Extraction of redox extracellular vesicles using exclusion-based sample preparation

Abstract

Studying specific subpopulations of cancer-derived extracellular vesicles (EVs) could help reveal their role in cancer progression. In cancer, an increase in reactive oxygen species (ROS) happens which results in lipid peroxidation with a major product of 4-hydroxynonenal (HNE). Adduction by HNE causes alteration to the structure of proteins, leading to loss of function. Blebbing of EVs carrying these HNE-adducted proteins as a cargo or carrying HNE-adducted on EV membrane are methods for clearing these molecules by the cells. We have referred to these EVs as Redox EVs. Here, we utilize a surface tension-mediated extraction process, termed exclusion-based sample preparation (ESP), for the rapid and efficient isolation of intact Redox EVs, from a mixed population of EVs derived from human glioblastoma cell line LN18. After optimizing different parameters, two populations of EVs were analyzed, those isolated from the sample (Redox EVs) and those remaining in the original sample (Remaining EVs). Electron microscopic imaging was used to confirm the presence of HNE adducts on the outer leaflet of Redox EVs. Moreover, the population of HNE-adducted Redox EVs shows significantly different characteristics to those of Remaining EVs including smaller size EVs and a more negative zeta potential EVs. We further treated glioblastoma cells (LN18), radiation-resistant glioblastoma cells (RR-LN18), and normal human astrocytes (NHA) with both Remaining and Redox EV populations. Our results indicate that Redox EVs promote the growth of glioblastoma cells, likely through the production of H₂O₂, and cause injury to normal astrocytes. In contrast, Remaining EVs have minimal impact on the viability of both glioblastoma cells and NHA cells. Thus, isolating a subpopulation of EVs employing ESP-based immunoaffinity could pave the way for a deeper mechanistic understanding of how subtypes of EVs, such as those containing HNE-adducted proteins, induce biological changes in the cells that take up these EVs.

Keywords: 4-Hydroxynonenal (HNE); EV isolation; EV purification; Extracellular vesicles; Immunoaffinity; Redox EVs.

Figure 1.

Schematic of Exclusion-based Sample Preparation (ESP) technology for isolation of HNE-adducted Redox EVs

Figure 2.

LN18 cells were plated at 1.2×10⁷ cells in a 15cm dish. 24 hours after plating, one plate was radiateDd at 6 Gy. Both plates incubated for 48 hours. Media was collected and filtered to remove large debris. EVs were isolated (as described in section 2.2) and the number of EVs were counted using NTA. Radiation at 6 Gy induces EV production within LN18 cells. (A) Photograph of EV particles (white dots) from NTA. (B) Size distribution of EV. (C) EV concentration. (D) Size of EVs. (*, P<0.05)

Figure 3.

Initial Isolation of Redox EVs directly from media. The term “Remaining EVs” refers to EVs that are still present in the input sample after the process of Redox EVs isolation. (A) EVs size comparison (*,P<0.05) (B) EVs Zeta potential comparison (*, P<0.05) (C) HNE-adducted protein profile compared between the Remaining and Redox EVs. C.1 = Lane-view data of HNE band. C.2= Electropherogram data of HNE peaks (red arrow) at each molecular weight.

Figure 4.

Optimization of antibody concentration for efficient isolation of Redox EVs. (A) Concentration of Redox EVs increased with decreasing concentration of antibody bound to the beads. (*, P<0.05) (B) Average size of EVs decreases with decrease in the antibody concentration bound to the beads. (*, P<0.05) (C) HNE concentrations are higher in the EV with the use of 1.0 μg of antibody per mg of bead. (****, P<0.0001) (D) Albumin contamination remains low in either of the concentrations of antibody. (E) Flotillin-1 (Flot-1) was present mostly with the use of 0.2 μg of Antibody per mg of beads. Electropherogram data (area under the curve, green) and (F) Lane-view data represent level of Flot-1.

Figure 5.

Washing steps are studied to determine the amount of EVs lost and the albumin contamination. (A) NTA shows no EVs present in the washing step. The camera of NTA looks at an area of 640 × 480 pixels, which corresponds to 460 × 345μm. (B) Using 1 μg Ab/mg bead removed albumin in the wash compared to the other two concentrations. (C) HNE levels in wash step which are significantly less in 0.2 μg Ab/mg bead. (**, P<0.01) (D) Electropherogram data (green, area under curve) and (E) Lane-view data of Flotillin-1 (EV marker), which is marker for both Redox EVs and Remaining EVs, was slightly detected in the washing steps of 1 μg Ab/mg bead.

Figure 6.

Two populations of EVs were formed after using the ESP technique. Redox EVs which were isolated using HNE antibody conjugated beads and the EVs that were left behind in the isolation (Remaining EVs or non-Redox EVs). Comparing the two populations shows those that were isolated have characteristics of Redox EVs. (A) When EVs bleb from the surface, Redox EVs contain a higher amount of PtdSer on the outer leaflet giving them a more negative charge as shown by the Zeta Potential. (*, P<0.05) (B) Average size shows that the Redox EVs are smaller than those remaining. (*, P<0.05) (C) Electropherogram data (green, area under the curve) and Lane-view data of Flotillin-1 which show slightly shift in molecular weight in the Redox EVs (possibly due to the negative charge and salt content). (D) HNE is higher in Remaining EVs, however still present in Redox EVs. (***, P<0.001) (E) Representative EM photograph of immunogold staining with HNE antibody. (E.1) Normal mouse serum was used as control with no gold labeling of HNE presence. (E.2) Remaining EV show labeling of HNE (arrowhead) outside the EV, whereas (E.3) Redox EV show labeling of HNE (arrow heads) present on the outer membrane of EV. Arrows = EVs at the low magnification. Star = EV at high magnification.

Figure 7.

Treatment of Glioblastoma cells LN18, radiation resistance LN18 (RR-LN18), and NHA cells with Redox EVs show contrasting trends in cell viability. Cell viability using PrestoBlue was performed after EV treatments. (A) LN18 cells. (B) RR-LN18 cells (C) Normal Human Astrocytes (NHA) cells. (*, P<0.05) (****, P<0.0001)

Figure 8:

Increased extracellular H₂O₂ production by Redox EVs in GBM and NHA cells are correlating to cell viability. Cells were treated with EV Buffer (vehicle), Remaining EVs, or Redox EVs and incubated for 48 hours before measuring H₂O₂ production using the Amplex Red assay. (A) LN18 cells, (B) RR-LN18 cells, and (C) NHA cells were analyzed. Compared to Vehicle and Remaining EVs, treatment with Redox EVs significantly increased extracellular H₂O₂ production in LN18 cells, RR-LN18 cells, and NHA cells. NHA cells produced detectable levels of H₂O₂ (0.26 μM), while RR-LN18 cells demonstrated the highest H₂O₂ production (0.5 μM). (D-F) Cell number using Trypan blue were counted using hemocytometer after EV treatments. Polyethylene glycol catalase (PEG-CAT, 500 unit) were added to the cells 24 hr prior to EV treatments to scavenge H2O2 production inside the cells. * P<0.05, ** P<0.01 indicates statistically significant differences compared to controls.