EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open-source ImageJ/Fiji plugin.

Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno-)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross-reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid-based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell-single vesicle level, thereby enabling high content EV cell uptake assays and plate-based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open-source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.

A functional corona around extracellular vesicles enhances angiogenesis, skin regeneration and immunomodulation.

Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell-derived extracellular vesicles (EVs). Here we demonstrate that nano-sized EVs from therapy-grade human placental-expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell-secreted soluble factors via tangential flow-filtration (TFF) and subtractive tandem mass-tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein-based flow cytometry, super-resolution and electron microscopy verified EV identity. PLX-EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose-dependently inhibited T cell proliferation in vitro. Corona removal by size-exclusion or ultracentrifugation abrogated angiogenesis. Re-establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by super-resolution microscopy, electron microscopy and zeta-potential shift, and served as a proof-of-concept. Understanding EV corona formation will improve rational EV-inspired nano-therapy design.

Synergy of Human Platelet-Derived Extracellular Vesicles with Secretome Proteins Promotes Regenerative Functions.

Platelet-rich plasma is a promising regenerative therapeutic with controversial efficacy. We and others have previously demonstrated regenerative functions of human platelet lysate (HPL) as an alternative platelet-derived product. Here we separated extracellular vesicles (EVs) from soluble factors of HPL to understand the mode of action during skin-organoid formation and immune modulation as model systems for tissue regeneration. HPL-EVs were isolated by tangential-flow filtration (TFF) and further purified by size-exclusion chromatography (SEC) separating EVs from (lipo)protein-enriched soluble fractions. We characterized samples by tunable resistive pulse sensing, western blot, tandem mass-tag proteomics and super-resolution microscopy. We evaluated EV function during angiogenesis, wound healing, organoid formation and immune modulation. We characterized EV enrichment by TFF and SEC according to MISEV2018 guidelines. Proteomics showed three major clusters of protein composition separating TSEC-EVs from HPL clustering with TFF soluble fractions and TFF-EVs clustering with TSEC soluble fractions, respectively. HPL-derived TFF-EVs promoted skin-organoid formation and inhibited T-cell proliferation more efficiently than TSEC-EVs or TSEC-soluble fractions. Recombining TSEC-EVs with TSEC soluble fractions re-capitulated TFF-EV effects. Zeta potential and super-resolution imaging further evidenced protein corona formation on TFF-EVs. Corona depletion on SEC-EVs could be artificially reconstituted by TSEC late fraction add-back. In contrast to synthetic nanoparticles, which commonly experience reduced function after corona formation, the corona-bearing EVs displayed improved functionality. We conclude that permissive isolation technology, such as TFF, and better understanding of the mechanism of EV corona function are required to realize the complete potential of platelet-based regenerative therapies.

Scalable Enrichment of Immunomodulatory Human Acute Myeloid Leukemia Cell Line-Derived Extracellular Vesicles.

Acute myeloid leukemia (AML) cells can secrete trophic factors, including extracellular vesicles (EVs), instructing the stromal leukemic niche. Here, we introduce a scalable workflow for purification of immunomodulatory AML-EVs to compare their phenotype and function to the parental AML cells and their secreted soluble factors. AML cell lines HL-60, KG-1, OCI-AML3, and MOLM-14 released EVs with a peak diameter of approximately 80 nm in serum-free particle-reduced medium. We enriched EVs >100x using tangential flow filtration (TFF) and separated AML-derived soluble factors and cells in parallel. EVs were characterized by electron microscopy, immunoblotting, and flow cytometry, confirming the double-membrane morphology, purity and identity. AML-EVs showed significant enrichment of immune response and leukemia-related pathways in tandem mass-tag proteomics and a significant dose-dependent inhibition of T cell proliferation, which was not observed with AML cells or their soluble factors. Furthermore, AML-EVs dose-dependently reduced NK cell lysis of third-party K-562 leukemia targets. This emphasizes the peculiar role of AML-EVs in leukemia immune escape and indicates novel EV-based targets for therapeutic interventions.

Hypoxic Conditions Promote the Angiogenic Potential of Human Induced Pluripotent Stem Cell-Derived Extracellular Vesicles.

Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics.

Tumor angiogenesis and lymphangiogenesis: tumor/endothelial crosstalk and cellular/microenvironmental signaling mechanisms.

Tumor angiogenesis and lymphangiogenesis are key features of tumor progression and metastasis. The role of tumor cells-derived factors in the promotion of associated angiogenesis and lymphangiogenesis is much studied and, no doubt, very important for the understanding of cancer progression. This review aims to present and discuss the work done on the pro-angiogenic and lymphangiogenic cellular interactions within the tumor microenvironment and the signaling pathways that regulate this crosstalk. Such multifactor studies are critical for the development of future therapeutic approaches for cancer because they take into account the complexities of cellular interactions within the tumor microenvironment.