PS1007 NORMAL AND LEUKEMIC BONE MARROW STROMAL CELLS HAVE DIFFERENT CAPACITIES FOR MITOCHONDRIA TRANSFER TO ACUTE MYELOID LEUKEMIA CELLS
Authors: M.C. Do Nascimento, D.A. Pereira‐Martins, V.M. de Deus Wagatsuma, J.A. Machado‐Neto, E. Magalhães Rego
Journal: HemaSphere (2019)

Background: Recent data pointed to the association of disease progression and relapse in acute myeloid leukemia (AML) with the evidence of functional mitochondria (mt), and reactive oxygen species (ROS) transference from bone marrow stromal cells (BMSCs) to the blasts cells. Nevertheless, differential performances in this transference process among normal and leukemic‐BMSCs in co‐culture systems were not well established. Aims: To evaluate the presence of a favoring microenvironment for the AML's cells survival provided by leukemic‐BMSCs through the reduction of the cellular damage associated with mt transference process in co‐culture arrangements. Methods: BMSCs were isolated and expanded from bone marrow aspirates of 7 patients diagnosed with de novo AML (Based on European Leukemia Net risk stratification system: 1 favorable, 2 intermediate I and 4 adverse; median age: 51y; 57% male) and 4 age‐and sex‐matched hematologically healthy donors (median age: 30y; 66% males). By adherence, BMSCs were expanded until the fourth passage and immunophenotypically characterized for presence of CD73/CDw90/CD105 expression with CD34/CD45 negative staining. For co‐culture experiments, 3 AML cell lines (Kasumi‐1, OCI/AML3, and THP1) were used after synchronization by thymidine block and the collection of fractions following the protocol of Jing et al. for normal hematopoietic stem cells (Jing et al, Hematologica 2010). Cells were evaluated for total (H2DCFDA) and mitochondrial (MitoSOX) ROS production, mitochondrial content (Mitotracker) and proliferation index (Ki‐67) by flow cytometry after 3 and 5 days of co‐culture. For phase‐bright/dim cells, the analyses were conducted discerning CD45 + /CD73 + cells, being bright cells those in loosely adhered to the surface and dim those tightly attached to BMSCs. Gene expression was evaluated for genes related with BMSCs metabolism, such as TNFA , SLIT2 and PRGC1A. Results: BMSCs isolated from AML patients presented reduced CFU‐F capacity in comparison to healthy‐BMSCs ( P &lt; .05). In co‐culture, OCI/AML3 cells exhibited increased proliferation and reduced mtROS in non‐adherent cell phase after co‐culture with leukemic‐BMSCs ( P &lt; .05). No differences regarding proliferation, ROS production and mt levels were observed for the other cell lines in this population. Regarding phase‐bright, THP1 cells had increased proliferation rate at day 5 when co‐cultured with leukemic‐BMSCs (110%) than those normal‐BMSCs co‐culture (81%; P = .04). Analyzing all AML cells combined, the mtROS levels are reduced in the phase‐bright CD45 + fraction when co‐cultured with leukemic‐BMSCs compared to healthy‐BMSCs ( P = .005). In agreement, the AML CD73 + cells exhibited a reduction of 40% in the mt levels ( P = .014). THP1 cells in closely contact with leukemic‐BMSCs (phase‐dim) showed decreased total ROS levels in 5 days of co‐culture ( P = .005) associated with reduction in the mt content ( P &lt; .05). OCI/AML3 cells (CD45 + ) exhibited increased proliferation in phase‐dim compartment ( P &lt; .05). Take together, the AML cell lines showed reduced total/mtROS levels with increased mt number ( P &lt; .05). Equal to phase‐bright, leukemic phase‐dim cells (CD73 + ) had a reduction in mt levels ( P &lt; .05). Finally, the relative expression of SLIT2 and PRGC1A present a significantly decrease in AML‐BMSCs cells in comparison to healthy BMSCs ( P &lt; .05). Summary/Conclusion: Our data suggest in mt donation capacity from BMSCs to AML blasts in healthy and leukemic BMCS. Therefore providing a further understanding of the complex nature of tumor metabolism, not only in the blasts cells, but also within the microenvironment which supports it.