Abstract
Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5–100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).
Highlights
Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies
To overcome current limitations of the enrichment and purification of EVs and on-demand EV subpopulation characterization, we developed the immunomagnetic sequential ultrafiltration (iSUF) platform (Fig. 1 A) which includes three stages: (1) tangential flow filtration (TFF) for the enrichment and purification of EVs, (2) centrifugation for further enrichment of EVs, and (3) immunomagnetic affinity selection for desired EV subpopulation isolation
Our experiments showed that 500 kDa filter membranes were able to remove up to 99% of free proteins with over 99% EV recovery rate (Eq 1, Supplementary Fig. 1)
Summary
Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. We demonstrated the use of nanostructured polymeric brushes conjugated with epidermal growth factor receptor (EGFR) and integrated into a microfluidic channel to enhance specificity towards tumor-derived EVs isolated from glioblastoma (GBM) patients[30] This approach can achieve a remarkable 94% specificity towards tEVs, the limited amount of biofluid processed (1–1.5 mL of serum or plasma) and the retention of albumin, significantly limits its use for p roteomics[31]. We have demonstrated the use of iSUF to enrich EVs present in three different types of biofluids: cell culture media (CCM), serum, and urine for which the sample processing time was approximately 2 h Another feature of iSUF is that it can enrich EVs up to 1000 times with an EV recovery rate higher than 94%, which overcomes the limitations of other commercially available methods. To further validate the clinical utility of iSUF, we have processed serum samples from 10 metastatic breast cancer (BC) patients and demonstrated the presence of HER2, CD24 and miR21 biomarkers at significantly higher levels compared to healthy controls (p < 0.05)
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