Bone Marrow Stromal Cells May Have Genetic Aberrations and Are Capable to Gain Them in a Culture
Authors: Barkhatov Ildar , Tsvetkov Nikolai , Davydova Elena , Shakirova Alyona , Potter Alisa , Zubarovskaya Ludmila S , Afanasyev Boris V 
Published: October 2019

Abstract
Introduction. Stromal microenvironment posses a key role in the regulation of both normal hematopoiesis and its reconstitution after hematopoietic stem cell transplantation (HSCT). Recent data supports the idea that bone marrow stromal cells (BMSC) also have genetic aberrations and may tightly involved in the pathogenesis of HSCT complications. These findings justify the need for more detailed study of genetic aberrations in BMSC.
The aim of this study was to evaluate genetic aberrations inBMSC and check the ability to gain them in coculture system.
Materials and methods. The interaction of BMSC with hematopoietic tumor cell lines bearing specific genetic aberrations (BCR-ABL fusion transcript for K-562 and JAK2 V617F mutation for Uke-1 cell line) was investigated in stromal cells harvested from 17 patients and 8 healthy donors. We performed cultivation of BMSC monolayer and tumor cells suspension using semipermeable membrane plates inserts with different pore size (0,4 μm and 3,0 μm) in order to exclude direct cell-to-cell contact. We looked also forexisting specific genetic aberrations (point mutations and fusion transcripts) in BMSC of patients with the respective aberration in their leukemic clone. For this purpose we used both karyotyping (7 patients) and RQ-PCR method. BMSC were examined by flow cytometry to evaluate the possible contamination with cells of hematopoietic lineage.
Results.
We investigated the BMSC karyotype in seven patients and only one case led us to a remarkable finding. The clonal chromosomal rearrangement t(1;7) was detected in 25% of BMSC metaphases. Interestingly, this aberration was never detected in patient's leukemic cells. Moreover, at the moment of this investigation patient had full clinical and hematological remission, full donor chimerism and no signs of minimal residual disease (MRD).
We also examined BMSC from leukemia patients bearing recurrent genetic abnormalities and in one case the leukemia-specific marker was detected by RQ-PCR - we observed expression of ETV6-RUNX1 gene (≈0,02%) in BMSC by patient with t(12;21) acute lymphoblastic leukemia. At the moment of BMSC culture initiation ETV6-RUNX1 expression in patient's bone marrow was detected at high level (ETV6-RUNX1/ABL*100=321%). Before carrying out RNA extraction BMSC were harvested after the second passage and no contamination with CD45+/CD34+ cells by flow cytometry was observed (50,000 events collected from the sample). When BMSCs and Uke-1 cell line were cocultured by using of 3,0 μm pore culture dishes inserts, the BMSC population gained the Jak2V617F mutation (allele burden ≈ 30,39%). We reproduced similar experiments with the K-562 cell line and got similar results - CD45+ cells were also detected in BMSC population (≈ 30%). Moreover we detected CD45+ non-cellular particles by flow cytometry analysis. Implying K-562 cells are not likely to cross the semipermeable membrane (3,0 μm pores versus 20,0 μm cells as measured during microscopy). Besides BCR-ABL gene expression in BMSC was detected by RQ-PCR (BCR-ABL/ABL*100=19%). We repeated same test with 0,4 μm pore inserts and without them in order to check implication of cell-to-cell interaction. We didn't obtain any similar results with smaller pores, but the fusion transcript was detected in CD45- BMSC population when these two cell populations weren't devided. Both findings point out at possible horizontal gene transfer mediated by membrane vesicles larger than 0,4 μm and direct whole cell fusion.
Conclusion
Our data stands for the existence of horizontal gene transfer between leukemic clone and BMSC. This process seems to be mediated by membrane vesicles larger than 0,4 μm in size, though cell fusion can also take place. We also confirmed the fact BMSCs can bear clonal genetic rearrangements which are not specific to tumor cell populations. These findings show tight interaction between tumor and microenvironment cells and can partly explain nature of PCR-based MRD persistence in complete remission.

Disclosures
No relevant conflicts of interest to declare.