Exosomes are small sized vesicles (30-100nm) actively secreted by a wide variety of cells in body fluids. Exosomes are known carriers of mRNA, microRNA and proteins, and have been shown to transform the recipient cells. Exosomes are important in regulation of physiological processes in our body and depending on their content they can induce activation, proliferation, differentiation or apoptosis of the recipient cells. Increasingly, exosomes are studied for their potential as both indicators of disease initiation, progression, drug resistance and as a prospective new treatment approach. We hypothesize that identifying proteins that are differentially expressed on the exosomes among normal and leukemic cells will provide signature biomarkers and offer insight into the mechanism(s) contributing to the transformation of the leukemic niche. Exosomes were extracted from the serum samples of healthy and acute myeloid leukemia (AML) patients and were found to be more abundant in AML patients (p=0.049). Western blot analysis of the AML derived exosomes pellet confirmed the presence of CD63 and tumor susceptibility gene 101 (TSG101); two exosomal marker proteins indicating a good exosome preparation. Further, using ultracentrifugation, we prepared exosomes from the peripheral blood of two normal healthy individuals and MV411 (FLT3-ITD+) & KG1A cell lines after culturing in conditioned media in hypoxia for 48 hours. 2-Dimensional polyacrylamide gel electrophoresis (2D-PAGE) was performed from lysates of 100 µg of exosomes, and subsequently differently expressed spots were identified using 2D-PAGE analysissoftware. We identified 15 spots showing significantly up-regulated and 8 down-regulated patterns of expression in the exosomes from AML cell lines, relative to the pattern seen from the normal exosome lysates. In gel digestion with trypsin was performed, and proteins were identified in each differentially expressed spot using the Agilent 6510 QTOF Mass Spectrometer. The Scaffold Proteome Software was used to validate MS/MS based peptide and protein identifications. The Database for Annotation, Visualization and Integrated Discovery (DAVID ) v6.7 was used to interpret the biological functions of these differentially expressed proteins.
In up-regulated datasets, the top enriched pathways and annotations (p<0.05) were proteins involved in: the core histone H2A/H2B/H3/H, Ras protein signal transduction, DNA packaging, telomere maintenance, translational elongation, the G1 to S phase transition, Aminoacyl-tRNA synthetase, negative regulation of programmed cell death, Transforming protein RhoA (multidrug resistance protein), lung cancer oncogene 7(HLC-7), Growth factor receptor-bound protein 2(GRB2), Nucleoside diphosphate kinase A (NME1),Transforming growth factor-beta-induced protein(TGFBI), regulation of DNA repair, and secretary granule associated Rab proteins. All these leukemia exosomal up-regulated proteins have already been known to be involved in tumor transformation. The down-regulated leukemia exosomes proteins were involved in negative regulation of cytoskeleton organization, histone methyltransferase complex, Phosphatidylinositol transfer protein, EHD1(tumor suppressor gene), Cysteine and methionine metabolism, and calcium-dependent phospholipid binding. More functional studies on the protein content of exosomes are needed to decipher their potential functions in leukemogenesis. Identification of unique exosomal protein markers from leukemia patients will allow for better identification of their role to stratify patients according to their disease status, and enable us to elucidate their specific role and dysfunctional status in hematopoietic stem cells.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.