Exosome-mediated in vitro BCR-ABL p210 transcript horizontal transfer between leukemic and bone marrow stromal cells
Anna N. Parfenenkova, Ildar M. Barkhatov, Anton A. Kremlev, Boris V. Afanasyev
Raisa Gorbacheva Memorial Research Institute of Pediatric Oncology, Hematology and Transplantation, Pavlov First Saint Petersburg State Medical University, St. Petersburg, Russia
Contact: Dr. Ildar M. Barkhatov, PhD
E-mail: email@example.com, firstname.lastname@example.org
The key challenge of molecular genetics in oncohematology is determination of molecular markers, which can be applied to malignant cells in diagnostics of the disease is the analysis of molecular markers, which can be applied to malignant cells in diagnostics of the disease, its progression, and relapse risks. One of the most promising test subjects to approach this might be exosomes, extracellular membrane vesicles of 30-150 nm in size, that potentially contain such oncogenic markers. Exosomes are among important players in intercellular communication and are able to transfer a variety of biopolymers, which can potentially affect the results of minimal residual disease (MRD) determination. Since the main role in the regulation of hematopoiesis belongs to mesenchymal stromal cells (MSCs) of the bone marrow, we suggest that the exosomes that persist in the bone marrow, may be asource of the detectable transcript can be as and their migration into stromal microenvironment cells.
Materials and methods
Exosome isolation was performed by differential ultracentrifugation of К562 cell line (Chronic Myeloid Leukemia) conditional media. The isolated exosomal particles were analyzed using laser correlation spectroscopy approach including zeta-potential assessment to ensure electrokinetic capability to interact with biological systems. Transfer of BCR-ABL p210 transcript was performed in a 24-well plate. After that, 300 μl of serum-free growth medium with exosomes (isolated from about 70 millions cells per well) was added. Co-cultivation of K562 and MSC was performed in a 24-well plate with inserts of semi-permeable membrane (pore diameter 0.4 μm).
The largest number of vesicles in the sample corresponded to a presumed size of exosomes with average diameters in the range of 52÷107 nm and a mean value of 79±20 nm. The results of the study showed that the distribution of the average sample size in diameter is in the range of 51.8÷107.0 nm and a mean value of 78.96±20.03 nm. The zeta potential corresponds to stable particles and is on mean equal to 29.±7. mV. According to Real-Time PCR, the relative representation of the BCR-ABL p210 chimeric transcript in exosomes ranged between 44÷864 copies/ml of conditioned medium with a median value of 154 copies. After MSCs co-cultivation with K562, the relative content of BCR-ABL p210 mRNA in recipient mesenchymal cells was between 0.3 to 11.4% of ABL1 level, with a median of 0.29%. As a result of exosome-mediated transfection, the relative content of the chimeric transcript in the MSCs of healthy donors ranged from 0.01 to 15.88%, with a median value of 0.11%.
Exosomal fraction of microvesicules of K562 contain the chimeric BCR-ABL p210 mRNA transcript, a marker of CML, and are able to its transfer to the bone marrow MSCs of healthy donors. It has been confirmed during as experiments of co-cultivation, and direct transfection with exosomes. The amount of transcript transferred to stromal cells is comparable to the one in CML patients with a minimal residual disease, which proves the potential role of exosomes in contribution to the results of MRD determination.
Extracellular vesicles, exosomes, tumor exosomes, bone marrow stromal cells, cell-cell interactions.