Bone Marrow Mesenchymal Stromal Cells Protect Acute Lymphocytic Leukemia Cells From Cytotoxic Agents Via MAPK/Erk and Wnt/β-Catenin Signal Pathways
Authors: Hu Kaimin , Liu Lizhen , Wang Binsheng , Wu Kangni , Fu Shan , Gu Yanjun , Lai Xiaoyu , Yu Xiaohong , Huang He 
Published: October 2019

Abstract
Abstract 4736
Acute lymphoblastic leukemia (ALL) remains one of the greatest challenges in oncology. Relapsed ALL is a leading cause of death in young people, and further improvements in outcome will required the development of therapeutic approaches directed against rational therapeutic targets. Increasing evidence indicated that the bone marrow microenvironment plays a crucial role in the pathogenesis of leukemia by promoting tumor cell growth and survival as well as drug resistance. As a pivotal component of the bone marrow microenvironment, how bone marrow derived mesenchymal stromal cells (MSCs) affect ALL cells and the underlying mechanisms remain unclear.
In this study, bone marrow was obtained from healthy adult donors and MSCs were cultured and identified as previous report. Our study applied the co-culture system to mimic the bone marrow microenvironment. To study the effect of bone marrow stroma on ALL cells, three different sub-types of ALL cell lines including Sup-B15, Reh, and MOTL-4 were cultured with and without an underlying confluent layer of MSC. Survival of ALL cells was detected by CCK-8 kit and apoptosis was tested by PI/Annexin V assay. Cell cycle was evaluated by flow cytometry. In addition, Western-bloting was used in mechanism study.
The results demonstrated that MSCs improved the survival of ALL cells induced by the cytotoxic agents Idarubicin (IDA) and Etoposide (Vp-16). Number of viable ALL cells were increased remarkably in presence of MSCs. In Vp-16 treatment group, the ratios of viable ALL cell were 1:1.17±0.05 (Sup-B15: Sup-B15+MSC, P<0.05), 1:1.20±0.02 (Reh: Reh+MSC, P<0.05) and 1:1.74±0.16 (MOLT-4: MOLT-4+MSC, P<0.05) respectively. In IDA treatment group, the ratios were 1:1.39±0.11 (Sup-B15: Sup-B15+MSC P<0.05), 1:1.67±0.05 (Reh: Reh+MSC, P<0.01) and 1:1.06±0.07 (MOLT-4: MOLT-4+MSC) respectively. Our data also indicated that coculture with normal MSCs protected ALL cells from apoptosis induced by IDA and Vp-16. The propotion of apoptotic cells was reduced in presence of MSCs both in Vp-16 treatment (Sup-B15: 26.46 ±0.70 vs. 22.00±5.35%, Reh: 18.16±2.09 vs. 16.94±4.34%, MOLT-4: 71.54 ±19.15 vs.62.59±35.63%) and IDA treatment (Sup-B15: 46.57±3.74 vs. 28.86±5.99, Reh: 48.58±6.73 vs. 36.49±5.33%, MOLT-4: 71.54 ±43.72±6.76 vs. 25.04±6.84, P<0.05 respectively).
To clarify the mechanisms of the protective role of MSCs, we compared the expression levels of apoptosis and survival related proteins in ALL cells cultured without or with MSCs. The data indicated that MAPK/Erk,Wnt/β-catenin signal pathways were activated in ALL cells co-cultured with MSC. ALL cells showed significantly higher expression of p-Erk and β-catenin in presence of MSCs. At the same time, the cycle inhibition protein p21, mitochondria mediated apoptosis proteins, and the oncogene c-myc were down-regulated.
Our study provides experimental basis for the exploring of novel ALL therapy that targets the microenvironment.

Disclosures:
No relevant conflicts of interest to declare.