Leukemia-Derived Fibroblast Cells Produce Vascular Endothelial Growth Factor Significantly in Chronic Myelogenous Leukemia Cases.

Objective: We reported recently that myofibroblast cells in bone marrow cells of chronic myelogenous leukemia (CML)-patients were partly derived from CML clone, which was identified after long-term bone marrow cell-cultures. The generated BCR-ABL-positive fibroblast cells produced various kinds of cytokines more than that observed in the normal clone-derived fibroblast cells. Among these cytokines, we focus on vascular endothelial growth factor (VEGF)-system in this report.

Materials and Methods: Bone marrow cells were collected from chronic myelogenous leukemia cases (20 of chronic phase (CP), 3 of accelerated phase (AP), 8 of blast crisis phase (BC)), which were separated with gravity sedimentation. Obtained non-adherent mononuclear cells were cultured in DMEM with 10% FCS in the humidified 5% CO₂ incubator. When cells showed morphological changes into fibroblastoid cells, cells were treated with trypsin and further cultured. The obtained stromal fibroblast cells were sub-cloned into culturing in 96 well plates, and from which RNAs were extracted. cDNAs were synthesized, and RT-PCR was performed to identify BCR-ABL fusion product. The selected clones were further analyzed on DNA levels with FISH to identify BCR-ABL. The concentration of VEGF in the patients’ sera and in the conditioned media of non-adherent mononuclear cells, of fibroblast cells derived from normal clone and of fibroblast cells with BCR-ABL was measured with ELISA kit (R&D Systems). The expression of VEGF, VEGF receptor type-1 and VEGF receptor type-2 was also compared at RNA levels. The effect of anti-VEGF antibody to the culturing CML cells was determined using ³H-incorporation assay system.

Results and Discussion: The levels of VEGF in sera from CML patients were elevated significantly. However, in patients with CP the expression of VEGF was not observed in non-adherent mononuclear cells. In contrast, VEGF production was observed in non-adherent mononuclear cells from the patients with AP and BC. Stromal fibroblasts produced VEGF in CP, AP and BC, in which BCR-ABL-positive fibroblast cells produced at the significant levels. The expression of VEGFR-1 and -2 was detected in non-adherent mononuclear cells from CP, AP and BC. When non-adherent mononuclear cells from CML patients were cultured onto the stromal fibroblast cells, significant additive effects were observed with ³H-thymidine incorporation assays. And when anti VEGF antibody was added to the cultures, the proliferation activity was significantly decreased. These observations indicate that in CML cases, especially in CP, VEGF plays an important role for the cellular proliferation with paracrine system.