CD34+ HSPCs-derived exosomes contain dynamic cargo and promote their migration through functional binding with the homing receptor E-selectin

Abstract

Exosomes are tiny vesicles released by cells that carry communications to local and distant locations. Emerging research has revealed the role played by integrins found on the surface of exosomes in delivering information once they reach their destination. But until now, little has been known on the initial upstream steps of the migration process. Using biochemical and imaging approaches, we show here that exosomes isolated from both leukemic and healthy hematopoietic stem/progenitor cells can navigate their way from the cell of origin due to the presence of sialyl Lewis X modifications surface glycoproteins. This, in turn, allows binding to E-selectin at distant sites so the exosomes can deliver their messages. We show that when leukemic exosomes were injected into NSG mice, they traveled to the spleen and spine, sites typical of leukemic cell engraftment. This process, however, was inhibited in mice pre-treated with blocking E-selectin antibodies. Significantly, our proteomic analysis found that among the proteins contained within exosomes are signaling proteins, suggesting that exosomes are trying to deliver active cues to recipient cells that potentially alter their physiology. Intriguingly, the work outlined here also suggests that protein cargo can dynamically change upon exosome binding to receptors such as E-selectin, which thereby could alter the impact it has to regulate the physiology of the recipient cells. Furthermore, as an example of how miRNAs contained in exosomes can influence RNA expression in recipient cells, our analysis showed that miRNAs found in KG1a-derived exosomes target tumor suppressing proteins such as PTEN.

Keywords: CD34+ hematopoietic progenitor stem cells; E-selectin; adhesion; cargo alterations; exosomes; migration; proteomics.

FIGURE 1

Mass spectrometry analysis reveals HSPCs-derived exosomes are enriched in proteins involved in adhesion and migration. Proteomics analysis of the cargo was performed to uncover putative functions of the isolated exosomes. The KEGG pathways and the Biological Processes from Gene Ontology analysis that were enriched in the KG1a (pink) and healthy HSPCs (blue)-derived exosomes were identified using the DAVID online tool. (A) Top hits of the KEGG pathways identified included pathways related to vehicle-trafficking and adhesion-migration functions. Furthermore, there was enrichment in signaling networks, highly related to the regulation of migration. The complete list of the identified KEGG pathways can be found in “HSPCs-derived exosomes proteome analysis.” Datasheet 2. (B) Top hits of Biological Processes identified included processes related to cell adhesion and signaling cascades, confirming the KEGG pathways. The complete list of identified Biological Processes can be found in “HSPCs-derive exosomes proteome analysis.” Datasheet 2. (C) KEGG map revealed proteins in KG1a (pink) and healthy HSPCs (blue frame)-derived exosomes related to Leukocyte Transendothelial Migration. Among the identified proteins, there were integrins (e.g., integrin-alpha), cytoskeleton-related proteins (e.g., actin, ezrin), and chemokines (e.g., IL-32).

FIGURE 2

KG1a-derived exosomes express E-selectin ligands. (A) Western blot analysis of lysates of KG1a-derived exosomes revealed the presence of CD34, CD43, CD44, and PSGL-1 at the expected molecular weights. For PSGL-1, two bands were detected as expected (Snapp et al., 1998), a lower band corresponding to the monomeric form and a higher band corresponding to the dimeric form. (B) Western blot analysis of KG1a-derived exosome lysates stained with rE-selectin-IgG revealed several potential E-selectin ligands. (C) rE-selectin-IgG was used to immunoprecipitate (IP) proteins from lysates of KG1a-derived exosomes either in the presence of Ca²⁺ (2 mM) or EDTA (20 mM). Western blot analysis revealed the presence of CD34, CD44, and PSGL-1 (blue arrows) in samples where Ca²⁺ was added but not in samples containing EDTA confirming the selective, calcium-dependent binding of E-selectin to its ligands. In western blots for CD43, no band was observed at the expected MW (see red rectangle). (D) CD44, CD34, PSGL-1, and CD43 were immunoprecipitated (IP) from the lysates of KG1a-derived exosomes using antibodies against each potential E-selectin ligand. The IP product was blotted against the rE-selectin-IgG (upper) and each respective ligand (lower, positive control). A clear band corresponding to CD43 was not detected (see red rectangle). Note that at in the upper blots, double the amount of sample was loaded compared to the lower blots in order to establish that although a substantial amount of CD43 protein is present (lower), the lack of E-selectin binding (upper) is likely a result of the lack of proper glycosylation. (E) CD44 and CD43 were Immunoprecipitated (IP) from lysates of KG1a-derived exosomes using antibodies against each ligand. The IP product was blotted for sLe^x/a using the HECA-452 antibody (upper) and each specific ligand (lower). No band corresponding to CD43 was detected with HECA-452 (see red rectangle) even though CD43 was present (see lower CD43 blot) suggesting that this ligand does not express sLe^x/a structures. Results shown are representative of n = 3 independent experiments.

FIGURE 3

E-selectin is able to pull down intact exosomes expressing E-selectin ligands. (A) Cartoon illustrating the experimental workflow. rE-selectin-IgG was bound to magnetic beads coated with protein G. The complex was resuspended in PBS containing either 2 mM of Ca²⁺ or 20 mM of EDTA. Exosomes were added to that mixture, and after overnight incubation, the particles bound to the E-selectin-beads complex were eluted and lysates were analyzed by western blot. Refer to Materials and Methods for more details. Figure created with BioRender.com. (B) Western blot analysis of the eluted products from the E-selectin-bead complex. Control samples either containing EDTA to chelate out Ca⁺² or beads (without the E-selectin) were used to verify the specificity of the interactions. A bead alone control (without exosomes) was also included. Blots were stained for the common markers of exosomes: CD63 and CD81, but also from proteins revealed from the Mass Spectrometry data such as ezrin and β-actin. This is representative of n = 3 independent experiments.

FIGURE 4

KG1a-derived exosomes bind E-selectin with high affinity and avidity even under flow. (A) Cartoon illustrating the experimental flow assay imaging setup showing labeled KG1a-derived exosomes flowing over rhE-selectin, that is, pre-deposited onto a microfluidics chamber. Figure created with BioRender.com. (B, C) DiD labeled KG1a-derived exosomes were introduced into a microfluidics chamber coated with rhE-selectin. Following 30 min of incubation, the chambers were washed and imaged using fluorescence microscopy. (B) In the chambers where Ca⁺² was present (upper images), a plethora of exosomes were observed. However, in chambers where EDTA was present (lower images), exosomes were scarce. (C) The observed differences were quantified and expressed as number of exosomes observed per area, student’s t-test, p-value (**) < 0.01. (D) DiD labeled KG1a-derived exosomes were introduced into microfluidics chambers coated with rhE-selectin at a constant flow of 100 μl min⁻¹ and increased up to 2000 μl min⁻¹. Several frames were recorded to illustrate the binding of exosomes to the deposited E-selectin in real-time. In the 1st frame, one exosome is observed to be bound to the E-selectin. In the 2nd frame, another exosome entered the recording field (white line indicated by a blue arrow) bound firmly to the E-selectin by the 3rd frame (indicated by the blue arrow). This exosome remained bound stably to the E-selectin as shown in the 4th frame and even after 1000 frames (1000th frame) when the flow rate reached up to 2000 μl min⁻¹. Furthermore, a new exosome bound at the recoding area (red arrow). Scale bar = 5 μm. (E, F) Microscale Thermophoresis (MST) assay between KG1a-derived exosomes and rE-selectin. (E) A Binding Check assay was performed using labeled rE-selectin and unlabeled KG1a-derived exosomes. As illustrated, a clear difference was apparent between the signal detected from the sample with the labeled E-selectin alone (Target, blue dots) and the sample with the labeled E-selectin mixed with the KG1a-derived exosomes (Complex, green dots), indicating binding detectable by the MST. (F) Binding Affinity assay performed using labeled rE-selectin and unlabeled KG1a-derived exosomes. The Kd was found to be in the level of pMolar, indicating a strong interaction between exosomes and rE-selectin. Results shown are representative of n = 3 independent experiments.

FIGURE 5

In vivo biodistribution of exosomes is influenced by E-selectin. KG1a-derived exosomes were prestained with VivoTrack 680 and delivered to NSG mice intravenously via the tail vein. NSG recipients were either pretreated with blocking anti-E-selectin antibody 3 h prior to exosome delivery (exosomes + anti-E-sel) or left untreated (exosomes). Control group mice that did not receive exosomes. (A) Quantitative analysis of fluorescence intensity, statistically analyzed by student’s t-test, using IVIS imaging of the whole body of NSG mice in the dorsal position at 2, 6, 12, 24, and 48 h is shown. The dorsal position is helpful to detect the fluorescence signal from the spine (p-value (*) <0.05, Exosomes group vs. Exosomes + anti-E-sel group). (B) Quantitative analysis of fluorescence intensity, statistically analyzed by student’s t-test, using IVIS imaging of the whole body of NSG mice in the left-lateral position at 2, 6, 12, 24, and 48 h is shown. The left-lateral position is helpful to detect the fluorescence signal from the spleen (p-value (*) <0.05, Exosomes group vs. Exosomes + anti-E-sel group). (C) Representative IVIS images of organs after 48 h of whole body IVIS imaging (at 12 and 24 h) following injection of VivoTrack 680-labeled KG1a-derived exosomes. Live IVIS imaging of mice from the dorsal (to show spine) and the lateral left (to show spleen) positions are shown at 12 and 24 h post-delivery of KG1a-derived exosomes or control group. Mice pretreated with anti-E-selectin blocking antibody 3 h prior to KG1a-derived exosome delivery are also shown. After imaging at 48 h, mice were sacrificed and organs were imaged ex vivo (spleen, liver, leg bones and spine). Representative images of these organs are shown. (D) Quantitative analysis of the fluorescence intensity of the leg bones, spine, spleen and liver are shown the exosomes accumulation in different organs and were statistically analyzed by student’s t -test (p-value (*) <0.05, Exosomes group vs. Exosomes + anti-E-sel group).

FIGURE 6

Exosomes may alter their cargo during transport to recipient cells in a non-stochastic manner. (A) Freshly derived KG1a exosomes were treated with E-selectin in the presence of Ca²⁺ to mediate binding. Subsequently, lysates were prepared and several signaling proteins were analyzed by western blot including ezrin, phospho-ezrin, Rac-1/Cdc42 and NHERF-1. Interestingly, there was an overall increase in ubiquitination observed in response to E-selectin binding but not all proteins analyzed were perturbed. CD81 blot was used as a loading control for the western blots illustrated. Results shown are representative of n = 3 independent experiments. (B) The proteomics analysis of the KG1a (pink) and healthy HSPCs (blue frame)-derived exosomes revealed the presence of proteins related to the proteosome indicating that exosomes contain the machinery to enable them towards controlled degradation of proteins.