Abstract 2606: Bone marrow microenvironment contributes to stem cell phenotype in acute myeloid leukemia.
Authors: Battula Venkata Lokesh , Chen Ye , Yang Frank , Konopleva Marina , Andreeff Michael 
Published: August 2014

Acute myeloid leukemia (AML) is a heterogeneous disease that originates in the bone marrow (BM). BM consists of a complex hypoxic micro-environment that includes osteoblasts, osteoclasts, and mesenchymal stromal cells (MSCs).We previously reported that co-culture with BM-derived MSCs induces anti-apoptotic and drug resistant genes in leukemic cells. As drug resistance is a characteristic of cancer stem cells (CSCs), we hypothesized that components of the micro-environment such as MSCs and hypoxia induce stem cell phenotype in leukemic cells. To test the hypothesis, AML cell lines including OCI-AML3 and KG1 cells were cultured with or without stromal cells at 1% (hypoxia) or 21% (normoxia) oxygen for 1 or 3 or 5 or 7 days and tested for stem cell related functions. Co-culture of leukemic cells with bone marrow-derived MSCs induced Aldehyde dehydrogenase (ALDH) activity in ‘attached fraction’ of OCI-AML3 cells from 5.8%±3.3% to 36.9%±4.7% in a time dependent manner. Interestingly, ALDH activity was also increased in ‘floating fraction’ of OCI-AML3 cells from 2.52%±0.9% to 38.5%±5.7% when co-cultured with MSCs in a time dependent manner, suggesting that cell to cell contact is not necessary to observe this phenotype. In addition, culture of OCI-AML3 cells in 1% oxygen alone increased ALDH+ population from 0.5%±0.1% to 25.6%±3.2% after 7 days and no-further increase was observed when co-cultured with MSCs in hypoxic conditions suggesting that stroma or hypoxia alone are sufficient for induction of stem cell activity in leukemia cells. Another well characterized property of hematopoietic stem cells is generation of side population (SP). KG1 cells, when co-cultured with MSCs, induced side population cells (from 0.2%±0.1% to 2.6%±0.8%); Similarly, OCI-AML3 cells induced SP cells (from 0.2%±0.1% to 10.2%±2.4%) when cultured under hypoxia conditions, indicating that the micro-environment induces stemness in leukemia cells. Interestingly, the size of leukemic cells was reduced from 19.5±2.3μm to 14.4±1.7μm when cultured in 1% oxygen, suggesting low metabolic activity of these cells. Cell cycle analysis revealed that hypoxia inhibited cell cycle at G0/G1 phase of the cell cycle. In case of OCI-AML3 cells, S-phase cells were decreased from 14.5%±0.1% to 5.02±0.1% and the cells in G1-phase were increased from 68.02%±3.1% to 87.62%±2.7%. In case of KG1 cells, S-phase cells were decreased from 26.24%±2.2% to 17.07±1.3% and the cells in G1-phase were increased from 68.02%±3.1% to 77.44%±4.7% indicating cells became more quiescent when cultured under micro-environmental conditions Gene expression analysis revealed that embryonic stem cell markers, including oct4, nanog and sox2, were significantly up regulated by 2-3 fold when cells were cultured at 1% oxygen. These findings indicate that components of the micro-environment induce stem cell phenotype in leukemic cells which may help their survival during chemotherapy.

Citation Format: Venkata Lokesh Battula, Ye Chen, Frank Yang, Marina Konopleva, Michael Andreeff. Bone marrow microenvironment contributes to stem cell phenotype in acute myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2606. doi:10.1158/1538-7445.AM2013-2606