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Acute lymphoblastic leukaemia cells produce large extracellular vesicles containing organelles and an active cytoskeleton.

ABSTRACT: Extracellular vesicles have been described in non-paracrine cellular interactions in cancer. We report a similar phenomenon in B-cell precursor (BCP) acute lymphoblastic leukaemia (ALL). Using advanced microscopy and high throughput screening, we further characterise a subset of large vesicles (LEVs) identified in cell lines, murine models of human BCP-ALL and clinical samples. Primary ALL blasts and cell lines released heterogeneous anucleate vesicles <6 micron into extracellular fluids. Larger LEVs were enclosed in continuous membranes, contained intact organelles and demonstrated an organised cytoskeleton. An excess of circulating CD19-positive LEVs were observed in diagnostic samples and isolated from mice engrafted with BCP-ALL primary cells. LEVs exhibited dynamic shape change in vitro and were internalised by other leukaemic cell lines leading to phenotypic transformation analogous to the cell of origin. In patient-derived xenografts, LEVs were released by primary ALL cells into extracellular spaces and internalised by murine mesenchymal cells in vivo. Collectively these data highlight the heterogeneity but accessibility of LEVs in clinical samples and their potential to provide a unique insight into the biology of the cell of origin and to their development as novel biomarkers to aid diagnosis and improve therapeutic outcomes.

SUBMITTER: Johnson SM

PROVIDER: S-EPMC5373679 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Acute lymphoblastic leukaemia cells produce large extracellular vesicles containing organelles and an active cytoskeleton.

Johnson Suzanne M SM Dempsey Clare C Parker Catriona C Mironov Aleksandr A Bradley Helen H Saha Vaskar V

Journal of extracellular vesicles 20170310 1

Extracellular vesicles have been described in non-paracrine cellular interactions in cancer. We report a similar phenomenon in B-cell precursor (BCP) acute lymphoblastic leukaemia (ALL). Using advanced microscopy and high throughput screening, we further characterise a subset of large vesicles (LEVs) identified in cell lines, murine models of human BCP-ALL and clinical samples. Primary ALL blasts and cell lines released heterogeneous anucleate vesicles <6 micron into extracellular fluids. Larger ...[more]

PMID: 28386390

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Project description:Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatus IMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

Project description:PurposeThe bone marrow niche plays an important role in leukemia development. However, the contributions of different niche components to leukemia development and their underlying mechanisms remain largely unclear.MethodCre/LoxP-based conditional knockout technology was used to delete VPS33B or ANGPTL2 gene in niche cells. Murine B-ALL model was established by overexpressing the N-Myc oncogene in hematopoietic stem progenitor cells. The frequency of leukemia cells and immunophenotypic B220+ CD43+ LICs was detected by flow cytometry. SEVs was isolated by sequential centrifugation and mass spectrometry was performed to analyze the different components of SEVs. Immunoprecipitation and western blot were used to measure the interaction of VPS33B and ANGPTL2.ResultsHere, we showed that specific knockout of vascular protein sorting 33b (Vps33b) in endothelial cells (ECs), but not megakaryocytes or mesenchymal stem cells, resulted in a significant decrease in the secretion of small extracellular vesicles (SEVs) and a delay in the development of B-cell lymphoblastic leukemia (B-ALL). Vps33b knockdown endothelial cells contained much lower levels of SEVs that contained angiopoietin-like protein 2 (ANGPTL2) than the control cells. Importantly, conditional knockout of Angptl2 in ECs significantly delayed B-ALL progression. Moreover, C-terminal region of ANGPTL2 (aa247-471) could directly interact with Sec1-like domain 1 of VPS33B (aa1-aa146). We further demonstrated that the point mutations R399H and G402S in ANGPTL2 led to a dramatic decrease in the secretion of ANGPTL2-SEVs. We also showed that wild-type ANGPTL2-containing SEVs, but not mutant ANGPTL2-containing SEVs, significantly enhanced B-ALL development.ConclusionIn summary, our findings indicate that the secretion of ANGPTL2-containing SEVs in ECs sustains the leukemogenic activities of B-ALL cells, which is fine-tuned by the direct interaction of VPS33B and ANGPTL2. These findings reveal that niche-specific SEVs play an important role in B-ALL development.

Dataset Information

Acute lymphoblastic leukaemia cells produce large extracellular vesicles containing organelles and an active cytoskeleton.

Publications

Acute lymphoblastic leukaemia cells produce large extracellular vesicles containing organelles and an active cytoskeleton.

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