Flow cytometry-based quantification of cytokine levels in AML patients plasma and cell culture supernatants

Bone marrow stromal cells (BMSCs) have been found to support leukemic cell survival; however, the mechanisms responsible are far from being elucidated yet. The main aim of the current study is to identify particular cytokine/chemokine patterns of acute myeloid leukemia (AML) cells, and, on a longer term, to correlate them with the patient outcome and response to therapy. Therefore, the influence of BMSCs on in vitro modulation of cytokine secretion (IL-1beta, TNF-alpha, IL-10, IFN-gamma, IL-4, IL-5, and IL-2) by AML cells as well as the AML cells supportive capacity of BMSCs-derived soluble factors was investigated.

With this purpose, we used an in vitro experimental model consisting in the evaluation of the effect of BMSC confluent layers-conditioning medium (BMSC-CM) on M4/5 AML cell cultures.

Our results show that BMSC-CM from both AML patients and healthy subjects conferred a substantial beneficial effect on AML cells throughout the culture (p=0.0002 and 0.0020 respectively at 24 hours and p=0,0013 and 0,0030 respectively at 72 hours), with a temporary increase in AML cell viability conferred by BM plasma from AML patients. Significant differences were observed with respect to IL1 b secretion which was upregulated in AML cell cultures both after 24 and 72 hours following the addition of AML-BMSC-CM, in contrast to control-BMSC-CM.

Our results suggest the contribution of BMSCs from AML patients to the generation of particular factors which may be key players involved in the in vivo maintenance of the malignant clone.