Multimode chromatography-based techniques for high purity isolation of extracellular vesicles from human blood plasma.

Extracellular vesicles (EVs) play a pivotal role in various biological pathways, such as immune responses and the progression of diseases, including cancer. However, it is challenging to isolate EVs at high purity from blood plasma and other biofluids due to their low abundance compared to more predominant biomolecular species such as lipoprotein particles and free protein complexes. Ultracentrifugation-based EV isolation, the current gold standard technique, cannot overcome this challenge due to the similar biophysical characteristics of such species. We developed several novel approaches to enrich EVs from plasma while depleting contaminating molecular species using multimode chromatography-based strategies. On average, we identified 716 ± 68 and 1054 ± 35 protein groups in EV isolates from 100 µL of plasma using multimode chromatography- and ultracentrifugation-based techniques, respectively. The developed methods resulted in similar EV isolates purity, providing significant advantages in simplicity, throughput, scalability, and applicability for various downstream analytical and potential clinical applications.

KLRG1 Cell Depletion as a Novel Therapeutic Strategy in Patients with Mature T-Cell Lymphoma Subtypes.

PURPOSE: Develop a novel therapeutic strategy for patients with subtypes of mature T-cell and NK-cell neoplasms. EXPERIMENTAL DESIGN: Primary specimens, cell lines, patient-derived xenograft models, commercially available, and proprietary anti-KLRG1 antibodies were used for screening, target, and functional validation. RESULTS: Here we demonstrate that surface KLRG1 is highly expressed on tumor cells in subsets of patients with extranodal NK/T-cell lymphoma (ENKTCL), T-prolymphocytic leukemia (T-PLL), and gamma/delta T-cell lymphoma (G/D TCL). The majority of the CD8+/CD57+ or CD3-/CD56+ leukemic cells derived from patients with T- and NK-large granular lymphocytic leukemia (T-LGLL and NK-LGLL), respectively, expressed surface KLRG1. The humanized afucosylated anti-KLRG1 monoclonal antibody (mAb208) optimized for mouse in vivo use depleted KLRG1+ TCL cells by mechanisms of ADCC, ADCP, and CDC rather than apoptosis. mAb208 induced ADCC and ADCP of T-LGLL patient-derived CD8+/CD57+ cells ex vivo. mAb208 effected ADCC of subsets of healthy donor-derived KLRG1+ NK, CD4+, CD8+ Tem, and TemRA cells while sparing KLRG1- naïve and CD8+ Tcm cells. Treatment of cell line and TCL patient-derived xenografts with mAb208 or anti-CD47 mAb alone and in combination with the PI3K-δ/γ inhibitor duvelisib extended survival. The depletion of macrophages in vivo antagonized mAb208 efficacy. CONCLUSIONS: Our findings suggest the potential benefit of a broader treatment strategy combining therapeutic antibodies with PI3Ki for the treatment of patients with mature T-cell and NK-cell neoplasms. See related commentary by Varma and Diefenbach, p. 2300.

Human red blood cells release microvesicles with distinct sizes and protein composition that alter neutrophil phagocytosis.

Extracellular vesicles (EVs) are membrane-bound structures released by cells and tissues into biofluids, involved in cell-cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell-type in the body, generating daily large numbers of microvesicles. In vitro, RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187. The fate of microvesicles released during in vivo aging of RBCs and their interactions with circulating cells is hitherto unknown. Using SEC plus DEG isolation methods, we have found that human RBCs generate microvesicles with two distinct sizes, densities, and protein composition, identified by flow cytometry, and MRPS, and further validated by immune TEM. Furthermore, proteomic analysis revealed that RBC-derived microvesicles (RBC-MVs) are enriched in proteins with important functions in ion channel regulation, calcium homeostasis, and vesicular transport, such as of sorcin, stomatin, annexin A7, and RAB proteins. Cryo-electron microscopy identified two separate pathways of RBC-MV-neutrophil interaction, direct fusion with the plasma membrane and internalization, respectively. Functionally, RBC-MVs decrease neutrophil ability to phagocytose E. coli but do not affect their survival at 24 hrs. This work brings new insights regarding the complexity of the RBC-MVs biogenesis, as well as their possible role in circulation.

Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection.

MicroRNAs are short, non-coding RNA sequences involved in gene expression regulation. Quantification of miRNAs in biological fluids involves time consuming and laborious methods such as Northern blotting or PCR-based techniques. Molecular beacons (MB) are an attractive means for rapid detection of miRNAs, although the need for sophisticated readout methods limits their use in research and clinical settings. Here, we introduce a novel method based on delayed electrophoretic mobility, as a quantitative means for detection of miRNAs-MB hybridization. Upon hybridization with the target miRNAs, MB form a fluorescent duplex with reduced electrophoretic mobility, thus bypassing the need for additional staining. In addition to emission of light, the location of the fluorescent band on the gel acts as an orthogonal validation of the target identity, further confirming the specificity of binding. The limit of detection of this approach is approximately 100 pM, depending on the MB sequence. The method is sensitive enough to detect specific red blood cell miRNAs molecules in total RNA, with single nucleotide specificity. Altogether, we describe a rapid and affordable method that offers sensitive detection of single-stranded small DNA and RNA sequences.

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation.

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.