Tumor-derived Extracellular Vesicles Research Articles

Abstract 1968: Comparable detection of large tumor-derived vesicles (oncosomes) by known CTC platforms: Potential new analyte for biomarker monitoring?

Abstract Background: Monitoring tumors and detecting biomarkers through blood analysis remains challenging. Large oncosomes (LOs) are tumor-derived extracellular vesicles that offer a promising avenue for understanding tumor biology and biomarker status, potentially surpassing circulating tumor cells (CTCs) in accuracy. Validated LO detection methods are required to unlock LOs analytical potential. We evaluated two LO detection methods using known CTC platforms, the FDA-approved CellSearch, and RareCyte to compare their performance. In addition, we assessed the potential of LOs for HER2 detection in metastatic breast cancer (MBC) patients. Methods: LO counts were analyzed in parallel blood samples from 96 MBC patients using either the CellSearch/ACCESS tdEV analysis package, or the RareCyte method with in-house LO analysis. Overall survival (OS) was defined as the time from baseline blood draw to death or last follow-up. HER2 scores from primary tumor biopsies were retrieved from patient charts. HER2 overexpression in LOs and CTCs was determined using the RarePlex HER2/ER CTC panel kit (n=9). Correlations between LO and CTC counts were assessed using Spearman’s Rho test, and comparability of LO counts between platforms was evaluated with the Wilcoxon Signed Ranks test. Cutoffs for favorable vs. unfavorable LO counts were determined using an online tool. Survival curves for these groups were compared using the log-rank test. Correlation of HER2+ analytes with tissue biopsy HER2 scores was also analyzed using Spearman’s Rho test. Results: A strong positive correlation was observed between LO counts from the two platforms (r = 0.49, p < 0.001). LO counts also correlated with their respective CTC counts (CellSearch: r = 0.88, p < 0.001; RareCyte: r = 0.93, p = 0.001). Higher LO counts were associated with worse survival outcomes (CellSearch: r = 0.35, p < 0.001; RareCyte: r = 0.39, p = 0.006). Patients with unfavorable LO counts had significantly shorter survival compared to patients with favourable LO counts (CellSearch: HR = 3.5, p < 0.001; RareCyte: HR = 3.6, p = 0.02). RareCyte detected significantly more LOs than CellSearch (median LO count: 132 vs. 41, p = 0.01). HER2 signal was detected in 5/9 patients with CTCs and in 8/9 patients with LOs. Notably, the proportion of HER2+ LOs positively correlated with tissue biopsy HER2 score (r=0.799, p=0.01). In contrast, the proportion of HER2+ CTCs did not correlate with tissue biopsy. Conclusions: While RareCyte captured more LOs than CellSearch, LO counts were highly concordant across platforms, validating both methods. The association of LO counts with overall survival suggests that LOs reflect tumor progression, and may complement CTC-based monitoring. Furthermore, the correlation between LO HER2 status and tumor biopsy HER2 scores underscores the potential of LO analysis for biomarker monitoring in metastatic breast cancer. Citation Format: Eszter Papp, Pieter Mestdagh, Luc Y. Dirix, Peter Vermeulen, Mark Kockx. Comparable detection of large tumor-derived vesicles (oncosomes) by known CTC platforms: Potential new analyte for biomarker monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1968.

Abstract 2005: ROR1-specific extracellular vesicle tetraspanin-8 levels predict distant metastasis in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC), comprising 15-20% of all breast cancer cases, is the most aggressive subtype, with limited efficacy from targeted therapies and immunotherapies. Metastasis drives TNBC-related mortality, emphasizing the urgent need for predictive methods to identify high-risk cases early, guide clinical management, and facilitate timely interventions. Extracellular vesicles (EVs) play a pivotal role in cancer progression and metastasis, offering a promising avenue for metastasis research. However, isolating tumor-derived EVs from the total EV population remains a significant clinical challenge, underscoring the necessity for precise detection strategies. Methods: EVs were isolated from MCF-10A cells, MCF-7 cells, and MDA-MB-231 cells using ultracentrifugation, followed by morphological characterization and surface marker protein validation. Mass spectrometry and UniProt gene ontology analyses were conducted to identify TNBC-specific EV proteins, classified by molecular functions or biological processes. TSPAN8 expression in breast cancer cells was analyzed using the CCLE and TCGA databases. Cholesterol-PEG-Biotin and Streptavidin-APC (Cholesterol-PEG-APC) were employed to anchor EV membranes, enabling concurrent detection of quantum dot-labeled EV proteins and membrane signals. Results: ROR1, a transmembrane protein expressed in TNBC cells and EVs, was identified as a specific capture protein for TNBC-derived EVs. Database analysis revealed elevated TSPAN8 expression in highly metastatic TNBC MDA-MB-231 cells and in the tissue of TNBC-LAR subtype, where TSPAN8 plays a decisive role in metastasis. Using ROR1 to capture MDA-MB-231 EVs demonstrated that the fluorescence intensity from quantum dot-labeled QD605 anti-TSPAN8 and APC increased with EV concentration. Furthermore, higher average EV-TSPAN8/APC levels were associated with greater metastatic risk in patients. Correlation analysis using the TCGA database showed a positive relationship between ROR1 and TSPAN8 expression. VCAM1 was identified within the interaction network of ROR1 and TSPAN8, with positive correlations to both, providing a theoretical basis for leveraging ROR1-specific EV-TSPAN8 detection in metastasis research. Conclusions: ROR1 effectively captures tumor-derived EVs and holds significant potential for enhancing EV-TSPAN8 detection, providing a promising strategy for predicting metastasis in TNBC. Evaluating ROR1-specific EV-TSPAN8 levels in TNBC blood samples and its link to disease progression warrants further investigation. Additionally, exploring its clinical utility across diverse TNBC subtypes and treatment regimens could refine its application, ultimately improving the precision of early metastasis prediction in TNBC. Research Sponsor: National Natural Science Foundation of China (No. 82202603). Citation Format: Yinchao Zhang, Linze Xu, Jin Zhang, Dandan Wu, Yang Liu. ROR1-specific extracellular vesicle tetraspanin-8 levels predict distant metastasis in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2005.

Abstract 4505: Differential protein expression in platelets collected from glioma patients and healthy controls: A comparative study

Abstract Background: Glioblastoma (GBM) is the most aggressive type of primary brain tumor in adults. Standard treatment fosters event free survival for approximately 7 months and extends overall survival to only 15 months. Due to the risk of biopsy complications, the verification of the tumor is not always possible. Therefore, in many cases the treatment is based on MRI findings and exclusion of other malignancies. A confirmative liquid biopsy would provide additional and useful support for clinical decision making and patient counselling. To date no liquid biopsy for GBM is available. Platelets interact with tumor cells in multiple ways, including direct contact, release of cytokines, and uptake of tumor-derived extracellular vesicles. These interactions cause modifications in platelet RNA and protein profiles, yielding tumor educated platelets that reflect specific tumor characteristics. Our study aimed to use proteomic analysis to identify differential protein expression in platelets collected from glioma patients compared to healthy controls, identifying potential biomarkers for glioma diagnosis and therapeutic monitoring Methods: Platelet samples were collected from 14 glioma patients and 14 matching healthy controls. Platelets were isolated using differential centrifugation to minimize contamination with other blood components. Protein extraction and quantification were performed, followed by analysis using mass spectrometry-based proteomics. Protein identification and quantification were conducted with Label-Free Quantification techniques, and differentially expressed proteins were statistically validated. Bioinformatic tools were employed to analyze pathways and functional annotations related to identified proteins. Results: In total 3444 proteins were identified and quantified. Of these, 463 proteins were differentially expressed between platelets in glioma patients and healthy controls. Reactome Pathway, gene ontology enrichment and protein protein interactions analyses revealed proteins associated with pathways linked to Vesicle-mediated transport, Apoptosis, Metabolism of Lipids, Cell Cycle Checkpoints, Glutathione synthesis/recycling, Platelet Activation and Signal transduction. Conclusion: The study identifies a distinct proteomic profile in platelets from glioma patients compared to healthy individuals, suggesting that platelets in glioma may harbor unique proteins characteristics related to tumor biology. These findings support the potential of platelets as a source of biomarkers for glioma detection, confirmation of diagnosis and monitoring. Future studies with larger cohorts are warranted to validate these proteins as diagnostic markers and explore their mechanistic roles in glioma progression. Citation Format: Vibecke Markhus, Katarina Fritz-Wallace, Olav Mjaavatten, Einar Kleboe Kristoffersen, Rupavathana Mahesparan, Hrvoje Miletic, Frode Selheim, Dorota Goplen. Differential protein expression in platelets collected from glioma patients and healthy controls: A comparative study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4505.

The Ceramide-Dependent EV Secretome Differentially Affects Prostate Cancer Cell Migration.

Tumor-derived extracellular vesicles (EVs) play an important role in cancer progression. Neutral sphingomyelinases (nSMases) are lipid-modifying enzymes that modulate the secretion of EVs from cells. How nSMase activity and therefore ceramide generation affect the composition and functionality of secreted EVs is not fully understood. Here, we aimed to investigate the expression of nSMases 1 and 2 in prostate cancer (PCa) tissue and their role in EV composition and secretion for prostate cancer cell migration. Reduced nSMase 1 and 2 expression was found in prostate cancer and correlated with the age of the patient. When nSMase 2 was inhibited by GW4869 in PCa cells (PC3 and DU145), the EV secretome was significantly altered, while the number of EVs and the total protein content of released EVs were not significantly changed. Using proteomic analysis, we found that extracellular matrix proteins, such as SDC4 (Syndecan-4) and SRPX-2, were differentially secreted on EVs from GW4869-treated PC3 cells. In scratch wound migration assays, GW4869 significantly increased migration compared to control PC3 cells but not DU145 cells, while SDC4 knockdown significantly reduced the migration of PC3 cells. These and other nSMase-2-dependent secreted proteins are interesting candidates for understanding the role of stress-induced EVs in the progression of prostate cancer.

P-Pev: micelle-like complexes transformed from tumor extracellular vesicles by PEG-PE for personalized therapeutic tumor vaccine.

P-Pev: micelle-like complexes transformed from tumor extracellular vesicles by PEG-PE for personalized therapeutic tumor vaccine.

Enrichment and Detection of HER2-Expressing Extracellular Vesicles Based on DNA Tetrahedral Nanostructures: A New Strategy for Liquid Biopsy in Breast Cancer.

Extracellular vesicles (EVs) play a crucial role as important mediators of intercellular communication in the progression of tumors. The capture and analysis of tumor-derived EVs offer new possibilities for the application of cancer liquid biopsies. This study aims to construct a DNA tetrahedral nanostructure that specifically recognizes HER2 and CD63, enabling the effective enrichment and detection of HER2-expressing EVs (HEVs). We enriched HEVs from cell lines and 13 random clinical samples and validated their characteristics by dynamic light scattering, transmission electron microscopy, and Western blotting. Further, we detected HEVs levels in clinical samples. The HEVs levels in HER2-positive breast cancer patients were significantly higher than those in healthy/benign controls (mean, 4.737 vs 4.160 vs 4.144 U/μL, P < 0.0001), displaying a concentration gradient across different HER2 expression levels. This study establishes an approach for HEV detection, thus providing a new tool for the diagnosis of HER2-positive breast cancer.

Simple and Rapid Tumor EV Enrichment Enabled by Long DNA Probe-Guided Entanglement.

Specific subpopulations of extracellular vesicles (EVs) hold significant clinical potential for biomarker discovery, disease diagnosis, and therapeutic agents. However, this field remains underutilized due to the lack of straightforward and versatile techniques for isolating EV subpopulations from biofluids. Here, we present LODGE, a long DNA probe-guided EV entanglement strategy for the simple, rapid, and selective enrichment of tumor-derived EVs (tEVs) from clinical specimens. LODGE uses two long DNA affinity probes to recognize specific subpopulations, causing them to aggregate with the assistance of splint strands, thereby achieving non-destructive, high-yield, and high-purity separation of tEVs within a short period. Proteomic analysis revealed that the isolated tEVs contributed to the identification of tumor-associated biomarkers compared to total EVs. Additionally, by incorporating a split G-quadruplex-containing molecular trap domain, a novel structure that significantly improves the fluorescence emission of thioflavin T (ThT), into DNA affinity probes, we developed an innovative LODGE-ThT sensing strategy for the highly sensitive profiling of multiple tEV subpopulations. Using data from the tEVs alongside clinical indicators processed with machine learning algorithms, we effectively classified five tumor types. Our results show that LODGE is a promising tool for identifying specific EV subpopulations, fostering their biomedical applications.

Profiling Nascent Tumor Extracellular Vesicles via Metabolic Timestamping and Aptamer-Driven Specific Click Chemistry.

Tumor-derived extracellular vesicles (tEVs) are essential mediators of tumor progression and therapeutic resistance, yet their secretion dynamics and cargo composition in response to therapies remain poorly understood. Here, we present STAMP, specific click-tagging driven by aptamer for tEV labeled with a metabolic timestamp, which exploits the unique kinetics and thermodynamics of aptamer to significantly enhance the local concentration of clickable probes on tEVs for their covalent attachment to the timestamp, resulting in the selective microfluidic isolation of nascent tEVs following stimulation. In a PD-L1 antibody-treated model, we demonstrated the feasibility of STAMP and revealed a robust positive correlation between the nascent EpCAM+ EV levels and tumor volume. Proteome profiling of isolated nascent tEVs identified previously unknown upregulated vesicle proteins following immunotherapy, including key regulators of immune activation and suppression, suggesting that tumors orchestrate an intricate dual adaptive response through tEV secretion modulation to simultaneously elicit therapeutic sensitivity and resistance. Notably, among the upregulated proteins, we identified HSP70, whose enhanced presentation on tEVs promotes antitumor immunity and inhibits tumor growth. Thus, STAMP provides an effective gateway for studying EV dynamics with cell-origin accuracy and for identifying potential therapeutic targets based on EV transitions.

Tumor-derived extracellular vesicles: key drivers of immunomodulation in breast cancer.

Breast cancer (BC) remains a significant global health challenge characterized by its heterogeneity and treatment complexities. Extracellular vesicles (EVs) are small membranous particles released by cells, facilitating intercellular communication by transporting bioactive molecules such as proteins, lipids, and nucleic acids. Tumor-derived EVs have emerged as pivotal regulators in the tumor microenvironment (TME) and drivers of BC progression. These EVs carry diverse cargoes of bioactive molecules, influencing critical processes such as immune modulation, angiogenesis, and metastasis. By altering the behaviors of immune cells including macrophages, dendritic cells, and T cells, tumor-derived EVs contribute to immune evasion and tumor growth. Furthermore, Tumor-derived EVs play a role in mediating drug resistance, impacting the effectiveness of therapeutic interventions. Understanding the multifaceted roles of BC tumor-derived EVs is essential for the development of innovative therapeutic strategies. Targeting pathways mediated by EVs holds promise for enhancing the efficacy of cancer treatments and improving patient outcomes. This comprehensive review provides insights into the intricate interactions of tumor-derived EVs in immune modulation and BC progression, highlighting potential therapeutic targets and avenues for novel cancer therapies.

Bushen Jiedu formula alleviates colorectal cancer progression through reducing lncRPPH1 in tumor-derived extracellular vesicles.

Bushen Jiedu formula alleviates colorectal cancer progression through reducing lncRPPH1 in tumor-derived extracellular vesicles.

Tumor-derived extracellular vesicles: Hijacking T cell function through exhaustion.

Tumor-derived extracellular vesicles: Hijacking T cell function through exhaustion.

Dual-color fluorescence detection of tumor-derived extracellular vesicles using a specific and serum-stable membrane-fusion approach.

Dual-color fluorescence detection of tumor-derived extracellular vesicles using a specific and serum-stable membrane-fusion approach.

Tumor-derived extracellular vesicle PD-1 promotes tumor immune evasion via disruption of peripheral T cell homeostasis.

Tumor-derived extracellular vesicle PD-1 promotes tumor immune evasion via disruption of peripheral T cell homeostasis.

Tumor-derived arachidonic acid reprograms neutrophils to promote immune suppression and therapy resistance in triple-negative breast cancer.

Tumor-derived arachidonic acid reprograms neutrophils to promote immune suppression and therapy resistance in triple-negative breast cancer.

1581 Quantifying Subpopulations of Tumor-Derived Extracellular Vesicles through Targeting Druggable Surface Proteins for Early Detection of Epithelial Ovarian Cancer

1581 Quantifying Subpopulations of Tumor-Derived Extracellular Vesicles through Targeting Druggable Surface Proteins for Early Detection of Epithelial Ovarian Cancer

B7-H3-liquid biopsy for the characterization and monitoring of the dynamic biology of prostate cancer.

B7-H3-liquid biopsy for the characterization and monitoring of the dynamic biology of prostate cancer.

HBV-associated hepatocellular carcinomas inhibit antitumor CD8+ T cell via the long noncoding RNA HDAC2-AS2

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Extracellular vesicles (EV) are critical mediators of intercellular communication within the tumor microenvironment, and cancer-cell-secreted EVs often facilitate cancer progression. Here we show that in HBV-associated HCC, tumor-cell-derived EVs contain a TGFβ-inducible long noncoding RNA, termed HDAC2-AS2. EVs enriched with HDAC2-AS2 facilitate cancer progression by suppressing cytotoxicity of intra-tumor CD8+ T cells. Mechanistically, in activated cytotoxic CD8+ T cells, translocation of the transcription factor cyclin-dependent kinase 9 (CDK9), to the cytoplasm is critical for functional integrity. HDAC2-AS2 targets and blocks cytosolic CDK9, and this results in exhaustion of PD-1+CD8+ T cells and suppression of IFN-γ+CD8+ T cell cytotoxicity. Notably, we demonstrate that low CDK9 and high HDAC2-AS2 expressions are associated with poor survival of HCC, which can be rescued by anti-PD-1 therapy. These findings emphasize the significance of tumor-derived EVs in suppressing antitumor CD8+ T cell immunity to promote tumorigenesis, and highlight extracellular HDAC2-AS2 as a promising biomarker and therapeutic target for HCC.

Rapid in situ mutation detection in extracellular vesicle DNA.

Aim: A PCR- and sequencing-free mutation detection assay facilitates cancer diagnosis and reduces over-reliance on specialized equipment. This benefit was highlighted during the pandemic when high demand for viral nucleic acid testing often sidelined mutation analysis. This shift led to substantial challenges for patients on targeted therapy in tracking mutations. Here, we report a 30-min DNA mutation detection technique using Cas12a-loaded liposomes in a microplate reader, a fundamental laboratory tool. Methods: CRISPR-Cas12a complex and fluorescence-quenching (FQ) probes are introduced into tumor-derived extracellular vesicles (EV) through membrane fusion. When CRISPR-RNA hybridizes with the DNA target, activated Cas12a can trans-cleave FQ probes, resulting in fluorescence signals for the quantification of DNA mutation. Results: This method enables the detection of EGFR L858R mutation in EV DNA within 30 min. Laborious extraction, purification, and other preparation steps for EV DNA are eliminated. The need for advanced data processing is also dispensed with. In a cohort study involving 10 healthy donors and 30 patients with advanced non-small cell lung cancer (NSCLC), the assay achieved a sensitivity of 86.7%, a specificity of 90%, and an accuracy of 87.5%. Conclusion: The limit of detection of our Cas12 assay was ~ 8 × 105 EVs, corresponding to a mutation allele frequency (MAF) of ~ 10%. The MAF in late-stage cancers varies widely but often falls within 5%-50%. Therefore, without amplification of targets, this Cas12 assay can detect mutations in patients with advanced lung cancer. Future advancements in multiplex and high-throughput mutation detection using this assay will streamline self-diagnosis and treatment monitoring at home.

Tumor extracellular vesicle-derived PD-L1 promotes T cell senescence through lipid metabolism reprogramming.

The limited success of cancer immunotherapy has posed challenges in treating patients with cancer. However, promising strides could be made with a deeper understanding of the factors that cause T cell dysfunction within the tumor microenvironment and by developing effective strategies to counteract tumor-induced immune suppression. Here, we report that tumor-derived extracellular vesicles (tEVs) can induce senescence and suppression in T cells. Programmed death ligand 1 (PD-L1), a key component within tEVs, induced DNA damage and hyperactive lipid metabolism in both human and mouse T cells. This caused an elevated expression of lipid metabolic enzymes and an increase in cholesterol and lipid droplet formation, leading to cellular senescence. At a molecular level, PD-L1 derived from tEVs activated the cAMP-response element binding protein (CREB) and signal transducer and activator of transcription (STAT) signaling, which promoted lipid metabolism and facilitated senescence in human and mouse T cells. Inhibiting EV synthesis in tumors or blocking CREB signaling, cholesterol synthesis, and lipid droplet formation in effector T cells averted the tEV-mediated T cell senescence in vitro and in vivo in cell adoptive transfer and melanoma mouse models. The same treatments also bolstered the antitumor efficacy of adoptive transfer T cell therapy and anti-PD-L1 checkpoint immunotherapy in both human and mouse melanoma models. These studies identified mechanistic links between tumor-mediated immune suppression and potential immunotherapy resistance, and they provide new strategies for cancer immunotherapy.

Digital Profiling of Tumor Extracellular Vesicle-Associated RNAs Directly from Unprocessed Blood Plasma.

Tumor-derived extracellular vesicle (tEV)-associated RNAs hold promise as diagnostic biomarkers, but their clinical use is hindered by the rarity of tEVs among nontumor EVs. Here, we present EV-CLIP, a highly sensitive droplet-based digital method for profiling EV RNA. EV-CLIP utilizes the fusion of EVs with charged liposomes (CLIPs) in a microfluidic chip. Optimized CLIP surface charge enables exceptional sensitivity and selectivity for EV-derived miRNAs and mRNAs. This approach streamlines detection with minimal plasma volume (20 μL) and eliminates the need for prior EV isolation or RNA preparation, preventing loss of EVs or RNA. In testing with 83 patient samples, EV-CLIP detected EGFR L858R and T790M mutations with high AUC values of 1.0000 and 0.9784, respectively. Its success in serial monitoring during chemotherapy highlights its potential for precise quantification of rare EV subpopulations, facilitating the exploration of single EV RNA content and enhancing understanding of diverse EV populations in various disease states.