Microvesicles Transcripts As Hallmark and Vector From Leukemic Parental Cells

Abstract 1459

Microvesicles (MVs) are nano-sized lipid bodies (100–1000 nm in diameter) that are released by cells, both in vitro and in vivo. MVs are secreted in the extracellular space and as carriers of proteins, mRNA and miRNA represent vectors from donor to target cells involved in intercellular communication. MVs modulate the functional state of receiving cells through fusion with the target cell. In leukemia MVs were suggested to modulate the hematopoietic niche.

In this study, we investigated the transcriptome of MVs released from leukemic cell lines. In particular, we analyzed K562, a BCR-ABL positive human erythromyeloblastoid leukemia cell line, and we collected RNA both from cells and MVs released in culture. Since many different methods have been described for microvesicles isolation and description of MV populations are often ambiguous, an accurate protocol has been developed in order to select a defined MVs population. In detail, for MVs isolation cell culture medium was centrifuged at 2500g for 15 minutes. These centrifugations allowed to delete cells and bigger bodies. Then supernatant was filtered by means of a 1.2um filter, in order to keep only vesicles of defined physical measure and to eliminate residual bigger vesicles, such as apoptotic bodies (>1000nm). The filtration allowed an accurate selection of a well defined MVs population. The filtered medium was then centrifuged at 18000g for 1h at 4°C. MVs were resuspended in Trizol for RNA isolation. Also RNA of cells, from which MVs have been released, was extracted using the Trizol method, according to manufacturer instructions. Cell line RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by means of NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.). To check MVs RNA integrity we determined the ratio of 5' amplicons to 3' amplicons of housekeeping transcripts by Real Time PCR. We screened the presence of several housekeeping transcripts in MVs and selected GAPDH as reference. The GAPDH 5':3' amplicon ratio should equal 1 to ensure MVs RNA quality. Afterwards, reverse transcription–polymerase chain reaction (RT-PCR) amplification was performed. cDNA was synthesized from 1mg of total RNA.

We analyzed the gene expression profile of K562 cell line and MVs, released from these cells, using GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix). Gene expression data have been compared between cells and MVs. Ninety-one percent of probe sets showed a similar expression (fold-change lower than 1.5) between cell line and MVs. Thirteen percent of these probes were recognized as present call probe sets in both cell line and MVs. Analysis of probe sets using DAVID Functional Annotation Bioinformatics Microarray Analysis revealed a conservation of MVs gene transcripts involved in pathways of cell function such as RNA processing, protein translation, aminoacid metabolism and cell respiration. Remarkably, in MVs we observed a high presence of gene transcripts coding for protein belonging to the Chronic Myeloid Leukemia pathway that are expressed downstream of the BCR-ABL tyrosin kinase fusion protein. The maintenance of this pathway in MVs highlights the intrinsic peculiarity of BCR-ABL positive K562 cells apparently also conserved in MVs mRNA outfit representing a hallmark of the parental cell from which they have been released.

Furthermore, 3.8% of the probe sets resulted to be up-regulated in MVs compare to the cell line (fold-change higher than 1.5). In MVs, we observed a higher expression of genes belonging to cell communication pathways, adhesion and migration processes, membrane and ionic channels signals.

In conclusion, we isolated MVs released by K562 leukemic cells using an accurate selection of the MV population based on physical measures and for the first time a whole transcriptome gene expression analysis has been performed comparing K562 cells and MVs. Moreover, we identified an enrichment of transcripts coding for proteins involved in several essential pathways in the MVs supporting the hypothesis of a functional selection from the parental cell transcriptome and underlining the relevant role of MVs as vehicles of messages to target cells.