarity in their EV metabolic profile, further separated from COPD and healthy controls. Interestingly, some pathways were specifically enriched in sarcoidosis EVs, unraveling manifestations of a disrupted metabolism detected in EVs.</p><p dir="ltr">Besides their role in disease progression, EVs can also be used as markers to help guide diagnosis. Study III investigated urinary EVs from patients with urinary bladder cancer and healthy controls, characterized by proximity extension assay. A protein signature specific for bladder cancer patients was identified and additional proteins were correlated with muscle-invasiveness. Furthermore, a classifier was able to distinguish muscle-invasive samples from the rest with an accuracy of 92% based on the EV protein profile, paving the way for future EV biomarker studies in urinary bladder cancer.</p><p dir="ltr">Finally, Study IV focused on improving the use of EVs as cancer immunotherapy. Combining antigen-loaded EVs from bone marrow derived dendritic cells, with checkpoint blockade inhibitors (anti-PD1 and anti-PD-L1) did not improve the treatment of established tumors, but using the EVs as a prophylactic cancer vaccine had a synergistic effect with anti-PD-L1 therapy, inducing an anti-tumor response in a checkpoint refractory melanoma mouse model.</p><p dir="ltr">In conclusion, the work in this thesis focuses on the various roles of EVs in lung diseases and cancer by studying their function in disease progression, their cargo, or tuning their immune-stimulatory effect in a therapeutic setting.</p><h3>List of scientific papers</h3><p dir="ltr">I. Casper J. E. Wahlund, Gözde Güçlüler Akpinar, Loïc Steiner, Ahmed Ibrahim, Elga Bandeira, Rico Lepzien, Ana Lukic, Anna Smed- Sörensen, Susanna Kullberg, Anders Eklund, Johan Grunewald, Susanne Gabrielsson</p><p dir="ltr">Sarcoidosis exosomes stimulate monocytes to produce pro- inflammatory cytokines and CCL2.</p><p dir="ltr">Scientific Reports 10, 15328 (2020)<br><a href="https://doi.org/10.1038/s41598-020-72067-7" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41598-020-72067-7<br></a><br></p><p dir="ltr">II. Loïc Steiner, Hasnat Noor, Ralph Evaristo Claret Monte, Elga Bernardo Bandeira De Melo, Chantal Reinhardt, Carl Wengse, Maria Eldh, Begum Horuluoglu, Anders Linden, Lena Palmberg, Ingrid Lundberg, Susanna Kullberg, Carl Wengse, Gözde Güçlüler Akpinar, and Susanne Gabrielsson</p><p dir="ltr">BALF extracellular vesicles from patients with Sarcoidosis and Anti- synthetase Syndrome exhibit metabolic similarities, differing from those in patients with Chronic Obstructive Pulmonary Disease and healthy individuals.</p><p dir="ltr">[Manuscript]</p><p dir="ltr">III. Loïc Steiner, Maria Eldh, Annemarijn Offens, Rosanne E. Veerman, Markus Johansson, Tammer Hemdan, Hans Netterling, Ylva Huge, Abdul-Sattar Aljabery Firas, Farhood Alamdari, Oskar Liden, Amir Sherif and Susanne Gabrielsson</p><p dir="ltr">Protein profile in urinary extracellular vesicles is a marker of malignancy and correlates with muscle invasiveness in urinary bladder cancer.</p><p dir="ltr">Cancer Letters 609, 217352 (2025)<br><a href="https://doi.org/10.1016/j.canlet.2024.217352" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.canlet.2024.217352<br></a><br></p><p dir="ltr">IV. Rosanne E. Veerman, Gözde Güclüler Akpinar, Annemarijn Offens, Loïc Steiner, Pia Larssen, Andreas Lundqvist, Mikael C. I. Karlsson, Susanne Gabrielsson</p><p dir="ltr">Antigen-Loaded Extracellular Vesicles Induce Responsiveness to Anti-PD-1 and Anti-PD-L1 Treatment in a Checkpoint Refractory Melanoma Model.</p><p dir="ltr">Cancer Immunology Research 11 217-227 (2023)<br><a href="https://doi.org/10.1158/2326-6066.cir-22-0540" rel="noreferrer" target="_blank">https://doi.org/10.1158/2326-6066.cir-22-0540</a></p>

<p dir="ltr">Extracellular vesicles (EVs) are small mediators of cellular interactions, secreted by all cell types, and abundantly found in body fluids. They specifically package a wide variety of bioactive molecules, such as proteins, nucleic acids, or metabolites, which are often altered in the context of diseases.</p><p dir="ltr">In lung diseases and particularly sarcoidosis, a granulomatous disease with unknown etiology, EVs have been shown to have an altered protein signature. In Study I, EVs originating from sarcoidosis patients were used to stimulate monocytic cells, which secreted more pro-inflammatory cytokines than healthy controls, suggesting a role of EVs in sustaining a pro-inflammatory environment. Study II focused on the characterization of the metabolomic content of EVs, comparing sarcoidosis to two other lung diseases (chronic obstructive pulmonary disease, COPD, and a variant of Myositis, anti-synthetase syndrome, ASyS). In line with clinical manifestations, patients with sarcoidosis and ASyS showed similarity in their EV metabolic profile, further separated from COPD and healthy controls. Interestingly, some pathways were specifically enriched in sarcoidosis EVs, unraveling manifestations of a disrupted metabolism detected in EVs.</p><p dir="ltr">Besides their role in disease progression, EVs can also be used as markers to help guide diagnosis. Study III investigated urinary EVs from patients with urinary bladder cancer and healthy controls, characterized by proximity extension assay. A protein signature specific for bladder cancer patients was identified and additional proteins were correlated with muscle-invasiveness. Furthermore, a classifier was able to distinguish muscle-invasive samples from the rest with an accuracy of 92% based on the EV protein profile, paving the way for future EV biomarker studies in urinary bladder cancer.</p><p dir="ltr">Finally, Study IV focused on improving the use of EVs as cancer immunotherapy. Combining antigen-loaded EVs from bone marrow derived dendritic cells, with checkpoint blockade inhibitors (anti-PD1 and anti-PD-L1) did not improve the treatment of established tumors, but using the EVs as a prophylactic cancer vaccine had a synergistic effect with anti-PD-L1 therapy, inducing an anti-tumor response in a checkpoint refractory melanoma mouse model.</p><p dir="ltr">In conclusion, the work in this thesis focuses on the various roles of EVs in lung diseases and cancer by studying their function in disease progression, their cargo, or tuning their immune-stimulatory effect in a therapeutic setting.</p><h3>List of scientific papers</h3><p dir="ltr">I. Casper J. E. Wahlund, Gözde Güçlüler Akpinar, Loïc Steiner, Ahmed Ibrahim, Elga Bandeira, Rico Lepzien, Ana Lukic, Anna Smed- Sörensen, Susanna Kullberg, Anders Eklund, Johan Grunewald, Susanne Gabrielsson</p><p dir="ltr">Sarcoidosis exosomes stimulate monocytes to produce pro- inflammatory cytokines and CCL2.</p><p dir="ltr">Scientific Reports 10, 15328 (2020)<br><a href="https://doi.org/10.1038/s41598-020-72067-7" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41598-020-72067-7<br></a><br></p><p dir="ltr">II. Loïc Steiner, Hasnat Noor, Ralph Evaristo Claret Monte, Elga Bernardo Bandeira De Melo, Chantal Reinhardt, Carl Wengse, Maria Eldh, Begum Horuluoglu, Anders Linden, Lena Palmberg, Ingrid Lundberg, Susanna Kullberg, Carl Wengse, Gözde Güçlüler Akpinar, and Susanne Gabrielsson</p><p dir="ltr">BALF extracellular vesicles from patients with Sarcoidosis and Anti- synthetase Syndrome exhibit metabolic similarities, differing from those in patients with Chronic Obstructive Pulmonary Disease and healthy individuals.</p><p dir="ltr">[Manuscript]</p><p dir="ltr">III. Loïc Steiner, Maria Eldh, Annemarijn Offens, Rosanne E. Veerman, Markus Johansson, Tammer Hemdan, Hans Netterling, Ylva Huge, Abdul-Sattar Aljabery Firas, Farhood Alamdari, Oskar Liden, Amir Sherif and Susanne Gabrielsson</p><p dir="ltr">Protein profile in urinary extracellular vesicles is a marker of malignancy and correlates with muscle invasiveness in urinary bladder cancer.</p><p dir="ltr">Cancer Letters 609, 217352 (2025)<br><a href="https://doi.org/10.1016/j.canlet.2024.217352" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.canlet.2024.217352<br></a><br></p><p dir="ltr">IV. Rosanne E. Veerman, Gözde Güclüler Akpinar, Annemarijn Offens, Loïc Steiner, Pia Larssen, Andreas Lundqvist, Mikael C. I. Karlsson, Susanne Gabrielsson</p><p dir="ltr">Antigen-Loaded Extracellular Vesicles Induce Responsiveness to Anti-PD-1 and Anti-PD-L1 Treatment in a Checkpoint Refractory Melanoma Model.</p><p dir="ltr">Cancer Immunology Research 11 217-227 (2023)<br><a href="https://doi.org/10.1158/2326-6066.cir-22-0540" rel="noreferrer" target="_blank">https://doi.org/10.1158/2326-6066.cir-22-0540</a></p>

Extracellular Vesicles in Cancer

The sustained growth, invasion, and metastasis of cancer cells depend upon bidirectional cell-cell communication within complex tissue environments. Such communication predominantly involves the secretion of soluble factors by cancer cells and/or stromal cells within the tumour microenvironment (TME), although these cell types have also been shown to export membrane-encapsulated particles containing regulatory molecules that contribute to cell-cell communication. These particles are known as extracellular vesicles (EVs) and include species of exosomes and shed microvesicles. EVs carry molecules such as oncoproteins and oncopeptides, RNA species (for example, microRNAs, mRNAs, and long non-coding RNAs), lipids, and DNA fragments from donor to recipient cells, initiating profound phenotypic changes in the TME. Emerging evidence suggests that EVs have crucial roles in cancer development, including pre-metastatic niche formation and metastasis. Cancer cells are now recognized to secrete more EVs than their nonmalignant counterparts, and these particles can be isolated from bodily fluids. Thus, EVs have strong potential as blood-based or urine-based biomarkers for the diagnosis, prognostication, and surveillance of cancer. In this Review, we discuss the biophysical properties and physiological functions of EVs, particularly their pro-metastatic effects, and highlight the utility of EVs for the development of cancer diagnostics and therapeutics.

Extracellular vesicles (EVs) play an integral role in cancer biology, influencing tumor progression, metastasis, and tumor microenvironment. Due to their distinctive molecular composition, including proteins, nucleic acids, and lipids, EVs present a promising candidate for cancer diagnostics and precision therapeutics. This review was conducted by looking up recent studies obtained through PubMed, Scopus, and Web of Science databases using targeted keywords such as "Extracellular Vesicles," "Cancer Therapy," "Biomarkers," "Exosomes," "Tumor Microenvironment," and "Precision Medicine." From an initial 4,320 articles identified, 427 were screened after applying publication filters, resulting in the inclusion of 298 articles relevant to EV isolation, characterization, diagnostic sensitivity, specificity, and therapeutic efficacy. Biomarkers derived from EVs derived across various cancers showed high diagnostic performance. For example, four miRNA EVs showing sensitivity and specificity of 98% and 96% respectively was found in breast cancer. EV-RNA and surface antigen analyses for hepatocellular carcinoma with 93.8% sensitivity and 74.5% specificity. Additionally, EV biomarker cancers of the colorectal microRNA miR-23a and miR-301a had 89% sensitivity and >70% specificity. EVs in a therapeutic context were an effective drug delivery system for enhancing precision of chemotherapy and immunotherapy with reduced systemic toxicity. The theranostics of EVs provide great capacity for early cancer diagnosis and personalized treatment based on their high diagnostic sensitivity and specificity. Future standardization protocols are essential to translate EV technologies into clinical oncology.

Small extracellular vesicles in cancer

Cancer-derived extracellular vesicles (EVs) are regarded as having promising potential to be used as therapeutics and disease biomarkers. Mechanistically, EVs have been shown to function in most, if not all, steps of cancer progression. Cancer EVs, including small EVs (sEVs), contain unique biomolecular cargo, consisting of protein, nucleic acid and lipids. Through progress in the identification of this specific cargo, cancer biomarkers have been identified and developed, opening up novel and interesting opportunities for cancer diagnosis and prognosis. Intriguingly, we still lack a comprehensive understanding of the cancer-specific pathways that govern EV biogenesis in cancer cells. Filling this knowledge gap will rapidly improve cancer EV biomarkers, as it will also allow discrimination of the procancer and anticancer actions of those EVs. Even more promising is uncovering therapeutically targetable, tumour-specific EV pathways and content, which will generate novel classes of cancer therapies. This Review highlights the progress the cancer sEV field has made in the areas of biomarker discovery and validation as well as sEV-based therapeutics, highlights the challenges we are facing and identifies gaps in our knowledge, which currently prevent us from developing the full potential of sEVs in cancer diagnostic and therapy.

Extracellular vesicles (EVs) encompass a multitude of lipid bilayer-delimited particles, of which exosomes are the most widely studied. Bidirectional cell-cell communications via EVs have a pivotal role in the physiology of multicellular organisms. EVs carry biological cargoes (including proteins, RNA, DNA, lipids and metabolites) capable of mediating a range of pleiotropic cellular functions. Over the past decade, EVs released by cancer cells (onco-EVs) have been shown to promote cancer progression including tumour outgrowth and metastatic dissemination. Furthermore, the innate ability of EVs to protect vulnerable molecular cargoes (such as RNA, DNA or proteins) from enzymatic degradation, their presence in most biofluids and the ability to transverse biological barriers to reach distant organs make them ideal targeted drug delivery systems, including in patients with cancer. Many of these properties also support investigations of EVs as biomarkers with potential roles in both diagnosis and treatment monitoring. In this Review, we describe advances in the development of EVs as cancer therapeutics or biomarkers, including cancer vaccines, targeted drug delivery systems and immunotherapies, as well as potential roles in early cancer detection, diagnosis and clinical management. We also describe the potential of emerging technologies to support further discoveries as well as the clinical translation of EVs into diagnostic and therapeutic clinical tools. We highlight the potential of single-EV and onco-EV detection and discuss how advances in multi-omic and artificial intelligence-enabled integration are providing new biological insights and driving clinical translation.

Extracellular vesicles (EVs) are nano-sized particles secreted by most cells. They transport different types of biomolecules (nucleic acids, proteins, and lipids) characteristic of their tissue or cellular origin that can mediate long-distance intercellular communication. In the case of cancer, EVs participate in tumor progression by modifying the tumor microenvironment, favoring immune tolerance and metastasis development. Consequently, EVs have great potential in liquid biopsy for cancer diagnosis, prognosis and follow-up. In addition, EVs could have a role in cancer treatment as a targeted drug delivery system. The intense research in the EV field has resulted in hundreds of patents and the creation of biomedical companies. However, methodological issues and heterogeneity in EV composition have hampered the advancement of EV validation trials and the development of EV-based diagnostic and therapeutic products. Consequently, only a few EV biomarkers have moved from research to clinical laboratories, such as the ExoDx Prostate IntelliScore (EPI) test, a CLIA/FDA-approved EV prostate cancer diagnostic test. In addition, the number of large-scale multicenter studies that would clearly define biomarker performance is limited. In this review, we will critically describe the different types of EVs, the methods for their enrichment and characterization, and their biological role in cancer. Then, we will specially focus on the parameters to be considered for the translation of EV biology to the clinic laboratory, the advances already made in the field of EVs related to cancer diagnosis and treatment, and the issues still pending to be solved before EVs could be used as a routine tool in oncology.

Extracellular vesicles (EVs), a class of nanoscale, membrane-bound vesicles secreted by various cell types, have emerged as rapidly advancing fields of research in recent years. This heterogeneous vesicle is a versatile carrier system for a variety of biomolecules, including proteins, nucleic acids, and metabolites. EVs play pivotal roles in intercellular communication, immune regulation, and disease pathogenesis, with particular implications for cancer biology. On the one hand, EVs promote tumor progression and metastasis by facilitating communication between cancer cells and their microenvironment. On the other hand, EVs carry noncoding RNAs, such as miRNAs and other regulatory RNAs, which directly modulate immune cell function or exert antitumor effects by influencing cancer cell proliferation and apoptosis. In addition to their biological roles, EVs show great potential as drug delivery systems because of their ability to be effectively taken up by target cells and stably deliver therapeutic payloads. In the context of cancer therapy, natural EVs demonstrate inherent therapeutic potential, particularly in targeting highly metabolically active organs. Furthermore, engineered EVs, which serve as both therapeutic vehicles and molecular imaging probes, have demonstrated significant potential for cancer theranostics. This review focuses on elucidating the dynamic changes and biological functions of EVs in vivo, with the aim of exploring the translational potential of EV-based molecular imaging and tracing technologies in cancer treatment. This work seeks to provide critical insights that may enhance the precision and efficacy of tumor therapies, offering a foundation for future clinical applications.

Extracellular vesicles (EVs), including exosomes and microvesicles, are extracellular nanovesicles released by most cells. EVs play essential roles in intercellular communication via the transport of a large variety of lipids, proteins, and nucleic acids to recipient cells. Nucleic acids are the most commonly found molecules inside EVs, and due to their small size, microRNAs and other small RNAs are the most abundant nucleic acids. However, longer molecules, such as messenger RNAs (mRNAs), have also been found. mRNAs encapsulated within EVs have been shown to be transferred to recipient cells and translated into proteins, altering the behavior of the cells. Secretion of EVs is maintained not only through multiple normal physiological conditions but also during aberrant pathological conditions, including cancer. Recently, the mRNAs carried by EVs in cancer have attracted great interest due to their broad roles in tumor progression and microenvironmental remodeling. This review focuses on the biological functions driven by mRNAs carried in EVs in cancer, which include supporting tumor progression by activating cancer cell growth, migration, and invasion; inducing microenvironmental remodeling via hypoxia, angiogenesis, and immunosuppression; and promoting modulation of the microenvironment at distant sites for the generation of a premetastatic niche, collectively inducing metastasis. Furthermore, we describe the potential use of mRNAs carried by EVs as a noninvasive diagnostic tool and novel therapeutic approach.

Simple Summary3D cell cultures are a qualitative improvement in cancer research because these models preserve cancer physiological characteristics better than traditional bi-dimensional cultures. Moreover, they facilitate the study of complex 3D interactions using extracellular matrices and the co-culture of different cell types. In this manner, the cells can contact themselves in a fully physiological but also controlled arrangement. In the context of tumor interactions, extracellular vesicles are essential in number of key aspects in oncology: as major interactors with extracellular matrix, as cell-to-cell messengers, as carriers of diagnostic-valuable biomarkers, and as target-specific treatment-deliver agents. The present article aims to discuss the findings achieved using 3D culture models in oncology. We further review the involvement of extracellular vesicles in the pathogenesis of cancer as well as their potential use in diagnostics and therapeutics.The improvement of culturing techniques to model the environment and physiological conditions surrounding tumors has also been applied to the study of extracellular vesicles (EVs) in cancer research. EVs role is not only limited to cell-to-cell communication in tumor physiology, they are also a promising source of biomarkers, and a tool to deliver drugs and induce antitumoral activity. In the present review, we have addressed the improvements achieved by using 3D culture models to evaluate the role of EVs in tumor progression and the potential applications of EVs in diagnostics and therapeutics. The most employed assays are gel-based spheroids, often utilized to examine the cell invasion rate and angiogenesis markers upon EVs treatment. To study EVs as drug carriers, a more complex multicellular cultures and organoids from cancer stem cell populations have been developed. Such strategies provide a closer response to in vivo physiology observed responses. They are also the best models to understand the complex interactions between different populations of cells and the extracellular matrix, in which tumor-derived EVs modify epithelial or mesenchymal cells to become protumor agents. Finally, the growth of cells in 3D bioreactor-like systems is appointed as the best approach to industrial EVs production, a necessary step toward clinical translation of EVs-based therapy.