Exploiting extracellular vesicles for ultrasensitive detection of cancer biomarkers from liquid biopsies

Extracellular Vesicles
/References

Notarangelo, Michela, Chiara Zucal, Angelika Modelska, Isabella Pesce, Giorgina Scarduelli, Cristina Potrich, Lorenzo Lunelli, et al. 2019. “Ultrasensitive Detection of Cancer Biomarkers by Nickel-Based Isolation of Polydisperse Extracellular Vesicles from Blood.” EBioMedicine 43 (May): 114–26. https://doi.org/10.1016/j.ebiom.2019.04.039.

Extracellular vesicles (EVs) are small membrane-surrounded structures containing transmembrane proteins and enclosing cytosolic proteins and nucleic acids. They are released in the extracellular space by both normal and neoplastic cells and play an important role in cell-cell communication in numerous physiological processes and pathological conditions, through the transfer of their functional cargo to recipient cells. EVs are highly abundant in biological fluids, and even more represented in cancer patients’ biofluids, therefore many studies suggested that they can be instrumental in liquid biopsies as prognostic markers or for early detection of tumors. Moreover, being secreted by potentially all the cells, they can serve in oncology to represent the tumor heterogeneity, which is underestimated by the current diagnostic tools. Given their small size, EVs are difficult to isolate in a high-throughput way and, therefore, one of the main obstacles to their clinical application, is that the existing isolation methods are impractical. During these years, I worked at the development and optimization of a novel technique that allows purification of heterogeneous EVs from biological fluids in an efficient, fast and reproducible way. This technique, named Nickel-Based Isolation (NBI), is a biochemical assay that allows obtaining polydisperse EVs in a physiological pH solution, therefore, preserving their morphology, heterogeneity, and stability. We tested and optimized this assay in protein-enriched systems and comparing it to the techniques currently used to characterize and measure EVs, such as flow cytometry and Tunable Resistive Pulse Sensing. We challenged the reproducibility of this method by isolating EVs from different biological fluids. Interestingly, the EVs purified with NBI result more intact and stable compared to the ones obtained with other methods, and can be studied in a clinical setting and used as an innovative tool for detection of molecules associated with diseases. We demonstrated the specificity of the procedure by using individual isolated vesicles in biochemical and molecular assay, optimized to characterize the biological content of EVs. We were able to detect picomolar concentration of PSMA on 105 EVs isolated from plasma of prostate cancer patients and BRAF-V600E transcript in just 103 EVs from the plasma of colon cancer patients, reaching unprecedented matching with tissue biopsy results. We also investigated the transcriptome of EVs isolated from glioblastoma cancer stem cells, in order to exploit the potential of EVs as diagnostic markers.

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Recent Publications

Extracellular Vesicles

Smoking induces different expression of miR-320b and miR-10b-5p in plasma extracellular vesicles of non-small cell lung cancer patients

Background Previous studies found that cigarette smoke (CS) exposure could induce NSCLC malignancy and miRNA dysregulation. Yet, the association of CS-induced miRNA dysregulation and NSCLC malignancy has not been clearly understood. This study aimed to evaluate the effect of CS exposure in smokers on the expression of miR-10b-5p and miR-320b in extracellular vesicles (EVs) from NSCLC patients. Material and methods Bioinformatic analysis was conducted to validate miRNA candidates. Blood and tissue samples were collected from NSCLC patients (n = 21) with smoking and non-smoking history. EVs were isolated from plasma and miRNAs were extracted from the isolated EVs. The miRNAs relative expression was analyzed and compared. Results In silico analysis identified miR-320b and miR-10b-5p as potential biomarkers for diagnosing NSCLC in smokers. Experimental analysis revealed differential expression of EVs-associated miRNAs in NSCLC patients with smoking and non-smoking histories. EVs-associated miR-10b-5p was significantly overexpressed in smoker NSCLC patients (p = 0.000), while miR-320b expression was significantly lower in this group (p = 0.018). Additionally, smoking intensity influenced miRNA expression, with higher smoking intensity correlating with increased miR-10b-5p expression and decreased miR-320b expression. ROC analysis demonstrated that EVs were a superior source of miRNAs compared to plasma for NSCLC diagnostics. miR-10b-5p and miR-320b in EVs showed higher diagnostic performance (AUC 0.878; 0.739) compared to plasma (AUC 0.628; 0.559). Conclusion CS exposure induces different expression of miR-10b-5p and miR-320b in EVs of NSCLC patients with smoking history. EV-related miR-10b-5p and miR-320b showed potential to be utilized as prognostic biomarker for smokers NSCLC patients.

2025
Extracellular Vesicles

Targeted isolation of extracellular vesicles from cell culture supernatant using immuno-affinity chromatography

Extracellular vesicles (EVs) have emerged as promising therapeutics with broad clinical applications as diagnostic biomarkers and therapeutic drug delivery systems. Yet, these biopharmaceuticals pose a challenge in terms of manufacturing due to their complexity and heterogeneity. Despite advancements in the field, current purification technologies lack scalability and/or selectivity. Affinity chromatography (AC) − coupling unmatched specificity and scalability − could be used to simplify purification processing and generate clinical-grade EVs with higher titers and purity. In the present work, we report the implementation of an immuno-AC resin to capture and purify EVs directly from clarified cellular feedstocks. Firstly, to guide and support marker selection, vesicle phenotype characterization was conducted using single particle interferometric reflectance image sensing (SP-IRIS) coupled with immunofluorescence. CD81 was the marker which shown to be more present and more likely to have the other markers (CD63 and CD9). Thus, anti-CD81 VHH ligand was generated and evaluated towards recombinant CD81 protein and CD81 bearing EV particles using surface plasmon resonance (SPR). Different chromatographic studies with Anti-CD81 ligand immobilized onto agarose beads resin were conducted to optimize the process parameters (residence time, dynamic binding capacity and impurity clearance). At residence time of 2 min, on average 40 % of pure triple tetraspanin-positive EV fraction was recovered. The enrichment in EV particles herein obtained, based on scale-up calculations, it would be possible to produce 1 × 1013 EVs from a 1L cell culture, while meeting impurity requirements in a single-step purification process (impurity removal over 2 log reduction value). A single-step purification process is possible, enabling the successful isolation of homogeneous EVs population, counting with a final HCP titer of 60 ng/mL and 9 ng/mL of dsDNA impurities. EV’s morphological integrity and internalization ability were also demonstrated, showcasing elution’s efficiency under mild conditions. Overall, this work contributes to the development of a novel, highly specific, AC technology using a camelid-derived affinity ligand which, bridging the scalability requirements demanded of large-scale production, could potentiate the advent of EV-based therapies.

2025
Extracellular Vesicles

Targeted isolation of extracellular vesicles from cell culture supernatant using immuno-affinity chromatography

Extracellular vesicles (EVs) have emerged as promising therapeutics with broad clinical applications as diagnostic biomarkers and therapeutic drug delivery systems. Yet, these biopharmaceuticals pose a challenge in terms of manufacturing due to their complexity and heterogeneity. Despite advancements in the field, current purification technologies lack scalability and/or selectivity. Affinity chromatography (AC) − coupling unmatched specificity and scalability − could be used to simplify purification processing and generate clinical-grade EVs with higher titers and purity. In the present work, we report the implementation of an immuno-AC resin to capture and purify EVs directly from clarified cellular feedstocks. Firstly, to guide and support marker selection, vesicle phenotype characterization was conducted using single particle interferometric reflectance image sensing (SP-IRIS) coupled with immunofluorescence. CD81 was the marker which shown to be more present and more likely to have the other markers (CD63 and CD9). Thus, anti-CD81 VHH ligand was generated and evaluated towards recombinant CD81 protein and CD81 bearing EV particles using surface plasmon resonance (SPR). Different chromatographic studies with Anti-CD81 ligand immobilized onto agarose beads resin were conducted to optimize the process parameters (residence time, dynamic binding capacity and impurity clearance). At residence time of 2 min, on average 40 % of pure triple tetraspanin-positive EV fraction was recovered. The enrichment in EV particles herein obtained, based on scale-up calculations, it would be possible to produce 1 × 1013 EVs from a 1L cell culture, while meeting impurity requirements in a single-step purification process (impurity removal over 2 log reduction value). A single-step purification process is possible, enabling the successful isolation of homogeneous EVs population, counting with a final HCP titer of 60 ng/mL and 9 ng/mL of dsDNA impurities. EV’s morphological integrity and internalization ability were also demonstrated, showcasing elution’s efficiency under mild conditions. Overall, this work contributes to the development of a novel, highly specific, AC technology using a camelid-derived affinity ligand which, bridging the scalability requirements demanded of large-scale production, could potentiate the advent of EV-based therapies.

2025
Extracellular Vesicles
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