Pubblicazioni
Addressing Heterogeneity in Direct Analysis of Extracellular Vesicles and Their Analogs by Membrane Sensing Peptides as Pan-Vesicular Affinity Probes
Gori, R. Frigerio, P. Gagni, J. Burrello, S. Panella, A. Raimondi, G. Bergamaschi, G. Lodigiani, M. Romano, A. Zendrini, A. Radeghieri, L. Barile, M. Cretich. Adv. Sci. 2024, 11,2400533 https://doi.org/10.1002/advs.202400533
Extracellular vesicles (EVs), crucial mediators of cell-to-cell communication, hold significant diagnostic potential due to their ability to concentrate protein biomarkers in bodily fluids. However, challenges in isolating EVs from biological specimens hinder their widespread use. The preferred strategy involves direct analysis, integrating isolation and analysis solutions, with immunoaffinity methods currently dominating. Yet, the heterogeneous nature of EVs poses challenges, as proposed markers may not be as universally present as thought, raising concerns about biomarker screening reliability. This issue extends to EV-mimics, where conventional methods may lack applicability. Addressing these challenges, the study reports on Membrane Sensing Peptides (MSP) as pan-vesicular affinity ligands for both EVs and their non-canonical analogs, streamlining capture and phenotyping through Single Molecule Array (SiMoA). MSP ligands enable direct analysis of circulating EVs, eliminating the need for prior isolation. Demonstrating clinical translation, MSP technology detects an EV-associated epitope signature in serum and plasma, distinguishing myocardial infarction from stable angina. Additionally, MSP allow analysis of tetraspanin-lacking Red Blood Cell-derived EVs, overcoming limitations associated with antibody-based methods. Overall, the work underlines the value of MSP as complementary tools to antibodies, advancing EV analysis for clinical diagnostics and beyond, and marking the first-ever peptide-based application in SiMoA technology.
A High-Sensitivity approach for detection of breast cancer-derived extracellular vesicles using single molecule array technology
Arianna Bonizzi, Paola Gagni, Alessandro Gori, Serena Mazzucchelli, Lorena Signati, Roberta Cazzola, Marta Truffi, Francesca Piccotti, Fabio Corsi, Carlo Morasso
Autumn Meeting for Young Chemists in the Biomedical Sciences 23-25 June 2025, Palermo, Italy
Introduction: Extracellular vesicles (EVs) are small membrane-bound particles released by cells that carry a variety of molecular contents such as proteins, lipids, and nucleic acids. These vesicles play crucial roles in cell-to-cell communication and can reflect the physiological and pathological state of their cells of origin. Furthermore, EVs are considered promising in cancer research due to their carriage of tumour-related molecules. However, current methods for quantifying EVs often lack the sensitivity and specificity to distinguish them from those derived from normal cellular processes. Additionally, markers from cancer cells are present at significantly lower concentrations compared to those found on general EVs and a sensitive and easy-to-use method is needed to prove their real clinical potential.
Methods: Single Molecule Array (SiMoA) technology, a new ultrasensitive digital ELISA immunoassay, emerged as a powerful tool for the sensitive detection of biomarkers at single-molecule level directly in unprocessed plasma. SiMoA immunoassays were established based on the use of magnetic beads conjugated with an anti-CD63 capture antibody to specifically isolated EVs, followed by the use of biotinylated anti-CD9 or anti-HER2 detector antibodies to identify general and tumor-specific EVs populations. Additional SiMoA assays were developed and tested based on the use of magnetic beads conjugated with a membrane sensing peptide (MSP) able to bind EVs for their curvature. The results obtained with the new MSP-beads coupled with the two detector antibodies previously described were thus compared with the one obtained using traditional antibody based beads.
Results: Two SiMoA assay platforms (anti-CD63 and MSP-conjugated beads) were comparatively evaluated for their sensitivity in detecting EVs isolated from breast cancer patient-derived organoids (PDOs). Both assays demonstrated clear dose-response relationships to the increasing amounts of EVs. Furthermore, the MSP-based assay also demonstrated significantly enhanced sensitivity and superior overall analytical performance compared to the CD63 assay in distinguishing tumor-related EVs from cancerous PDOs.
The two detection strategies were also tested directly on human plasma samples collected from metastatic breast cancer patients (MBC, n=49), including 17 HER2+ and 32 HER2- metastatic patients, and healthy controls (HC, n=30). Results obtained from both assays showed that HER2-expressing EVs can be detected by SiMoA at very low concentrations in patients’ plasma and that HER2+ EVs are enriched in HER2+ MBC patients compared to HER2- patients but do not differ from levels detected in HC.
Conclusion: This study demonstrates the feasibility of SiMoA technology for detecting low-abundance cancer-released EVs biomarkers, with its high sensitivity and highlight how the use of magnetic beads conjugated with a MSP can further improve the specificity of the assays.
Ultra-Sensitive Detection of Breast Cancer-Derived Extracellular Vesicles Using Single Molecule Array Technology: A Comparative Study of Capture Strategies
Arianna Bonizzi, Paola Gagni, Alessandro Gori, Serena Mazzucchelli, Lorena Signati, Roberta Cazzola, Marta Truffi, Francesca Piccotti, Fabio Corsi, Carlo Morasso
Annual Meeting International Society for Extracellular Vesicles ISEV2025, 24-27 April 2025, Vienna
Introduction: Extracellular vesicles (EVs) have gained significant interest as promising liquid biomarkers for breast cancer (BC). However, many aspects of their biological roles remain unexplored due to technical limitations in current EVs isolation and characterization protocols. A critical challenge is identifying markers enriched in cancer-released EVs to distinguish them from the general EVs population. Cancer-specific markers are present on EVs at minimal concentrations, underscoring the need for sensitive, user-friendly methods to unlock their full clinical potential.
Methods: Here we leverage Single Molecule Array (SiMoA) technology, enabling ultra-sensitive biomarker detection, to detect HER2-positive EVs in BC patients. Two distinct SiMoA immunobeads were developed: magnetic beads functionalized with either an anti-CD63 antibody or a membrane-sensing peptide (MSP) with specific affinity for EVs curvature. These approaches were evaluated and compared as strategies for capturing EVs in biological samples, using CD9 and HER2 detector antibodies to differentiate between general and tumor-specific EVs populations, respectively.
Results: The two assays were tested on EVs isolated from patient-derived organoid (PDO) lines: one from a healthy sample and the other from a tumor sample. Both assays exhibited a clear dose-dependent response to the loaded EVs. Importantly, the MSP/HER2 assay demonstrated superior performance compared to the CD63/HER2 assay, effectively distinguishing EVs from the two PDOs lines and revealing significantly higher HER2 expression in tumor-derived EVs compared to those from the healthy sample.
The two detection strategies were also evaluated directly on plasma samples collected from metastatic breast cancer (MBC) patients and healthy controls (HC). The results indicate that the signals obtained with MSP-functionalized beads were higher than those obtained with CD63-functionalized beads across HER2 assays.
In both assays, general EVs levels appeared lower in MBC patients compared to HC, and HER2-expressing EVs showed an enrichment trend in MBC patients.
Conclusion: This study demonstrates the feasibility of SiMoA technology for detecting cancer released EVs, with its high sensitivity and highlight how the use of magnetic beads conjugated with MSP can further improve the specificity of the assay. Coupling SiMoA technology with MSP thus offers a rapid and effective method for the detection of cancer released EVs in BC patients.ity and highlight how the use of magnetic beads conjugated with a MSP can further improve the specificity of the assays.