Circulating tumor DNA (ctDNA) has emerged as a dynamic biomarker in cancer, as evidenced by its increasing integration into clinical practice. Carrying tumor specific characteristics, ctDNA can be used to inform treatment selection, monitor response, and identify drug resistance. In this review, we provide a comprehensive, up-to-date summary of ctDNA in monitoring treatment response with a focus on lung, colorectal, and breast cancers, and discuss current challenges and future directions.

Naturally occurring extracellular vesicles (EVs) and synthetic nanoparticles like liposomes have revolutionized precision diagnostics and medicine. EVs excel in biocompatibility and cell targeting, while liposomes offer enhanced drug loading capacity and scalability. The clinical translation of EVs is hindered by challenges including low yield and heterogeneity, whereas liposomes face rapid immune clearance and limited targeting efficiency. To bridge these gaps, biomimetic synthetic vesicles (SVs) have emerged as innovative platforms, combining the advantageous properties of EVs and liposomes. This review emphasizes critical aspects of EV biology, such as mechanisms of EV-cell interaction and source-dependent functionalities in targeting, immune modulation, and tissue regeneration, informing biomimetic SV engineering. We reviewed a broad array of biomimetic SVs, with a focus on lipid bilayered vesicles functionalized with proteins. These include cell-derived nanovesicles, protein-functionalized liposomes, and hybrid vesicles. By addressing current challenges and highlighting opportunities, this review aims to advance biomimetic SVs for transformative biomedical applications.

Extracellular vesicles (EVs) have emerged as key players in intercellular communication, facilitating the transfer of crucial cargo between cells. Liquid biopsy, particularly through the isolation of EVs, has unveiled a rich source of potential biomarkers for health and disease, encompassing proteins and nucleic acids. A milestone in this exploration occurred a decade ago with the identification of extracellular vesicle-associated DNA (EV-DNA) in the bloodstream of a patient diagnosed with pancreatic cancer. Subsequent years have witnessed substantial advancements, deepening our insights into the molecular intricacies of EV-DNA emission, detection, and analysis. Understanding the complexities surrounding the release of EV-DNA and addressing the challenges inherent in EV-DNA research are pivotal steps toward enhancing liquid biopsy-based strategies. These strategies, crucial for the detection and monitoring of various pathological conditions, particularly cancer, rely on a comprehensive understanding of why and how EV-DNA is released. In our review, we aim to provide a thorough summary of a decade’s worth of research on EV-DNA. We will delve into diverse mechanisms of EV-DNA emission, its potential as a biomarker, its functional capabilities, discordant findings in the field, and the hurdles hindering its clinical application. Looking ahead to the next decade, we envision that advancements in EV isolation and detection techniques, coupled with improved standardization and data sharing, will catalyze the development of novel strategies exploiting EV-DNA as both a source of biomarkers and therapeutic targets.

Background: Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. Methods: We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCβ4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). Results: The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. Conclusions: We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.

Lymph nodes (LNs) are frequently the first sites of metastasis. Currently, the only prognostic LN assessment is determining metastatic status. However, there is evidence suggesting that LN metastasis is facilitated by the formation of a pre‐metastatic niche induced by tumour derived extracellular vehicles (EVs). Therefore, it is important to detect and modify the LN environmental changes. Earlier work has demonstrated that neutrophil extracellular traps (NETs) can sequester and promote distant metastasis. Here, we first confirmed that LN NETs are associated with reduced patient survival. Next, we demonstrated that NETs deposition precedes LN metastasis and NETs inhibition diminishes LN metastases in animal models. Furthermore, we discovered that EVs are essential to the formation of LN NETs. Finally, we showed that lymphatic endothelial cells secrete CXCL8/2 in response to EVs inducing NETs formation and the promotion of LN metastasis. Our findings reveal the role of EV‐induced NETs in LN metastasis and provide potential immunotherapeutic vulnerabilities that may occur early in the metastatic cascade.