Interleukin (IL)-1 Beta and IL-6 Activated Acute and Chronic Myelogenous Leukemia-Derived Myofibroblasts to Express Leukemia-Specific Transcripts

Background and Aims: We previously reported that interleukin 1-beta (IL-1-b) stimulated bone-marrow stromal myofibroblasts from normal individuals to express CD34. And, when myofibroblasts were cultured with IL-1-b and IL-6, GATA-2 and CD45 expressed. We report here that bone marrow-derived myofibroblasts from acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) patients were also activated to express leukemia-specific molecules when IL-1-b and IL-6 were added to the cultures.

Materials and Methods: Bone marrow samples were collected from informed 7 AML patients (2 cases of M2 with t(8;21), 3 of M0, M2, and M7 positive for FLT-ITD mutation, 2 of Philadelphia-positive bi-phenotypic acute leukemia (Ph-AL)), and 3 CML ones, whose mononuclear cells were separated, and were cultured, split, and re-cultured for one month to eliminate non-adherent cells, vascular cells, and monocytes/macrophages to prepare myofibroblasts. The obtained cells were further cultured for one more month to obtain myofibroblasts with stably growth, and then leukemia-specific molecules (FLT3-ITD, BCR-ABL1 fusion (major and minor), RUNX1-RUNX1T1 fusion) were analyzed at DNA and RNA levels. Cells were further cultured with IL-1-b and IL-6 for two weeks, and morphological changes and expressions of specific molecules were observed.

Results: When bone marrow-derived myofibroblast obtained from myeloid leukemia patients were analyzed, leukemia-specific molecules were detected at DNA levels (FISH analysis detected 0.5-5% BCR-ABL1 fusion in CML- and Ph-AL-derived myofibroblast-cultures, 1-2% RUNX1- RUNX1T1 in AML with t(8;21)-cultures, and positive FLT3-ITD mutation at genomic PCR in AML-cultures); however, RT-PCR analyses revealed that leukemia-specific transcripts were not detected in all cells. When myofibroblasts were cultured with IL-1-b and IL-6, leukemia-specific transcripts were detected with RT-PCR analysis in all cases. Also, CD45, and GATA-2 expressed in these cultures.

Discussion: We reported previously that a few populations of bone marrow-derived myofibroblasts obtained from AML and CML patients expressed leukemia-specific transcripts and proteins when cultured as a separated single colony; however, when myofibroblasts were cultured not as a fractionated single colony but a whole stromal cells for a long term, expression of leukemia-specific molecules weakened and no longer confirmed. Myofibroblasts with leukemic character seem to stop their proliferation and keep dormant. One interesting point is that normal stromal myofibroblasts derived from myeloid leukemia patients can inhibit the growth of myofibroblasts that have leukemic characters. Some mechanisms may work on this observation. Also, an important issue is that myofibroblasts reportedly have an ability of antigen presentation. If some myofibroblasts can express leukemia-specific molecules as well as HLA when cultured with IL-1-b and IL-6, they can act as a leukemia-specific antigen-presenting cell. We now attempt to research this theme to develop a new cell-mediated vaccine for myeloid leukemia.