Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jul;5(7):678-689.
doi: 10.1038/s41551-021-00752-7. Epub 2021 Jun 28.

An integrated magneto-electrochemical device for the rapid profiling of tumour extracellular vesicles from blood plasma

Jongmin Park #  1   2   3 , Jun Seok Park #  1   2   4 , Chen-Han Huang  1   2   5 , Ala Jo  1   2 , Kaitlyn Cook  6 , Rui Wang  6   7 , Hsing-Ying Lin  1   2 , Jan Van Deun  1   2 , Huiyan Li  1   2 , Jouha Min  1   2 , Lan Wang  8 , Ghilsuk Yoon  9 , Bob S Carter  8 , Leonora Balaj  8 , Gyu-Seog Choi  4 , Cesar M Castro  1   10 , Ralph Weissleder  11   12   13 , Hakho Lee  14   15
Affiliations

An integrated magneto-electrochemical device for the rapid profiling of tumour extracellular vesicles from blood plasma

Jongmin Park et al. Nat Biomed Eng. 2021 Jul.

Abstract

Assays for cancer diagnosis via the analysis of biomarkers on circulating extracellular vesicles (EVs) typically have lengthy sample workups, limited throughput or insufficient sensitivity, or do not use clinically validated biomarkers. Here we report the development and performance of a 96-well assay that integrates the enrichment of EVs by antibody-coated magnetic beads and the electrochemical detection, in less than one hour of total assay time, of EV-bound proteins after enzymatic amplification. By using the assay with a combination of antibodies for clinically relevant tumour biomarkers (EGFR, EpCAM, CD24 and GPA33) of colorectal cancer (CRC), we classified plasma samples from 102 patients with CRC and 40 non-CRC controls with accuracies of more than 96%, prospectively assessed a cohort of 90 patients, for whom the burden of tumour EVs was predictive of five-year disease-free survival, and longitudinally analysed plasma from 11 patients, for whom the EV burden declined after surgery and increased on relapse. Rapid assays for the detection of combinations of tumour biomarkers in plasma EVs may aid cancer detection and patient monitoring.

PubMed Disclaimer

Conflict of interest statement

Competing interests

R.W. declares that he has received consultancy payments from Accure Health, and that he is a shareholder of Lumicell. H.L. declares that he has received consultancy payments from Accure Health. Patents: all patents associated with R.W. and H.L. have been assigned to and handled by Massachusetts General Hospital.

Figures

Fig. 1 |
Fig. 1 |. HiMEX approach for clinical EV analyses.
a, Study design. We collected blood samples from colorectal cancer (CRC) patients (n = 91) during their standard clinical care. Extracellular vesicles (EVs) as well as conventional markers (i.e., CEA, CA19-9) were analysed from blood samples and cross-compared with clinical outcomes, including immunohistology, radiologic reports, and survival. b, Two-step HiMEX assay protocol. EVs are enriched via immunomagnetic capture. Bead-bound EVs are then labelled with probing antibodies for signal generation through electrochemical reaction. The assay is simple and fast (<1 hour), directly analysing plasma samples without requiring purification steps. c, We developed a compact HiMEX reader with a touchscreen interface. The reader accommodated an array of 96 electrodes in a conventional 96-well plate. Under each electrode were push-pin connectors to make electrical contacts and a magnet to concentrate bead-bound EVs. PCB, printed circuit board. d, The HiMEX reader was designed to carry out 96 parallel measurements within 2 minutes. Each electrode was connected its own potentiostat. A microcontroller applied electrical potential through a digital-to-analogue converter (DAC), initiating electrochemical reaction. Resulting currents from the electrode were read out by an analogue-to-digital converter (ADC) via rapid multiplexing (MUX). The microcontroller processed and displayed data on a touchscreen. The reader also communicated with an external device (e.g., smartphones) for data logging. PGA, programmable gain amplifier.
Fig. 2 |
Fig. 2 |. HiMEX assay optimization and characterization.
a, Scanning electron micrograph of microbeads after incubation with EV samples. The beads (diameter, 3 μm), functionalized with antibodies against CD63, captured EVs isolated from a cell culture media (HCT116 cell line). A representative image is selected from technical duplicate samples. b, Three types of magnetic beads, with each type specific to a different tetraspanin, and their mixture (Mix) were used to capture EVs. Key tetraspanin expression was then measured on captured EVs. The bead cocktail most efficiently overcame EV heterogeneity. A representative image is selected from duplicate measurements. Full western blot images are shown in Supplementary Information. c, EVs (107/mL) from HT29 cell lines were captured and further labelled for EGFR. Mixed bead types led to the highest HiMEX signal. The data are displayed as mean ± SD from technical triplicates. d, The HiMEX assay had superior sensitivity and wider dynamic range than conventional ELISA. The limit of detection was ~104 EVs/mL for HiMEX and ~107 EVs/mL for ELISA. a.u., arbitrary unit. EV numbers were estimated from nanoparticle tracking analysis. The data points represent mean from technical duplicates. e, EVs from different CRC cell lines were profiled to a set of protein markers. The HiMEX results showed a good match (Pearson correlation coefficient, r = 0.82) with those from ELISA. To obtain the HiMEX expression (ξ) of a target protein marker (M), the marker-associated current level was normalized against the loading control, the current level of CD63. For ELISA, the same amount of EVs (109 EVs/mL) was used for each marker. Data were plotted in log scales to better display small values. The blue-dashed line indicates the best linear fit in the log-log scale, and the shaded grey area 95% confidence band. HiMEX data represent mean values from technical duplicates.
Fig. 3 |
Fig. 3 |. EV profiling for CRC detection.
a, Tumour tissue and plasma EV analyses. Three key CRC protein markers (EpCAM, EGFR, CD24) were assessed in tumour tissues (immunohistochemistry) and blood samples (HiMEX) from each patient. Expression profiles showed a qualitative match. Data from two representative patients are shown. The bar graphs show mean ± SEM from technical triplicate measurements. Full images of these samples are shown in Supplementary Information. b, Tumour tissue and plasma EV samples from 12 CRC patients were analysed for EpCAM, EGFR, and CD24. Tissue staining was graded as the fraction of marker-positive cells among total cancer cells. The pathological score and the EV expression profile showed significant correlation (Spearman rank coefficient ρs = 0.65, p < 0.0001; two-sided test). c, HiMEX analyses for CRC diagnosis. As a training set, plasma samples from 58 CRC patients before surgery and 25 non-CRC controls were analysed. The expression of five CRC markers (EpCAM, EGFR, CD24, CD133, and GPA33) was measured by HiMEX. The diagnostic metric, EVCRC, was determined as a weighted sum of four marker levels (EpCAM, EGFR, CD24, GPA33) through logistic regression. d, The average level of each marker was higher in CRC patients than in non-CRC controls. The marker distribution, however, overlapped between patients and controls, reducing the classification power of single markers. e, For each CRC marker and EVCRC, receiver operating characteristic (ROC) curves were constructed. The area under curve (AUC) of EVCRC was 0.98, significantly larger than those of single markers (all p < 0.001). Cutoff levels that maximized the sum of sensitivity and specificity were determined from the ROC curves. f–g, EVCRC effectively differentiated CRC patients from controls (p = 0.0009; unpaired two-sided t-test). In the training cohort, the diagnostic accuracy was 98%. The cutoff value from the EVCRC ROC curve was 4.96.
Fig. 4 |
Fig. 4 |. Analyses of prospective cohorts for CRC diagnosis.
a, Plasma EVs from 33 CRC patients, 9 healthy donors, and 6 non-CRC patients (gastrointestinal stromal tumour, n = 2; small bowel internal herniation, n = 2; appendicitis, n = 2) were analysed for the expression EpCAM, EGFR, CD24, and GPA33. EVCRC was calculated according to the same formula as with the training cohorts. b, EVCRC levels were significantly higher in CRC patients than in healthy controls, validating the EV-based diagnostic algorithm. The same cutoff value (4.96) from the training set was applied. c, EVCRC remained superior in CRC detection, with its AUC significantly larger than those of single markers. Detailed statistics are in Table 2.
Fig. 5 |
Fig. 5 |. HiMEX analyses of longitudinal CRC patient samples.
a, CRC patient blood samples were analysed before and after surgery (n = 13). Molecular EV profiling revealed that EVCRC values decreased in all patients (p < 0.0001; paired two-sided t-test), whereas total EV concentrations (p = 0.59; paired two-sided t-test) and the levels of conventional serum markers, CEA (p = 0.19; paired two-sided t-test) and CA19-9 (p = 0.19; paired two-sided t-test), showed no significant changes. Each data point in the graph represents the mean value from technical duplicates. b, Plasma EVs were further monitored after surgery, as some patients underwent chemotherapy. EVCRC values rebounded in all patients (n = 7) with recurrent tumours. By contrast, in patients without recurrent tumour (n = 4), EVCRC continued to decrease or stabilized from the post-surgery level. Each data point in the graph represents mean ± SEM from technical duplicates. c, EVs from recurrent (n = 3) and non-recurrent (n = 3) CRC patients were analysed for mRNA expression. Highly variable genes were plotted. Genes involved in DNA repair, protection against oxidative pressure, and chemoresistance (5-FU) showed higher levels in recurrent patients’ EVs. d, CRC patients (n = 90) were monitored up to five years for disease-free survival (DFS). Kaplan-Meier estimator for DFS was plotted, stratified by preoperative EVCRC levels. High EVCRC values (≥ 53.4) were associated with poor prognostics (p = 0.02, two-sided log-rank test).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Heitzer E, Haque IS, Roberts CES & Speicher MR Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat. Rev. Genet 20, 77–88 (2018). - PubMed
    1. Siravegna G, Marsoni S, Siena S & Bardelli A Integrating liquid biopsies into the management of cancer. Nat. Rev. Clin. Oncol 14, 531–548 (2017). - PubMed
    1. Chi KR The tumour trail left in blood. Nature 532, 269–271 (2016). - PubMed
    1. Pantel K & Alix-Panabieres C Real-time liquid biopsy in cancer patients: fact or fiction? Cancer Res. 73, 6384–6388 (2013). - PubMed
    1. Théry C, Ostrowski M & Segura E Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol 9, 581–593 (2009). - PubMed

Publication types

MeSH terms