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Pediatric B-acute lymphoblastic leukemia (B-ALL) is a disease of abnormally growing B lymphoblasts. Here we hypothesized that extracellular vesicles (EVs), which are nanosized particles released by all cells (including cancer cells), could be used to monitor B-ALL severity and progression by sampling plasma instead of bone marrow. EVs are especially attractive as they are present throughout the circulation regardless of the location of the originating cell. First, we used nanoparticle tracking analysis to compare EVs between non-cancer donor (NCD) and B-ALL blood plasma; we found that B-ALL plasma contains more EVs than NCD plasma. We then isolated EVs from NCD and pediatric B-ALL peripheral blood plasma using a synthetic peptide-based isolation technique (Vn96), which is clinically amenable and isolates a broad spectrum of EVs. RNA-seq analysis of small RNAs contained within the isolated EVs revealed a signature of differentially packaged and exclusively packaged RNAs that distinguish NCD from B-ALL. The plasma EVs contain a heterogenous mixture of miRNAs and fragments of long non-coding RNA (lncRNA) and messenger RNA (mRNA). Transcripts packaged in B-ALL EVs include those involved in negative cell cycle regulation, potentially suggesting that B-ALL cells may use EVs to discard gene sequences that control growth. In contrast, NCD EVs carry sequences representative of multiple organs, including brain, muscle, and epithelial cells. This signature could potentially be used to monitor B-ALL disease burden in pediatric B-ALL patients via blood draws instead of invasive bone marrow aspirates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Longjohn, Hudson, Peña-Castillo, Cormier, Hannay, Chacko, Lewis, Moorehead and Christian.)

B cell acute lymphoblastic leukemia (B-ALL) is the most prevalent pediatric cancer. MicroRNAs (miRNAs) are 18-22nt non-coding transcripts shown to be essential for the development of many cancers. While some miRNAs are reportedly expressed differentially between healthy and B-ALL, no studies have reported a consensus miRNA signature. Therefore, we performed a reanalysis of five miRNA datasets to identify differentially expressed miRNAs (DEmiRs) and a meta-analysis of previously identified DEmiRs from 25 studies. Overall, the re-analysis showed that the DEmiR data clustered by platform and not by disease state. The meta-analysis also did not reveal a consensus miRNA signature as there were many miRNAs upregulated in some studies and downregulated in others. However, eight promising miRNAs (miR-181b, miR-128b, miR-181a, miR-128, miR-128a, miR-181c, miR-155, miR-142-3p, and miR-451) were identified from the meta-analysis, which could be the basis of future investigations. These analyses reveal that standardization of miRNA isolation and analysis is needed in B-ALL to enable cross-study comparisons and identify a consensus signature., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Suspension cell lines grow free-floating in the cell culture media without any attachment to the culture plate/vessel. Suspension cells typically mimic cells that exist in the circulation of multicellular animals such as mouse and humans. Generally, cell lines derived from the blood such as lymphocytes, megakaryocyte, and neutrophils grow in suspension. These cell lines can be used for experimental studies to understand the biology/biochemistry of cancer cells. In this chapter, procedures for working with suspension cell lines are provided, including protocols for thawing, culturing, and cryopreserving cancer cell lines. Importantly, this chapter demonstrates the best practices required to work with suspension cell lines, to minimize the risk of contaminations from adventitious microorganisms or from other cell lines., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Extracellular vesicles (EVs) are nanosized lipid bound particles secreted by cells. EVs transfer biologic material and can be found in all body fluids. Accurately characterizing the size and concentration of EVs is difficult because of the nanoscale size of EVs. An evolving solution to this problem is tracking the Brownian motion of EVs in suspension, a technique known as nanoparticle-tracking analysis (NTA). This technique is used by many researchers in the EV field. The ability to accurately replicate data between studies and laboratories is critical to advancing the knowledge surrounding EVs for use in liquid biopsy and cancer studies, in general. Thus, this chapter provides a step-by-step guide on isolating EVs using a variety of methods, for characterizing EVs using the NS300 NTA instrument, troubleshooting tips, and a robust guide to reporting key parameters that will help improve cross-lab reproducibility of NTA data., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles that carry bioactive cargo, including proteins, lipids, and nucleic acids. Once taken up by target cells, EVs can modify the physiology of the recipient cells. In past studies, we reported that engagement of the glycophosphatidylinositol-anchored receptor CD24 on B lymphocytes (B cells) causes the release of EVs. However, a potential function for these EVs was not clear. Thus, we investigated whether EVs derived from CD24 or IgM-stimulated donor WEHI-231 murine B cells can transfer functional cargo to recipient cells. We employed a model system where donor cells expressing palmitoylated GFP (WEHI-231-GFP) were cocultured, after stimulation, with recipient cells lacking either IgM (WEHI-303 murine B cells) or CD24 (CD24 knockout mouse bone marrow B cells). Uptake of lipid-associated GFP, IgM, or CD24 by labeled recipient cells was analyzed by flow cytometry. We found that stimulation of either CD24 or IgM on the donor cells caused the transfer of lipids, CD24, and IgM to recipient cells. Importantly, we found that the transferred receptors are functional in recipient cells, thus endowing recipient cells with a second BCR or sensitivity to anti-CD24-induced apoptosis. In the case of the BCR, we found that EVs were conclusively involved in this transfer, whereas in the case in the CD24 the involvement of EVs is suggested. Overall, these data show that extracellular signals received by one cell can change the sensitivity of neighboring cells to the same or different stimuli, which may impact B cell development or activation., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Extracellular vesicles (EVs) are nanosized membrane-bound particles released from all living cells examined thus far. EVs can transfer information in the form of proteins, nucleic acids, and lipids from donor cells to recipient cells. Here we summarize recent advances in understanding the role(s) EVs play in hematological malignancies (HM) and outline potential prognostic and diagnostic strategies using EVs. EVs have been shown to promote proliferation and angiogenesis, and alter the bone marrow microenvironment to favour the growth and survival of diverse HM. They also promote evasion of anti-cancer immune responses and increase multi-drug resistance. Using knowledge of EV biology, including HM-specific packaging of cargo, EV based diagnostics and therapeutic approaches show substantial clinical promise. However, while EVs may represent a new paradigm to solve many of the challenges in treating and/or diagnosing HM, much work is needed before they can be used clinically to improve patient outcomes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)