Selective isolation of extracellular vesicles from minimally processed human plasma as a translational strategy for liquid biopsies

Abstract

Background: Intercellular communication is mediated by extracellular vesicles (EVs), as they enclose selectively packaged biomolecules that can be horizontally transferred from donor to recipient cells. Because all cells constantly generate and recycle EVs, they provide accurate timed snapshots of individual pathophysiological status. Since blood plasma circulates through the whole body, it is often the biofluid of choice for biomarker detection in EVs. Blood collection is easy and minimally invasive, yet reproducible procedures to obtain pure EV samples from circulating biofluids are still lacking. Here, we addressed central aspects of EV immunoaffinity isolation from simple and complex matrices, such as plasma.

Methods: Cell-generated EV spike-in models were isolated and purified by size-exclusion chromatography, stained with cellular dyes and characterized by nano flow cytometry. Fluorescently-labelled spike-in EVs emerged as reliable, high-throughput and easily measurable readouts, which were employed to optimize our EV immunoprecipitation strategy and evaluate its performance. Plasma-derived EVs were captured and detected using this straightforward protocol, sequentially combining isolation and staining of specific surface markers, such as CD9 or CD41. Multiplexed digital transcript detection data was generated using the Nanostring nCounter platform and evaluated through a dedicated bioinformatics pipeline.

Results: Beads with covalently-conjugated antibodies on their surface outperformed streptavidin-conjugated beads, coated with biotinylated antibodies, in EV immunoprecipitation. Fluorescent EV spike recovery evidenced that target EV subpopulations can be efficiently retrieved from plasma, and that their enrichment is dependent not only on complex matrix composition, but also on the EV surface phenotype. Finally, mRNA profiling experiments proved that distinct EV subpopulations can be captured by directly targeting different surface markers. Furthermore, EVs isolated with anti-CD61 beads enclosed mRNA expression patterns that might be associated to early-stage lung cancer, in contrast with EVs captured through CD9, CD63 or CD81. The differential clinical value carried within each distinct EV subset highlights the advantages of selective isolation.

Conclusions: This EV isolation protocol facilitated the extraction of clinically useful information from plasma. Compatible with common downstream analytics, it is a readily implementable research tool, tailored to provide a truly translational solution in routine clinical workflows, fostering the inclusion of EVs in novel liquid biopsy settings.

Keywords: Early-stage cancer; Enrichment; Extracellular vesicle; Immunoprecipitation; Liquid biopsy; Plasma; Platelet.

Fig. 1

Differential recovery of EV subpopulations in simple and complex matrices, between streptavidin-coated and covalently-conjugated beads. (A) CFSE-stained HEK293 EVs were isolated from PBS-BSA with MACS-STV and MACS. CD9 was the IP target and ISO/CD61 were negative controls. Recovery was plotted as % of input, obtained using fluorescent signals of samples and input. Fluorescence of IP flow-throughs (FT) allowed for an indirect calculation of recovery (yellow), whereas fluorescence on beads provided a direct recovery measure (blue). Specificity represents the difference (in percentage) between target and negative control signal. (B) Cryo-TEM images of triple-coated MACS beads (1), HEK293 EVs (2) and the IP complex formed between both in PBS-BSA (3). Respective scale bars are shown on the top right corner of each image. (C) S/N ratios of CFSE-stained HEK293 EVs recovered from plasma (donor 6) on MACS-STV and MACS. The IP target was CD9 and the negative controls were ISO/CD61. (D) After RNA isolation from MACS CD9 and CD61 samples obtained in (C), GAPDH and CA9 mRNA copies were measured by ddPCR. (E) S/N ratios of CFSE-stained HEK293 EVs recovered from plasma (donor 6) on MACS beads, coated with anti-CD9 and anti-PE antibodies. IP specificity was 78%

Fig. 2

Intact EV subpopulations spiked in plasma can be completely recovered using antibody-conjugated beads. (A) Single-particle analysis of fluorescent 22RV1-NIR EVs by nFCM. The dot plot on the left shows that the majority of 22RV1 EVs incorporated the NIR fluorophore, whereas the one on the right evidences the CD9-positive subpopulation of these NIR-EVs, determined after staining with CD9-PE. Percentages of fluorescent particles were background-corrected (BC) with buffer (PBS). (B) IP of 22RV1-NIR EVs spiked in plasma (donor 6). Recovery (% of input) was appreciated indirectly, through the NIR signal of IP flow-throughs (FT) and directly, by measuring the fluorescence of NIR EVs captured on beads. 22RV1-NIR EVs were used to plot a calibration curve correlating particle numbers with their fluorescent signal, which allowed to present percentages of input based on the actual number of particles recovered vs. input particles. Specificity (S) was calculated for both readouts. (C) IP of 22RV1-NIR-CD9-PE-stained EVs spiked in plasma (donor 6). Recovery, plotted as % of input, was assessed by indirect and direct fluorescence readouts. The NIR signal of 22RV1-NIR-CD9-PE EVs was used to plot a calibration curve, correlating particle numbers with their fluorescent signal, which allowed to present percentages of input based on the actual number of particles recovered vs. input particles. Recovery with each bead source was equivalent, as suggested by both readouts. (D) Cryo-TEM images of EVs captured from plasma (1) and plasma spiked with HEK293 EVs (2), using triple-coated beads. Respective scale bars are shown on the top right corner of each image

Fig. 3

IP efficiency is dependent on EV surface properties, complex matrix components and interactions between both. (A) Fluorescence S/N ratios were assessed on triple-coated beads upon the IP of 22RV1-NIR and HT29-CFSE spiked in either PBS-BSA or plasma (donor 6). Average recovery (S/N) differences between the two matrices were reported in percentage. (B) CFSE-labelled EVs were spiked in plasma (donor 7) or in PBS-BSA and recovered using triple-coated beads. Average recovery (S/N) differences between the two matrices were reported in percentage. (C) CFSE-labelled HEK293 (blue) and HT29 EVs (orange) were spiked in plasma samples of 3 different donors and recovered using triple-coated beads. CFSE S/N on beads was directly proportional to the amount of captured spike

Fig. 4

Direct EV subset detection and quantification on beads using fluorescently-tagged antibodies. (A) Increasing numbers of HT29 EVs captured from PBS-BSA with triple-coated beads were detected by staining IP complexes with CD9-PE. PE fluorescence signals highly correlated with the number of spiked and recovered EVs, evidenced by linear regression analysis (R² = 0,9992). (B) Scalar amounts of HT29 EVs were spiked in “EV-depleted plasma” (donor 7) and recovered using triple-coated beads, followed by staining with CD9-PE. A linear trendline showed correlation between the amount of spike and S/N obtained with CD9-PE, from 1 × 10⁸ to 1 × 10⁷ EVs (R² = 0,9783). An outlier mean S/N value at 5 × 10⁶ spiked EVs was presented in red. (C) Platelet-derived EVs were isolated from plasma samples of 3 different donors using anti-CD61 beads. Target subpopulations (CD41-PE) were detected against a negative control antibody (IgG1k-PE). (D) Cryo-TEM image of platelet-derived EVs recovered from plasma with anti-CD61 beads. Scale bars are shown on the top right corner of the image

Fig. 5

Double antibody staining of IP complexes allows for meaningful quantification of surface markers on plasma-recovered EVs. (A) Platelet EVs isolated from plasma with anti-CD61 beads were stained with CD41-PE and CD9-AF488 in single or double staining settings. Fluorescence signal for both markers was equivalent, despite the incubation with 1 or 2 antibodies simultaneously. (B) Triple-coated or anti-CD61 beads were incubated in untreated, thrombin treated or 56 °C heated plasma and double stained with CD41-PE and CD9-AF488. S/N ratios obtained with CD9-AF488 were plotted. A significant drop in fluorescent signal could be appreciated only upon 56 °C treatment, when CD61 beads were employed. (C) Measurements of CD41-PE, referring to the experiment described in (B). Substantial losses in CD41-PE signal were detected after staining IP complexes recovered with both beads, across treatments. Untreated and thrombin from triple-coated and CD61 beads, respectively, are shown as duplicates

Fig. 6

Differential expression (DE) analysis by DESeq2 between CD61+ and CD9, CD63 or CD81+ EV datasets on the healthy donor cohort. (A) Volcano plot showing DE genes between the two groups. Cut-offs were defined for adjusted p-values (0.05, Y axis) and log2 fold change (2, X axis). Upregulated and downregulated genes were depicted by green and red circles, respectively. (B) Supervised hierarchical clustering heatmap analysis based on the four DE genes discovered by DESeq2, samples and their respective group

Fig. 7

DESeq2 identified 47 DE genes between healthy and early-stage NSCLC cohorts, using platelet-derived EVs as biomarker source. (A) Volcano plot showing that no DE genes were found between the two cohorts, based on the triple-coated EV dataset. Cut-offs were defined for adjusted p-values (0.05, Y axis) and log2 fold change (2, X axis). (B) Volcano plot showing DE genes between the two cohorts, based on the platelet-derived EV dataset. Cut-offs were defined for adjusted p-values (0.05, Y axis) and log2 fold change (2, X axis). Upregulated and downregulated genes were depicted by green and red circles, respectively