Do Microenvironmental Factors Play Important Role in Supporting the Maintenance of Leukemic Stem Cells of Chronic Myelogenous Leukemia?

Background : In the era of tyrosine kinase inhibitor(TKI) for chronic myelogenou leukemia(CML), it is becoming more important to develop the way to get rid of the leukemic stem cells(LSC), as there is frequent relapse when TKI therapy is stopped even when molecular remission is achieved. Some researchers reported macrophages and Wnt signaling in bone marrow microenvironment might play significant role in the development and the progression of CML.

Methods : We isolated CD34⁺CD38⁻ LSC from bone marrow cells from 8 patients with CML chronic phase and also CD34⁺CD38⁻ normal hematopoietic stem cell from 7 normal controls using magnetic cell separation system. CD34⁺CD38⁻ cells were cultured on stromal cells for 5 weeks and transfered to methylcellulose media for long-term culture initiating cell(LTC-IC) assay. We checked whether there is a difference in LTC-IC capacity of LSC when cultured on leukemic or normal stromal feeder cells. CD45⁻ stromal supporting cells were established by 3-week culture after 12Gy irradiation of CD34⁻ bone marrow cells. We studied the expression of VCAM, ICAM, E-selectin in stromal cells and the presence of BCR-ABL transcript. Wnt and β-catenin, frizzled, Notch1 were analyzed in the LSCs. CD14⁺ cells were isolated and added in LTC-IC assay whether the presence of leukemic or normal CD14⁺ cells would make a change in the maintenance of LSCs.

Results : The number of CD34⁺CD38⁻ cells in CML was in the range of 1.3 × 10³ to 9.2 × 10⁴, that we could do only two sets of 2 × 2 LTC-IC assay that statistical analysis was impossible. LTC-IC frequency of LSCs was about 5-times higher compared to that of normal stem cells irrespective of the kind of stromal cells used. CD14⁺ cell co-culture did not changed significantly the LTC-IC frequency both in normal and LSCs. Wnt 2B, 5A and 10B were highly expressed in LSCs irrespective of the stromal cells. β-catenin was also strongly expressed in LSCs compared to normal stem cells. Notch1 was expressed only in CD34⁺CD38⁻ LSCs but not in normal stem cells. The kind of stromal cells or the addition of CD14⁺ cells in LTC-IC assay did not affected the Notch1 expression in LSCs.

Conclusion : CML CD34⁺CD38⁻ LSCs seem to have more LTC-ICs and wnt and notch pathway may play important role in the maintenance of LSCs that could be therapeutic targets.