Application of high-mannose-type glycan-specific lectin from Oscillatoria Agardhii for affinity isolation of tumor-derived extracellular vesicles

Tumors that lack T cell infiltration are less likely to respond to immune checkpoint inhibition and could benefit from cancer vaccination for the initiation of anti-tumor T cell responses. An attractive vaccine strategy is in vivo targeting of dendritic cells (DCs), key initiators of antigen-specific T cell responses. In this study we generated tumor-derived apoptotic extracellular vesicles (ApoEVs), which are potentially an abundant source of tumor-specific neo-antigens and other tumor-associated antigens (TAAs), and which can be manipulated to express DC-targeting ligands for efficient antigen delivery. Our data demonstrates that by specifically modifying the glycocalyx of tumor cells, high-mannose glycans can be expressed on their cell surface and on extracellular vesicles derived after the induction of apoptosis. High-mannose glycans are the natural ligands of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a dendritic cell associated C-type lectin receptor (CLR), which has the ability to efficiently internalize its cargo and direct it to both major histocompatibility complex (MHC)-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Compared to unmodified ApoEVs, ApoEVs carrying DC-SIGN ligands are internalized to a higher extent, resulting in enhanced priming of tumor-specific CD8+ T cells. This approach thus presents a promising vaccination strategy in support of T cell-based immunotherapy of cancer.

Download Full-text

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles

Scientific Reports ◽

10.1038/s41598-021-04512-0 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Mikkel Ø. Nørgård ◽

Lasse B. Steffensen ◽

Didde R. Hansen ◽

Ernst-Martin Füchtbauer ◽

Morten B. Engelund ◽

...

Keyword(s):

Mouse Model ◽

Extracellular Vesicles ◽

Fluorescent Protein ◽

Egfp Expression ◽

Affinity Isolation ◽

Reporter Mice ◽

Renal Tubular ◽

Co Precipitation ◽

Green Fluorescent

AbstractThe in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

Download Full-text

Quantitative proteomic analysis for evaluating affinity isolation of extracellular vesicles

Journal of Proteomics ◽

10.1016/j.jprot.2021.104359 ◽

2021 ◽

pp. 104359

Author(s):

Ai Nguyen ◽

Tingting Wang ◽

Illarion V. Turko

Keyword(s):

Extracellular Vesicles ◽

Proteomic Analysis ◽

Quantitative Proteomic Analysis ◽

Affinity Isolation

Download Full-text

Ultrastructural changes in isolated rat liver perfused with cyclic heptapeptide toxins from norwegian freshwater cyanobacteria (blue-green algae)

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128572 ◽

1987 ◽

Vol 45 ◽

pp. 858-859

Author(s):

A.S. Dabholkar ◽

W.W. Carmichael ◽

K. Berg ◽

J. Wyman

Keyword(s):

Ultrastructural Changes ◽

Sprague Dawley ◽

Isolated Rat Liver ◽

Blue Green Algae ◽

Sprague Dawley Rats ◽

Cyclic Heptapeptide ◽

Intracellular Changes ◽

Oscillatoria Agardhii ◽

Rat Livers ◽

Isolated Rat

Intracellular changes in the hepatocytes of isolated rat livers perfused with cyclic heptapeptide toxins are described. The toxins used are 1) -Ala-Leu- β-methyl isoAsp-Arg-ADDA-isoGlu-mdha (M.W. 944) from Microcystis aeruginosa- Lake Akersvatn, Norway; 2) -Ala-Arg-isoAsp-Arg-ADDA-isoGlu-mdha (M.W. 1023) from Oscillatoria agardhii var. - Lake Kolbatnvatn, Norway; 3) -Ala-Arg-isoAsp-Arg-ADDA-isoGlu-dha (M.W. 1009) from Oscillatoria agardhii var. isothrix - Lake Froylandsvatn, Norway. Approximate LD intraperitoneal mouse for the toxins is 50, 500 and 1000 μg/kg respectively.Livers were removed from male Sprague Dawley rats and perfused for 15 min with a blood-free perfusate (50 ml) followed by 60 min with perfusate containing i) 25, 50, or 200 μg of M. aeruginosa toxin ii) 50, 250, 500 or 1000 μg of O. agardhii var. toxin and iii) 1000, 2000, 2500 or 5000 μg of O. agardhii var. isothrix toxin. Control livers were perfused for 75 min with the blood-free perfusate.

Download Full-text