Dynamic regulation of CD24 expression and release of CD24-containing microvesicles in immature B cells in response to CD24 engagement.

The glycophosphatidylinositol-anchored cell surface receptor CD24 (also called heat-stable antigen) promotes the apoptosis of progenitor and precursor B-lymphocytes. However, the immediate proximal events that occur after engagement of CD24 in B cells are not precisely understood. Using a bioinformatics analysis of mouse (Mus musculus) gene expression data from the Immunological Genome Project, we found that known vesicle trafficking and cellular organization genes have similar expression patterns to CD24 during B-cell development in the bone marrow. We therefore hypothesized that CD24 regulates vesicle trafficking. We first validated that antibody-mediated engagement of CD24 induces apoptosis in the mouse WEHI-231 cell line and mouse primary bone marrow-derived B cells. We next found that CD24 surface protein expression is rapidly and dynamically regulated in both WEHI-231 cells and primary immature B cells in response to engagement of CD24. The change in surface expression was not mediated by classical endocytosis or exocytosis. However, we found that CD24-bearing plasma membrane-derived extracellular microvesicles were released in response to CD24 engagement. Furthermore, in response to CD24 engagement we observed a clear exchange of CD24 between different populations of B cells. Hence, we show that engagement of CD24 in immature B cells results in a dynamic regulation of surface CD24 protein and a redistribution of CD24 within the population.

A multiparametric extraction method for Vn96-isolated plasma extracellular vesicles and cell-free DNA that enables multi-omic profiling.

Extracellular vesicles (EVs) have been recognized as a rich material for the analysis of DNA, RNA, and protein biomarkers. A remaining challenge for the deployment of EV-based diagnostic and prognostic assays in liquid biopsy testing is the development of an EV isolation method that is amenable to a clinical diagnostic lab setting and is compatible with multiple types of biomarker analyses. We have previously designed a synthetic peptide, known as Vn96 (ME kit), which efficiently isolates EVs from multiple biofluids in a short timeframe without the use of specialized lab equipment. Moreover, it has recently been shown that Vn96 also facilitates the co-isolation of cell-free DNA (cfDNA) along with EVs. Herein we describe an optimized method for Vn96 affinity-based EV and cfDNA isolation from plasma samples and have developed a multiparametric extraction protocol for the sequential isolation of DNA, RNA, and protein from the same plasma EV and cfDNA sample. We are able to isolate sufficient material by the multiparametric extraction protocol for use in downstream analyses, including ddPCR (DNA) and 'omic profiling by both small RNA sequencing (RNA) and mass spectrometry (protein), from a minimum volume (4 mL) of plasma. This multiparametric extraction protocol should improve the ability to analyse multiple biomarker materials (DNA, RNA and protein) from the same limited starting material, which may improve the sensitivity and specificity of liquid biopsy tests that exploit EV-based and cfDNA biomarkers for disease detection and monitoring.

Characterization of miRNAs in Extracellular Vesicles Released From Atlantic Salmon Monocyte-Like and Macrophage-Like Cells.

Cell-derived extracellular vesicles (EVs) participate in cell-cell communication via transfer of molecular cargo including genetic material like miRNAs. In mammals, it has previously been established that EV-mediated transfer of miRNAs can alter the development or function of immune cells, such as macrophages. Our previous research revealed that Atlantic salmon head kidney leukocytes (HKLs) change their morphology, phagocytic ability and miRNA profile from primarily "monocyte-like" at Day 1 to primarily "macrophage-like" at Day 5 of culture. Therefore, we aimed to characterize the miRNA cargo packaged in EVs released from these two cell populations. We successfully isolated EVs from Atlantic salmon HKL culture supernatants using the established Vn96 peptide-based pull-down. Isolation was validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. RNA-sequencing identified 19 differentially enriched (DE) miRNAs packaged in Day 1 versus Day 5 EVs. Several of the highly abundant miRNAs, including those that were DE (e.g. ssa-miR-146a, ssa-miR-155 and ssa-miR-731), were previously identified as DE in HKLs and are associated with macrophage differentiation and immune response in other species. Interestingly, the abundance relative of the miRNAs in EVs, including the most abundant miRNA (ssa-miR-125b), was different than the miRNA abundance in HKLs, indicating selective packaging of miRNAs in EVs. Further study of the miRNA cargo in EVs derived from fish immune cells will be an important next step in identifying EV biomarkers useful for evaluating immune cell function, fish health, or response to disease.

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein-extraction methods.

Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of '-omics' research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

CD24 induces changes to the surface receptors of B cell microvesicles with variable effects on their RNA and protein cargo.

The CD24 cell surface receptor promotes apoptosis in developing B cells, and we recently found that it induces B cells to release plasma membrane-derived, CD24-bearing microvesicles (MVs). Here we have performed a systematic characterization of B cell MVs released from WEHI-231 B lymphoma cells in response to CD24 stimulation. We found that B cells constitutively release MVs of approximately 120 nm, and that CD24 induces an increase in phosphatidylserine-positive MV release. RNA cargo is predominantly comprised of 5S rRNA, regardless of stimulation; however, CD24 causes a decrease in the incorporation of protein coding transcripts. The MV proteome is enriched with mitochondrial and metabolism-related proteins after CD24 stimulation; however, these changes were variable and could not be fully validated by Western blotting. CD24-bearing MVs carry Siglec-2, CD63, IgM, and, unexpectedly, Ter119, but not Siglec-G or MHC-II despite their presence on the cell surface. CD24 stimulation also induces changes in CD63 and IgM expression on MVs that is not mirrored by the changes in cell surface expression. Overall, the composition of these MVs suggests that they may be involved in releasing mitochondrial components in response to pro-apoptotic stress with changes to the surface receptors potentially altering the cell type(s) that interact with the MVs.

Analysis of the structure, evolution, and expression of CD24, an important regulator of cell fate.

BACKGROUND: CD24 is a small, glycophosphatidylinositol-anchored cell surface receptor, expressed in a variety of cells types and tissues. CD24 gene and protein expression is highly dynamic in response to cellular differentiation and stimulation in a cell-specific manner. Furthermore, CD24 interacts with a diverse collection of ligands, including cell adhesion molecules such as P-selectin, and the immune-associated siglec family of transmembrane proteins. While much is known regarding the biological roles of CD24 in regulating cell survival, death and differentiation, little is known about the evolution and organization of CD24 across species or the relationship between CD24 expression and its known ligands. RESULTS: We analyzed the organization and evolution of the CD24 gene from 56 mammalian, avian and reptilian species. We further examined the mRNA expression of CD24 and its known ligands in Mus musculus in immune cells, immunologically privileged tissues, developing brain, and developing and regenerating liver. CD24 arose prior to the reptilian-avian divergence and is conserved across many mammalian species, although we were unable to identify CD24 in marsupials or monotremes. The CD24 genomic structure is diverse between and within species, with varying numbers of exons, introns, and the presence of untranslated regions. Of note, we found no obvious criteria distinguishing CD24 genes from those annotated as CD24-like. The expression of CD24 is similarly complex, with immune cells showing dynamic changes in mRNA levels during development, while immunologically privileged and developing tissues show a high, static expression level that decreases in mature tissues. Furthermore, the expression of CD24 correlated with some but not all of its known ligands in a tissues-specific manner, suggesting that novel ligands have yet to be identified and that cell-specific ligand expression can influence CD24 function. CONCLUSIONS: We find that CD24 arose prior to the divergence of reptiles, birds and mammals. Furthermore, the most highly conserved areas of the protein are the amino acids which can be glycosylated. We also find that CD24 expression is highly tissue-specific and in many cases, not well conserved with known CD24 ligands, suggesting yet-unknown CD24-ligand interactions. Together, these data are a valuable resource for furthering studies in CD24 biology.

Repression of CD24 surface protein expression by oncogenic Ras is relieved by inhibition of Raf but not MEK or PI3K.

CD24 is a dynamically regulated cell surface protein. High expression of CD24 leads to progression of lung, prostrate, colon, and pancreatic cancers, among others. In contrast, low expression of CD24 leads to cell proliferation and metastasis of breast cancer stem cells (BCSCs). Activating mutations in Ras are found in 30% of all human cancers. Oncogenic Ras constitutively stimulates the Raf, PI3K, and Ral GDS signaling pathways, leading to cellular transformation. Previous studies have shown that expression of oncogenic Ras in breast cancer cells generates CD24(-) cells from CD24(+) cells. However, the molecular mechanisms involved in the generation of CD24(-) cells were not determined. Here, we demonstrate that oncogenic Ras (RasV12) expression suppresses CD24 mRNA, protein, and promoter levels when expressed in NIH/3T3 cells. Furthermore, activation of only the Raf pathway was sufficient to downregulate CD24 mRNA and protein expression to levels similar to those seen in with RasV12 expression. In contrast, activation of the PI3K pathway downregulated mRNA expression with a partial effect on protein expression whereas activation of the RalGDS pathway only partially affected protein expression. Surprisingly, inhibition of MEK with U0126 only partially restored CD24 mRNA expression but not surface protein expression. In contrast, inhibition of Raf with sorafenib did not restore CD24 mRNA expression but significantly increased the proportion of RasV12 cells expressing CD24. Therefore, the Raf pathway is the major repressor of CD24 mRNA and protein expression, with PI3K also able to substantially inhibit CD24 expression. Moreover, these data indicate that the levels of CD24 mRNA and surface protein are independently regulated. Although inhibition of Raf by sorafenib only partially restored CD24 expression, sorafenib should still be considered as a potential therapeutic strategy to alter CD24 expression in CD24(-) cells, such as BCSCs.

Loss of CD24 in Mice Leads to Metabolic Dysfunctions and a Reduction in White Adipocyte Tissue.

CD24 is a glycophosphatidylinositol (GPI)-linked cell surface receptor that is involved in regulating the survival or differentiation of several different cell types. CD24 has been used to identify pre-adipocytes that are able to reconstitute white adipose tissue (WAT) in vivo. Moreover, we recently found that the dynamic upregulation of CD24 in vitro during early phases of adipogenesis is necessary for mature adipocyte development. To determine the role of CD24 in adipocyte development in vivo, we evaluated the development of the inguinal and interscapular subcutaneous WAT and the epididymal visceral WAT in mice with a homozygous deletion of CD24 (CD24KO). We observed a significant decrease in WAT mass of 40% to 74% in WAT mass from both visceral and subcutaneous depots in male mice, with no significant effect in female mice, compared to wild-type (WT) sex- and age-matched controls. We also found that CD24KO mice had increased fasting glucose and free fatty acids, decreased fasting insulin, and plasma leptin. No major differences were observed in the sensitivity to insulin or glucose, or in circulating triglycerides, total cholesterol, HDL-cholesterol, or LDL-cholesterol levels between WT and CD24KO mice. Challenging the CD24KO mice with either high sucrose (35%) or high fat (45%) diets that promote increased adiposity, increased WAT mass and fasting insulin, adiponectin and leptin levels, as well as reduced the sensitivity to insulin and glucose, to the levels of WT mice on the same diets. The CD24-mediated reduction in fat pad size was due to a reduction in adipocyte cell size in all depots with no significant reduction pre-adipocyte or adipocyte cell number. Thus, we have clearly demonstrated that the global absence of CD24 affects adipocyte cell size in vivo in a sex- and diet-dependent manner, as well as causing metabolic disturbances in glucose homeostasis and free fatty acid levels.