Microvesicles microRNAs Reflect and Affect Progression of Acute Myeloid Leukemia and Could Serve As a Biomarker of Disease Dynamics
Authors: Tzoran Inna , Rebibo-Sabbah Annie , Brenner Benjamin , Aharon Anat 
Published: October 2019

Abstract
Background: Acute myeloid leukemia (AML) is characterized by rapid growth of abnormal blast cells that accumulate in the bone marrow and interfere with the production of normal blood cells. Microvesicles (MVs) are shedding from various cells and express antigens reflecting their cellular origin. Our previous study has demonstrated a correlation between the level of MVs originating from blast cells (CD117+ MVs) in the peripheral blood and the amount of CD117 positive blast cells in the bone marrow (BM) at diagnosis and in remission (r=0.49, p=0.0025; r=0.6, p=0.01; respectively). Additional assessment of CD117 expression on MVs obtained at diagnosis in patients who died despite achieving a remission revealed significantly higher values compared to those found in patients who were alive at 3 years of follow-up (1.9 vs. 0.6; p=0.01). A similar trend was found in CD34 positive MVs (1.2 vs. 0.13; p=0.01). It has, therefore, been concluded that circulating MVs of AML patients might serve as a biomarker of leukemia progression. MVs also contain cytokines and micro-RNA (miRNA) that are critical for cell development, proliferation and apoptosis. MVs are the major transport vehicle for miRNAs, and serve as a unique mode of genetic exchange between cells. To this end, several studies have shown that cancer stem cells regulate tumor environment through MVs.
The current study aimed to explore the potential role of MV miRNAs as a biomarker of disease progression in AML and to study MV effects on the bone marrow leukemic niche.
Methods: Blood and bone marrow samples were collected from healthy controls and patients with AML at diagnosis and upon achievement of first remission. MV effects on the BM mesenchymal stem cells (BM-MSC) and endothelial cells were studied using confocal microscopy, migration and proliferation assays and the RT-PCR method. miRNA expression was screened by NanoString technology and validated by RT-PCR.
Results: The study was approved by the Institutional Review Board of the Rambam Health Care Campus (Approval #0351-10). Blood and bone marrow samples were collected from 43 AML patients and 4 random healthy volunteers after obtaining written informed consent. Sixty seven percent of patients remained alive and achieved remission following induction chemotherapy about one month after the diagnosis.
Co-incubation of fluorescent-labeled BM-MVs (CD33+/CD117+) of AML patients with unlabeled BM-MSC resulted in incorporation of >80% of MVs to the cells. Patient BM-MVs of obtained at diagnosis induced a higher migration rate of BM-MSC compared to MVs obtained from healthy controls (p<0.01). Patient BM-MVs also induced a significantly higher proliferation rate of BM-MSC (p<0.05).
Screening of patient BM-MVs demonstrated that the expression of some miRNAs was high at diagnosis but decreased in remission, while other miRNAs exhibited an opposite trend. Notably, these alterations were observed in miRNA-181a, that is known to play a role in normal and malignant hematopoiesis. Specifically, miRNA-181a levels in BM-MVs of AML patients were at least 10 times higher at diagnosis than in remission or in healthy controls (p<0.05). Similar results were found in miRNA-181a of circulating MVs of these patients, particularly in those who were alive at 1 year of follow-up. However, in patients that died within the first year, miRNA-181a expression at diagnosis was lower compared to healthy controls (2 times less, p=0.035) or to patients who were alive at the follow-up evaluation (7 times less, p= 0.0097). Co-incubation of BM-MSC with patient BM-MVs, obtained at diagnosis, resulted in a 4-time higher expression of miRNA-181a in these cells compared to untreated ones (p<0.05).
Conclusion: MV-miRNAs of AML patients are involved in the regulation of tumor BM microenvironment, affecting BM-MSC migration, proliferation and gene expression. MV-miRNAs reflect and affect AML progression and may serve as a biomarker of disease dynamics.

Disclosures
No relevant conflicts of interest to declare.