A Proteomic Approach to Understand the Clinical Significance of Acute Myeloid Leukemia-Derived Extracellular Vesicles Reflecting Essential Characteristics of Leukemia.

Extracellular vesicle (EV) proteins from acute myeloid leukemia (AML) cell lines were analyzed using mass spectrometry. The analyses identified 2450 proteins, including 461 differentially expressed proteins (290 upregulated and 171 downregulated). CD53 and CD47 were upregulated and were selected as candidate biomarkers. The association between survival of patients with AML and the expression levels of CD53 and CD47 at diagnosis was analyzed using mRNA expression data from The Cancer Genome Atlas database. Patients with higher expression levels showed significantly inferior survival than those with lower expression levels. ELISA results of the expression levels of CD53 and CD47 from EVs in the bone marrow of patients with AML at diagnosis and at the time of complete remission with induction chemotherapy revealed that patients with downregulated CD53 and CD47 expression appeared to relapse less frequently. Network model analysis of EV proteins revealed several upregulated kinases, including LYN, CSNK2A1, SYK, CSK, and PTK2B. The potential cytotoxicity of several clinically applicable drugs that inhibit these kinases was tested in AML cell lines. The drugs lowered the viability of AML cells. The collective data suggest that AML cell-derived EVs could reflect essential leukemia biology.

Exosome Classification by Pattern Analysis of Surface-Enhanced Raman Spectroscopy Data for Lung Cancer Diagnosis.

Owing to the role of exosome as a cargo for intercellular communication, especially in cancer metastasis, the evidence has been consistently accumulated that exosomes can be used as a noninvasive indicator of cancer. Consequently, several studies applying exosome have been proposed for cancer diagnostic methods such as ELISA assay. However, it has been still challenging to get reliable results due to the requirement of a labeling process and high concentration of exosome. Here, we demonstrate a label-free and highly sensitive classification method of exosome by combining surface-enhanced Raman scattering (SERS) and statistical pattern analysis. Unlike the conventional method to read different peak positions and amplitudes of a spectrum, whole SERS spectra of exosomes were analyzed by principal component analysis (PCA). By employing this pattern analysis, lung cancer cell derived exosomes were clearly distinguished from normal cell derived exosomes by 95.3% sensitivity and 97.3% specificity. Moreover, by analyzing the PCA result, we could suggest that this difference was induced by 11 different points in SERS signals from lung cancer cell derived exosomes. This result paved the way for new real-time diagnosis and classification of lung cancer by using exosome as a cancer marker.

Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma.

Isolation of pure extracellular vesicles (EVs), especially from blood, has been a major challenge in the field of EV research. The presence of lipoproteins and soluble proteins often hinders the isolation of high purity EVs upon utilization of conventional separation methods. To circumvent such problems, we designed a single-step dual size-exclusion chromatography (dSEC) column for effective isolation of highly pure EVs from bone marrow derived human plasma. With an aim to select appropriate column design parameters, we analyzed the physiochemical properties of the major substances in bone marrow derived plasma, which include EVs, lipoproteins, and soluble proteins. Based on these findings, we devised a novel dSEC column with two different types of porous beads sequentially stacked each other for efficient separation of EVs from other contaminants. The newly developed dSEC columns exhibited better performance in isolating highly pure EVs from AML plasma in comparison to conventional isolation methods.

The Potential of Exosomes Derived from Chronic Myelogenous Leukaemia Cells as a Biomarker.

BACKGROUND/AIM: Exosomes, derived from chronic myelogenous leukaemia (CML) cells, can be used as biomarkers and new targets for the detection of the BCR-ABL transcript. This study aimed to identify these possibilities. MATERIALS AND METHODS: Human CML cell line-derived exosomes and CML-patients-derived exosomes were isolated with a size-exclusion chromatography column and ExoQuick™ exosome precipitation solution, respectively. Isolated exosomes were analysed by nested PCR to detect the BCR-ABL transcript. RESULTS: Exosomes derived from the two human CML cell lines yielded a 250-bp band. RNA sequence analysis revealed 99% sequence homology with the partial mRNA for the human BCR-ABL chimeric protein. This ~250-bp band was also observed in the exosomes derived from patients with CML. However, only patients at the blast and accelerated phases showed the exosomal BCR-ABL transcript. CONCLUSION: CML-derived exosomes could act as novel targets for the detection of the BCR-ABL transcript.